Molecular Engineering of E. coli Bacterioferritin: A Versatile Nanodimensional Protein Cage.

Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering.

Microneedle Array Technique for the Longitudinal Extraction of Interstitial Fluid without Hair Removal.

Interstitial fluid (ISF) bathes the cells and tissues and is in constant exchange with blood. As an exchange medium for waste, nutrients, exosomes, and signaling molecules, ISF is recognized as a plentiful source of biomolecules. Many basic and pre-clinical small animal studies could benefit from an inexpensive and efficient technique that allows for the in vivo extraction of ISF for the subsequent quantification of molecules in the interstitial space. We have previously reported on a minimally invasive technique for the extraction of ISF using a 3D-printed microneedle array (MA) platform for comprehensive biomedical applications. Previously, hairless animal models were utilized, and euthanasia was performed immediately following the procedure. Here, we demonstrate the technique in Sprague Dawley rats, without the need for hair removal, over multiple extractions and weeks. As an example of this technique, we report simultaneous quantification of the heavy metals Copper (Cu), Lead (Pb), Lithium (Li), and Nickel (Ni) within the ISF, compared with whole blood. These results demonstrate the MA technique applicability to a broader range of species and studies and the reuse of animals, leading to a reduction in number of animals needed to successfully complete ISF extraction experiments.

Feasibility of Interstitial Fluid Ketone Monitoring with Microneedles.

Diabetic ketoacidosis (DKA) is one of the most dangerous and costly complications of diabetes, accounting for approximately 50% of deaths in diabetic individuals under 24 years. This results in over 130,000 hospital admissions yearly and costs the USA over USD 2.4 billion annually. Earlier diagnosis, treatment, and management of DKA are of critical importance to achieving better patient outcomes and preventing prolonged hospital admissions. Diabetic patients undergoing stress from illness or injury may not recognize early ketosis and often present advanced ketoacidosis, requiring intensive care admission. We have recently developed a microneedle-based technology to extract dermal interstitial fluid (ISF) from both animals and humans, which could enable wearable sensors to rapidly detect ketosis. Metabolite concentrations in ISF may differ in urine and blood and could likely represent local metabolic conditions in the surrounding tissue. Development of a wearable ketone detector will require an understanding of ketone concentrations and kinetics in ISF. Here, we report data that is first of its kind, with regard to the ketone concentrations present in the dermal ISF of rats, their correlation to blood, and the possible impact on the development of a wearable ISF "early warning system" to prevent morbidity from DKA. We extracted ISF, using minimally invasive microneedle arrays, from control Sprague Dawley rats and 17 h fasted rats. ISF and blood ketone levels were measured using a common glucose/ketone meter and strips. Local tissue concentrations of glucose were similar to those of blood, with an average blood to ISF glucose ratio of 0.99 ± 0.15 mg/dL. ISF ketones (0.4 ± 0.3 mM) were significantly higher (p = 4.2 × 10-9), compared with blood ketones (0.0 ± 0.0 mM). Although the fasted animals had slightly higher ISF ketones (1.3 ± 1.1 mM) compared with blood ketones (1.0 ± 1.0 mM), the difference was not significant (p = 0.3). This suggests ISF could possibly be useful as a surrogate for blood when determining ketone levels within a clinical setting.

Evaluation of the Hologic Aptima Combo 2 Assay for Detection of Neisseria gonorrhoeae from Joint Fluid Specimens.

Gonorrhea is a sexually transmitted bacterial infection caused by Neisseria gonorrhoeae. Nucleic acid amplification testing is the preferred method for routine diagnosis of gonorrhea from urogenital specimens, but culture is commonly used for diagnosis of disseminated infections, including gonococcal arthritis. The Hologic Aptima Combo 2 (AC2), a transcription-mediated amplification assay, is FDA and Health Canada licensed for detection of N. gonorrhoeae and Chlamydia trachomatis from urogenital, rectal, and pharyngeal specimens, but not joint fluid. In the current study, we compared the performance of microscopy, culture, and the AC2 for detection of N. gonorrhoeae from 170 joint fluid specimens. A total of five specimens were culture-positive, whereas 14 were AC2-positive. Gram-negative diplococci, characteristic of Neisseria, were observed in only two joint fluid specimens. Complementary testing confirmed the presence of N. gonorrhoeae in seven discordant (i.e., culture-negative/AC2-positive) specimens. These results indicate that the AC2 is more sensitive than culture for the diagnosis of gonococcal arthritis.

