Search

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates. The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

This study investigates the influence of microplastics on blood clotting. It addresses the lack of comprehensive research on the effects of microplastic size and surface modification on clotting dynamics in human whole blood. Thromboelastography was used to examine aminated (aPS), carboxylated (cPS), and non-functionalized (nPS) polystyrene particles with sizes of 50, 100, and 500  nm. Results show that cPS consistently activated the clotting cascade, demonstrating increased fibrin polymerization rates, and enhanced clot strength in a size and concentration-dependent manner. nPS had minimal effects on clotting dynamics except for 50  nm particles at the lowest concentration. The clotting effects of aPS (100  nm particles) resembled those of cPS but were diminished in the 500  nm aPS group. These findings emphasize the importance of microplastic surface modification, size, concentration, and surface area on in-vitro whole blood clotting dynamics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Christodoulides, Hall and Alves.)

Thrombosis can lead to a wide variety of life-threatening circumstances. As current thrombolytic drug screening models often poorly predict drug profiles, leading to failure of thrombolytic therapy or clinical translation, more representative clot substrates are necessary for drug evaluation. Utilizing a Chandler loop device to form clot analogs at high shear has gained popularity in stroke societies. However, shear-dependent clot microstructure has not been fully addressed and low shear conditions are often overlooked. We herein characterized the impact of wall shear rate (126 to 951 s -1 ) on clot properties in the Chandler loop. Different revolutions (20-60) per minute and tubing sizes (3.2 to 7.9 mm) were employed to create different sized clots to mimic various thrombosis applications. Increased shear resulted in decreased RBC counts (76.9 ± 4.3% to 17.6 ± 0.9%) and increased fibrin (10 to 60%) based on clot histology. Increased fibrin sheet morphology and platelet aggregates were observed at higher shear under scanning electron microscope. These results show the significant impact of shear and tubing size on resulting clot properties and demonstrate the capability of forming a variety of reproducible in-vivo-like clot analogs in the Chandler loop device controlling for simple parameters to tune clot characteristics., (© 2023. The Author(s).)

A great need exists for the development of a more representative in-vitro model to efficiently screen novel thrombolytic therapies. We herein report the design, validation, and characterization of a highly reproducible, physiological scale, flowing clot lysis platform with real-time fibrinolysis monitoring to screen thrombolytic drugs utilizing a fluorescein isothiocyanate (FITC)-labeled clot analog. Using this Real-Time Fluorometric Flowing Fibrinolysis assay (RT-FluFF assay), a tPa-dependent degree of thrombolysis was observed both via clot mass loss as well as fluorometrically monitored release of FITC-labeled fibrin degradation products. Percent clot mass loss ranged from 33.6% to 85.9% with fluorescence release rates of 0.53 to 1.17 RFU/min in 40 and 1000 ng/mL tPa conditions, respectively. The platform is easily adapted to produce pulsatile flows. Hemodynamics of human main pulmonary artery were mimicked through matching dimensionless flow parameters calculated using clinical data. Increasing pressure amplitude range (4-40 mmHg) results in a 20% increase of fibrinolysis at 1000 ng/mL tPA. Increasing shear flow rate (205-913 s -1 ) significantly increases fibrinolysis and mechanical digestion. These findings suggest pulsatile level affects thrombolytic drug activities and the proposed in-vitro clot model offers a versatile testing platform for thrombolytic drug screening., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of The American Institute of Chemical Engineers.)

Metabolic dysfunction of β-cells has been implicated as a contributor to diabetes pathogenesis, and efforts are ongoing to optimize analytical techniques that evaluate islet metabolism. High-resolution respirometry offers sensitive measurements of the respiratory effects of metabolic substrates and customizable manipulation of electron transport chain components, though the delicate nature of islets can pose challenges to conventional analyses. An affordable and reliable option for respirometry is the Oroboros Oxygraph-2 K system, which utilizes a stir bar to circulate reagents around cells. While this technique may be suitable for individual cells or mitochondria, the continual force exerted by the stir bar can have damaging effects on islet integrity. Herein, we demonstrate the protective benefits of a novel 3D-printed islet stabilization device and highlight the destructive effects of conventional Oxygraph analysis on islet integrity. Islet containment did not inhibit cellular responses to metabolic modulatory drugs, as indicated by robust fluctuations in oxygen consumption rates. The average size of wild-type mouse islets was significantly reduced following a standard Mito Stress Test within Oxygraph chambers, with a clear disruption in islet morphology and viability. Alternatively, containment of the islets within the interior chamber of the islet stabilization device yielded preservation of both islet morphology and increased cell viability/survival after respirometry analysis. Collectively, our study introduces a new and easily accessible tool to improve conventional Oxygraph respirometry of pancreatic islets by preserving natural islet structure and function throughout metabolic analysis.