In vitro susceptibility of common bacterial pathogens causing respiratory tract infections in Canada to lefamulin, a new pleuromutilin.

Background: Community-acquired pneumonia (CAP) is a significant global health concern. Pathogens causing CAP demonstrate increasing resistance to commonly prescribed empiric treatments. Resistance in Streptococcus pneumoniae, the most prevalent bacterial cause of CAP, has been increasing worldwide, highlighting the need for improved antibacterial agents. Lefamulin, a novel pleuromutilin, is a recently approved therapeutic agent highly active against many lower respiratory tract pathogens. However, to date minimal data are available to describe the in vitro activity of lefamulin against bacterial isolates associated with CAP. Methods: Common bacterial causes of CAP obtained from both lower respiratory and blood specimen isolates cultured by hospital laboratories across Canada were submitted to the annual CANWARD study's coordinating laboratory in Winnipeg, Canada, from January 2015 to October 2018. A total of 876 bacterial isolates were tested against lefamulin and comparator agents using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method, and minimum inhibitory concentrations (MICs) were interpreted using accepted breakpoints. Results: All S. pneumoniae isolates tested from both respiratory (n = 315) and blood specimens (n = 167) were susceptible to lefamulin (MIC ≤0.5 µg/mL), including isolates resistant to penicillins, clarithromycin, doxycycline, and trimethoprim-sulfamethoxazole. Lefamulin also inhibited 99.0% of Haemophilus influenzae isolates (regardless of ß-lactamase production) (99 specimens; MIC ≤2 µg/mL) and 95.7% of methicillin-susceptible Staphylococcus aureus (MSSA) (MIC ≤0.25 µg/mL; 70 specimens) at their susceptible breakpoints. Conclusions: Lefamulin demonstrated potent in vitro activity against all respiratory isolates tested and may represent a significant advancement in empiric treatment options for CAP.

Historique: La pneumonie communautaire est une préoccupation sanitaire importante dans le monde. Les agents pathogènes qui en sont responsables démontrent une résistance croissante envers des traitements empiriques souvent prescrits. La résistance du Streptococcus pneumoniae, la principale cause bactérienne de la pneumonie communautaire, augmente au Canada et dans le monde, ce qui fait ressortir l'importance d'agents antibactériens nouveaux et améliorés. La léfamuline, une nouvelle pleuromutiline, est un agent thérapeutique récemment homologué qui est très actif contre de nombreux agents pathogènes des voies respiratoires inférieures. Jusqu'à maintenant, peu de données sont toutefois disponibles pour décrire l'activité in vitro de la léfamuline contre les isolats bactériens associés à la pneumonie communautaire. Méthodologie: Les causes bactériennes courantes de la pneumonie communautaire déterminées à partir d'isolats des voies respiratoires inférieures et d'hémocultures dans des laboratoires canadiens mis en culture par des laboratoires hospitaliers du Canada et soumis à l'étude de surveillance canadienne annuelle dans les services hospitaliers du laboratoire coordonnateur de Winnipeg, au Canada, entre janvier 2015 et octobre 2018. Au total, les chercheurs ont testé 876 isolats bactériens au regard de la lémafuline et des agents comparatifs à l'aide de la méthode de référence de la microdilution dans un milieu de culture du Clinical and Laboratory Standards Institute (CLSI) et ont interprété les concentrations minimales inhibitrices (CMI) d'après les seuils acceptés. Résultats: La totalité des isolats de S. pneumoniae testés à partir de prélèvements des voies respiratoires (n = 315) et d'hémocultures (n = 167) était susceptible à la léfamuline (CMI ≤0,5 µg/mL), y compris les isolats résistants aux pénicillines, à la clarithromycine, à la doxycycline, au triméthoprime-sulfaméthoxazole et à des isolats multirésistants. La léfamuline inhibait également 99,0 % des isolats d'Haemophilus influenzae (quelle que soit leur production de ß-lactamases; n = 99; CMI ≤2 µg/mL) et 95,7 % de ceux de Staphylococcus aureus susceptibles à la méthicilline (SASM; n = 70; CMI ≤0,25 µg/mL) à leurs seuils susceptibles. La léfamuline a démontré des valeurs de CMI90 (concentration inhibant 90 % des isolats) de 0,25 µg/mL par rapport au SASM et au S. aureus résistant à la méthicilline (n = 130). Conclusion: La léfamuline a démontré une puissante activité in vitro au regard de tous les isolats respiratoires testés et peut représenter une avancée importante des traitements empiriques de la pneumonie communautaire.

Anisotropic Characterizations of Electrospun PAN Nanofiber Mats Using Design of Experiments.

This paper deals with the dielectric and mechanical characterizations of polyacrylonitrile (PAN)-aligned electrospun nanofiber mats. A two factor three level full factorial experiment is conducted to understand the effect of various parameters on dielectric and mechanical responses. These responses are recorded against randomly oriented and aligned nanofiber mats. Improved properties of electrospun mats have applications in the field of energy storage and nanocomposite reinforcement. Dielectric and mechanical characterizations of PAN mats are vital, as the aligned electrospun mats were found to be useful in advanced energy and mechanical reinforcement applications. Therefore, it is paramount to understand the effects of system parameters to these properties. The design of experiment (DoE) includes two factors and three level full factorial experiments with concentrations of PAN solutions at 8 wt.%, 9 wt.%, and 10 wt.%, and speed of the rotating mandrel (collector) at 3 volt (V), 4 V, and 5 V inputs. The electric field intensity used in the experiment is 1 kV/cm. DoE is conducted to understand the nonlinear interactions of parameters to these responses. The dielectric and mechanical characterizations of 8 wt.%, 9 wt.%, and 10 wt.% with different speeds for the original and improved systems are discussed. It was observed that at 9 wt.% and at all mandrel speeds, the dielectric and tensile properties are optimum.

Parametric study of 3D printed microneedle (MN) holders for interstitial fluid (ISF) extraction.

The need for novel, minimally invasive diagnostic, prognostic, and therapeutic biomedical devices has garnered increased interest in recent years. Microneedle (MN) technology has stood out as a promising new method for drug delivery, as well as extraction of interstitial fluid (ISF). ISF comprises a large portion of the extracellular fluid in living organisms yet remains inadequately characterized for clinical applications. Current MN research has focused on the fabrication of needles with different materials like silicone, carbon, and metals. However, little effort has been put forth into improving MN holders and patches that can be used with low cost MNs, which could effectively change how MNs are attached to the human body. Here, we describe different 3D-printed MN holders, printed using an MJP Pro 2500 3D printer, and compare the ISF extraction efficiencies in CD Hairless rats. We varied design parameters that may affect the skin-holder interface, such as throat thickness, tip curvature, and throat diameter. MN arrays, with insertion depths of 1500 µm, had extraction efficiencies of 0.44 ± 0.35, 0.85 ± 0.64, 0.32 ± 0.21, or 0.44 ± 0.46 µl/min when designed with flat, concave, convex, or bevel profile geometries, respectively. Our results suggest ISF extraction is influenced by MN holder design parameters and that a concave tip design is optimal for extracting ISF from animals. The future direction of this research aims to enable a paradigm in MN design that maximizes its efficiency and engineering performance in terms of volume, pressure, and wearability, thereby automatizing usage and reducing patient intervention to ultimately benefit remote telemedicine.

Extraction and biomolecular analysis of dermal interstitial fluid collected with hollow microneedles.

Dermal interstitial fluid (ISF) is an underutilized information-rich biofluid potentially useful in health status monitoring applications whose contents remain challenging to characterize. Here, we present a facile microneedle approach for dermal ISF extraction with minimal pain and no blistering for human subjects and rats. Extracted ISF volumes were sufficient for determining transcriptome, and proteome signatures. We noted similar profiles in ISF, serum, and plasma samples, suggesting that ISF can be a proxy for direct blood sampling. Dynamic changes in RNA-seq were recorded in ISF from induced hypoxia conditions. Finally, we report the first isolation and characterization, to our knowledge, of exosomes from dermal ISF. The ISF exosome concentration is 12-13 times more enriched when compared to plasma and serum and represents a previously unexplored biofluid for exosome isolation. This minimally invasive extraction approach can enable mechanistic studies of ISF and demonstrates the potential of ISF for real-time health monitoring applications.

Minimally-invasive, microneedle-array extraction of interstitial fluid for comprehensive biomedical applications: transcriptomics, proteomics, metabolomics, exosome research, and biomarker identification.