Micro/nanoplastics, whether manufactured or resulting from environmental degradation, can enter the body through ingestion, inhalation, or dermal pathways. Previous research has found that nanoplastics with diameters of ≤100 nm can translocate into the circulatory system in a dose-dependent manner and potentially impact thrombosis and hemostasis. To investigate the direct effects of microplastics on fibrin clot formation, a simplified ex vivo human thrombin/fibrinogen clot model was utilized. The 100 nm polystyrene particles (non-functionalized [nPS] and aminated [aPS]) were preincubated (0-200 µg/mL) with either thrombin or fibrinogen, and fibrin clot formation was characterized via turbidity and thromboelastography (TEG). When the particles were preincubated with fibrinogen, little effect was observed for aPS or nPS on turbidity or TEG up through 100 µg/mL. TEG results demonstrated a significant impact on clot formation rate and strength, in the case of nPS preincubated with thrombin exhibiting a significant dose-dependent inhibitory effect. In conclusion, the presence of microplastics can have inhibitory effects on fibrin clot formation that are dependent upon both particle surface charge and concentration. Negatively charged nPS exhibited the most significant impacts to clot strength, turbidity, and rate of fibrin formation when first incubated with thrombin, with its impact being greatly diminished when preincubated with fibrinogen in this simplified fibrin clot model.

There is an emerging interest in utilizing synthetic multivalent inhibitors that comprise of multiple inhibitor moieties linked on a common scaffold to achieve strong and selective enzyme inhibition. As multivalent inhibition is impacted by valency and linker length, in this study, we explore the effect of multivalent benzamidine inhibitors of varying valency and linker length on plasmin inhibition. Plasmin is an endogenous enzyme responsible for digesting fibrin present in blood clots. Monovalent plasmin(ogen) inhibitors are utilized clinically to treat hyperfibrinolysis-associated bleeding events. Benzamidine is a reversible inhibitor that binds to plasmin's active site. Herein, multivalent benzamidine inhibitors of varying valencies (mono-, bi- and tri-valent) and linker lengths (∼1-12 nm) were synthesized to systematically study their effect on plasmin inhibition. Inhibition assays were performed using a plasmin substrate (S-2251) to determine inhibition constants (Ki). Pentamidine (shortest bivalent) and Tri-AMB (shortest trivalent) were the strongest inhibitors with Ki values of 2.1±0.8 and 3.9±1.7 μM, respectively. Overall, increasing valency and decreasing linker length, increases effective local concentration of the inhibitor and therefore, resulted in stronger inhibition of plasmin via statistical rebinding. This study aids in the design of multivalent inhibitors that can achieve desired enzyme inhibition by means of modulating valency and linker length., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Background: Personal protective equipment (PPE) is essential to protect healthcare workers (HCWs). The practice of reusing PPE poses high levels of risk for accidental contamination by HCWs. Scarce medical literature compares practical means or methods for safe reuse of PPE while actively caring for patients., Methods: In this study, observations were made of 28 experienced clinical participants performing five donning and doffing encounters while performing simulated full evaluations of patients with coronavirus disease 2019. Participants' N95 respirators were coated with a fluorescent dye to evaluate any accidental fomite transfer that occurred during PPE donning and doffing. Participants were evaluated using blacklight after each doffing encounter to evaluate new contamination sites, and were assessed for the cumulative surface area that occurred due to PPE doffing. Additionally, participants' workstations were evaluated for contamination., Results: All participants experienced some contamination on their upper extremities, neck and face. The highest cumulative area of fomite transfer risk was associated with the hook and paper bag storage methods, and the least contamination occurred with the tabletop storage method. Storing a reused N95 respirator on a tabletop was found to be a safer alternative than the current recommendation of the US Centers for Disease Control and Prevention to use a paper bag for storage. All participants donning and doffing PPE were contaminated., Conclusion: PPE reusage practices pose an unacceptably high level of risk of accidental cross-infection contamination to healthcare workers. The current design of PPE requires complete redesign with improved engineering and usability to protect healthcare workers., (Copyright © 2022 The Healthcare Infection Society. All rights reserved.)