Interstitial fluid (ISF) has recently garnered interest as a biological fluid that could be used as an alternate to blood for biomedical applications, diagnosis, and therapy. ISF extraction techniques are promising because they are less invasive and less painful than venipuncture. ISF is an alternative, incompletely characterized source of physiological data. Here, we describe a novel method of ISF extraction in rats, using microneedle arrays, which provides volumes of ISF that are sufficient for downstream analysis techniques such as proteomics, genomics, and extracellular vesicle purification and analysis. This method is potentially less invasive than previously reported techniques. The limited invasiveness and larger volumes of extracted ISF afforded by this microneedle-assisted ISF extraction method provide a technique that is less stressful and more humane to laboratory animals, while also allowing for a reduction in the numbers of animals needed to acquire sufficient volumes of ISF for biomedical analysis and application.

Proteomic Characterization of Dermal Interstitial Fluid Extracted Using a Novel Microneedle-Assisted Technique.

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individual's physiological status using noninvasive techniques has grown. Microneedles have been proposed as a minimally invasive technique for sampling the dermal interstitial fluid (ISF) for clinical monitoring and diagnosis, but little is known about its composition. In this study, a novel microneedle array was used to collect dermal ISF from three healthy human donors and compared with matching serum and plasma samples. Using a shotgun quantitative proteomic approach, 407 proteins were quantified with at least one unique peptide, and of those, 135 proteins were differently expressed at least 2-fold. Collectively, these proteins tended to originate from the cytoplasm, membrane bound vesicles, and extracellular vesicular exosomes. Proteomic analysis confirmed previously published work that indicates that ISF is highly similar to both plasma and serum. In this study, less than one percent of proteins were uniquely identified in ISF. Taken together, ISF could serve as a minimally invasive alternative for blood-derived fluids with potential for real-time monitoring applications.

An Acyl-Linked Dimer of Daptomycin Is Strongly Inhibited by the Bacterial Cell Wall.

The lipopeptide antibiotic daptomycin is active against Gram-positive pathogens. It permeabilizes bacterial cell membranes, which involves the formation of membrane-associated oligomers. We here studied a dimer of daptomycin whose two subunits were linked through a bivalent aliphatic acyl chain. Unexpectedly, the dimer had very low activity on vegetative Staphylococcus aureus and Bacillus subtilis cells. However, activity resembled that of monomeric daptomycin on liposomes and on B. subtilis L-forms. These findings underscore the importance of the bacterial cell wall in daptomycin resistance.

Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture: a model system to understand cancer cell growth mechanics.

BACKGROUND: Spheroid based culture methods are gaining prominence to elucidate the role of the microenvironment in liver carcinogenesis. Additionally, the phenomenon of epithelial-mesenchymal transition also plays an important role in determining the metastatic potential of liver cancer. Tumor spheroids are thus important models to understand the basic biology of liver cancer. METHODS: We cultured, characterized and examined the formation of compact 3-D micro-tumor spheroids in five hepatocellular carcinoma (HCC) cell lines, each with differing TP53 mutational status (wt vs mutant vs null). Spheroid viability and death was systematically measured over a course of a 10 day growth period using various assays. We also examined the TP53 and E-cadherin (CDH1) mRNA and protein expression status in each cell line of the 2-D and 3-D cell models. RESULTS: A novel finding of our study was the identification of variable 3-D spheroid morphology in individual cell lines, ranging from large and compact, to small and unstable spheroid morphologies. The observed morphological differences between the spheroids were robust and consistent over the duration of spheroid culture growth of 10 days in a repeatable manner. Highly variable CDH1 expression was identified depending on the TP53 mutational status of the individual HCC cell line, which may explain the variable spheroid morphology. We observed consistent patterns of TP53 and CDH1 expression in both 2-D and 3-D culture models. CONCLUSIONS: In conclusion, we show that 3-D spheroids are a useful model to determine the morphological growth characteristics of cell lines which are not immediately apparent in routine 2-D culture methods. 3-D culture methods may provide a better alternative to study the process of epithelial-mesenchymal transition (EMT) which is important in the process of liver cancer metastasis.

Palliative Care Version 1.2016.

The NCCN Guidelines for Palliative Care provide interdisciplinary recommendations on palliative care for patients with cancer. The NCCN Guidelines are intended to provide guidance to the primary oncology team on the integration of palliative care into oncology. The NCCN Palliative Care Panel's recommendations seek to ensure that each patient experiences the best quality of life possible throughout the illness trajectory. Accordingly, the NCCN Guidelines outline best practices for screening, assessment, palliative care interventions, reassessment, and after-death care.

Palliative care, Version 1.2014. Featured updates to the NCCN Guidelines.