Objectives: There has long existed significant underrepresentation of minority students in STEM training and careers. Ongoing efforts to improve opportunities and participation for underrepresented minority students have focused on multiple areas, from increased funding to early exposure to research in STEM. We developed the novel Future Life Map career planning exercise with the goal of contributing to this multi-faceted approach. The exercise emphasizes on the consideration of multiple potential career destinations and routes to those destination. The exercise was designed with the goal of improving participant awareness of options and career planning self-efficacy to improve success and retention of underrepresented minority student participation and retention in STEM., Methods: We implemented the Future Life Map exercise with 2 separate groups of under-represented minority undergraduate students pursuing careers in STEM. Participants then completed an anonymous survey to evaluate the exercise and describe the value they derived from completing the Future Life Map., Results: The exercise presentation and its supporting documents were highly rated by participants with >81% of respondents rating it as "very informative" (4 or 5 on a 5-point Likert Scale). Participants reported that they were very likely to recommend the exercise to others (25 of 27 participants) and were likely to repeat the activity for their own future decision making (22 participants). Themes that emerged from participant reporting of the value of the exercise were: increased awareness of career and training options, improved understanding of the research required to make informed career/life decisions, and new awareness of specific information about career options under consideration., Conclusion: The Future Life Map exercise was successful in improving participant awareness of career options, career planning ability, and helped participants to feel more empowered. This is likely of particular benefit for improving participation and retention of under-represented minority students pursuing careers in STEM., Competing Interests: The authors have declared that no competing interests exist.

Acute pulmonary embolism (PE) does not always resolve after treatment and can progress to chronic thromboembolic disease (CTED) or the more severe chronic thromboembolic pulmonary hypertension (CTEPH). The mechanisms surrounding the likelihood of PE resolution or progress to CTED/CTEPH remain largely unknown. We have developed a rat model of CTEPH that closely resembles the human disease in terms of hemodynamics and cardiac manifestations. Embolization of rats with polystyrene microspheres followed by suppression of angiogenesis with the inhibitor of vascular endothelial growth factor receptor 2 (VEGF-R2) SU5416 results in transient, acute pulmonary hypertension that progresses into chronic PE with PH with sustained right ventricular systolic pressures exceeding 70 mmHg (chronic pulmonary embolism [CPE] model). This model is similar to the widely utilized hypoxia/SU5416 model with the exception that the "first hit" is PE. Rats with CPE have impaired right heart function characterized by reduced VO 2 Max, reduced cardiac output, and increased Fulton index. None of these metrics are adversely affected by PE alone. Contrast-mediated CT imaging of lungs from rats with PE minus SU5416 show large increases in pulmonary vascular volume, presumably due to an angiogenic response to acute PE/PH. Co-treatment with SU5416 suppresses angiogenesis and produces the CTEPH-like phenotype. We report here that treatment of CPE rats with agonists for soluble guanylate cyclase, a source of cGMP which is in turn a signal for angiogenesis, markedly increases angiogenesis in lungs, and ameliorates the cardiac deficiencies in the CPE model. These results have implications for future development of therapies for human CTEPH., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Plastic pollution has been a growing concern in recent decades due to the proliferation and ease of manufacturing of single use plastic products and inadequate waste and recycling management. Microplastic, and even smaller nanoplastic, particles are persistent pollutants in aquatic and terrestrial systems and are the subject of active and urgent research. This review will explore the current research on how exposure to plastic particles occurs and the risks associated from different exposure routes: ingestion, inhalation, and dermal exposure. The effects of microplastics on the cardiovascular system are of particular importance due to its sensitivity and ability to transport particles to other organ systems. The effects of microplastics and nanoplastics on the heart, platelet aggregation, and thrombus formation will all be explored with focus on how the particle characteristics modulate their effect. Plastic particle interactions are highly dependent on both their size and their surface chemistry and interesting research is being done with the interaction of particle characteristics and effect on thrombosis and the cardiovascular system. There is significant uncertainty surrounding some of the findings in this field as research in this area is still maturing. There are undoubtedly more physiological consequences than we are currently aware of resulting from environmental plastic exposure and more studies need to be conducted to reveal the full extent of pathologies caused by the various routes of microplastic exposure, with particular emphasis on longitudinal exposure effects. Further research will allow us to recognize the full extent of physiological impact and begin developing viable solutions to reduce plastic pollution and potentially design interventions to mitigate in-vivo plastic effects following significant or prolonged exposure., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Conducting in-vitro thrombosis research presents numerous challenges, the primary of which is working with blood products, whether whole blood or fractionated whole blood, that have limited functional shelf-lives. As a result, being able to significantly prolong the clotting functionality of whole blood via fractionation and recombination promises greater accessibility via resource minimization in the realm of thrombosis research. Whole blood with CPDA1 from healthy volunteers was fractionated and stored as frozen platelet-free plasma (PFP, -20°C), refrigerated packed red blood cells (pRBCs, 4°C) and cryopreserved platelets (-80°C). Subsequent recombination of the above components into their native ratios were tested via thromboelastography (TEG) to capture clotting dynamics over a storage period of 13 weeks in comparison to refrigerated unfractionated WB+CPDA1. Reconstituted whole blood utilizing PFP, pRCBs and cryopreserved platelets were able to maintain clot strength (maximum amplitude) akin to day-0 whole blood even after 13 weeks of storage. Clots formed by reconstituted whole blood exhibited quicker clotting dynamics with nearly two-fold shorter R-times and nearly 1.3-fold increase in fibrin deposition rate as measured by TEG. Storage of fractionated whole blood components, in their respective ideal conditions, provides a means of prolonging the usable life of whole blood for in-vitro thrombosis research. Cryopreserved platelets, when recombined with frozen PFP and refrigerated pRBCs, are able to form clots that nearly mirror the overall clotting profile expected of freshly drawn WB., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Impaired fibrinolysis has long been considered as a risk factor for venous thromboembolism. Fibrin clots formed at physiological concentrations are promising substrates for monitoring fibrinolytic performance as they offer clot microstructures resembling in vivo . Here we introduce a fluorescently labeled fibrin clot lysis assay which leverages a unique annular clot geometry assayed using a microplate reader. A physiologically relevant fibrin clotting formulation was explored to achieve high assay sensitivity while minimizing labeling impact as fluorescence isothiocyanate (FITC)-fibrin(ogen) conjugations significantly affect both fibrin polymerization and fibrinolysis. Clot characteristics were examined using thromboelastography (TEG), turbidity, scanning electron microscopy, and confocal microscopy. Sample fibrinolytic activities at varying plasmin, plasminogen, and tissue plasminogen activator (tPA) concentrations were assessed in the present study and results were compared to an S2251 chromogenic assay. The optimized physiologically relevant clot substrate showed minimal reporter-conjugation impact with nearly physiological clot properties. The assay demonstrated good reproducibility, wide working range, kinetic read ability, low limit of detection, and the capability to distinguish fibrin binding-related lytic performance. In combination with its ease for multiplexing, it also has applications as a convenient platform for assessing patient fibrinolytic potential and screening thrombolytic drug activities in personalized medical applications.