The NCCN Guidelines for Palliative Care provide interdisciplinary recommendations on palliative care for patients with cancer. These NCCN Guidelines Insights summarize the NCCN panel's discussions and guideline updates from 2013 and 2014. These include modifications/additions to palliative care screening and assessment protocols, new considerations for discussing the benefits and risks of anticancer therapy, and approaches to advance care planning. Recent updates focus on enhanced patient-centered care and seek to promote earlier integration of palliative care and advance care planning in oncology.

Influence of carbon chain length on the synthesis and yield of fatty amine-coated iron-platinum nanoparticles.

Iron oxide nanoparticles are among the most widely used and characterized magnetic nanoparticles. However, metal alloys such as superparamagnetic iron-platinum particles (SIPPs), which have better magnetic properties, are receiving increased attention. Scalable techniques to routinely synthesize SIPPs in bulk need further study. Here, we focus on the role played by the fatty amine ligand in the formation of the bimetallic FePt nanocrystal. More specifically, we compare the effect of varying lengths of fatty amine ligands on the shape, structure, uniformity, composition, and magnetic properties of the SIPPs. We synthesized SIPPs by employing a 'green' thermal decomposition reaction using fatty amine ligands containing 12 to 18 carbons in length. Greater fatty amine chain length increased the polydispersity, particle concentration, iron concentration, and the stability of the SIPPs. Additionally, longer reflux times increased the diameter of the particles, but decreased the iron concentration, suggesting that shorter reaction times are preferable. Fourier transform infrared spectroscopy of the SIPPs indicates that the ligands are successfully bound to the FePt cores through the amine group. Superconducting quantum interference device magnetometry measurements suggest that all of the SIPPs were superparamagnetic at room temperature and that SIPPs synthesized using tetradecylamine had the highest saturation magnetization. Our findings indicate that the octadecylamine ligand, which is currently used for the routine synthesis of SIPPs, may not be optimal. Overall, we found that using tetradecylamine and a 30-min reflux reaction resulted in optimal particles with the highest degree of monodispersity, iron content, stability, and saturation magnetization. PACS: 81.07.-b; 75.75.Fk; 61.46.Df.

Ethical principles and concepts in medicine.

Clinical ethics is the application of ethical theories, principles, rules, and guidelines to clinical situations in medicine. Therefore, clinical ethics is analogous to clinical medicine in that general principles and concepts must be applied intelligently and thoughtfully to unique clinical circumstances. The three major ethical theories are consequentialism, whereby the consequences of an action determine whether it is ethical; deontology, whereby to be ethical is to do one's duty, and virtue ethics, whereby ethics is a matter of cultivating appropriate virtues. In the real world of medicine, most people find that all three perspectives offer useful insights and are complementary rather than contradictory. The most common approach to clinical ethical analysis is principlism. According to principlism, the medical practitioner must attempt to uphold four important principles: respect for patient autonomy, beneficence, nonmaleficence, and justice. When these principles conflict, resolving them depends on the details of the case. Alternative approaches to medical ethics, including the primacy of beneficence, care-based ethics, feminist ethics, and narrative ethics, help to define the limitations of principlism and provide a broader perspective on medical ethics.

The James Supportive Care Screening: integrating science and practice to meet the NCCN guidelines for distress management at a Comprehensive Cancer Center.

BACKGROUND: Selecting a measure for oncology distress screening can be challenging. The measure must be brief, but comprehensive, capturing patients' most distressing concerns. The measure must provide meaningful coverage of multiple domains, assess symptom and problem-related distress, and ideally be suited for both clinical and research purposes. METHODS: From March 2006 to August 2012, the James Supportive Care Screening (SCS) was developed and validated in three phases including content validation, factor analysis, and measure validation. Exploratory factor analyses were completed with 596 oncology patients followed by a confirmatory factor analysis with 477 patients. RESULTS: Six factors were identified and confirmed including (i) emotional concerns; (ii) physical symptoms; (iii) social/practical problems; (iv) spiritual problems; (v) cognitive concerns; and (vi) healthcare decision making/communication issues. Subscale evaluation reveals good to excellent internal consistency, test-retest reliability, and convergent, divergent, and predictive validity. Specificity of individual items was 0.90 and 0.87, respectively, for identifying patients with DSM-IV-TR diagnoses of major depression and generalized anxiety disorder. CONCLUSIONS: Results support use of the James SCS to quickly detect the most frequent and distressing symptoms and concerns of cancer patients. The James SCS is an efficient, reliable, and valid clinical and research outcomes measure.