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients' hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

Background: We test if inhaled nitric oxide (NO) attenuates platelet functional and metabolic hyper-reactivity in subjects with submassive pulmonary embolism (PE)., Methods: Participants with PE were randomized to either 50 ppm NO + O2 or O2 only for 24 h with blood sampling at enrollment and after treatment; results were compared with healthy controls. Platelet metabolic activity was assessed by oxygen consumption (basal and uncoupled) and reactivity was assessed with agonist-stimulated thromboelastography (TEG) and fluorometric measurement of agonist-stimulated cytosolic [Ca ++ ] without and with pharmacological soluble guanylate (sGC) modulation., Results: Participants (N = 38 per group) were well-matched at enrollment for PE severity, comorbidities as well as TEG parameters and platelet O2 consumption. NO treatment doubled the mean plasma [NO3-] (P < 0.001) indicating successful delivery, but placebo treatment produced no change. After 24 h, neither TEG nor O2 consumption parameters differed significantly between treatment groups. Platelet cytosolic [Ca ++ ] was elevated with PE versus controls, and was decreased by treatment with cinaciguat (an sGC activator), but not riociguat (an sGC stimulator). Stimulated platelet lysate sGC activity was increased with PE compared with controls., Conclusions: In patients with acute submassive PE, despite evidence of adequate drug delivery, inhaled NO had no major effect on platelet O2 consumption or agonist-stimulated parameters on TEG. Pharmacological activation, but not stimulation, of sGC effectively decreased platelet cytosolic [Ca ++ ], and platelet sGC activity was increased with PE, confirming the viability of sGC as a therapeutic target., Competing Interests: Declaration of competing interest No authors have a conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thrombosis is a leading cause of death around the world. Fibrin, the protein primarily responsible for clot formation, is formed via cleaving soluble fibrinogen by thrombin with resulting properties varying under different clot forming conditions. This study sought to compare trends across thromboelastography (TEG) and turbidimetry utilizing a simplified fibrinogen/thrombin clot model. Turbidimetry is an optical measure (550 nm) of fibrin clot formation and is widely utilized due to its laboratory accessibility and ease of use. Thromboelastography (TEG) is a specialized viscoelastic technique that directly measures clot strength and is primarily utilized in the clinical setting to assess patients' hemostasis. In these experiments, human and bovine fibrin clots were formed in-vitro by mixing fibrinogen and thrombin under diverse clotting conditions. Increasing thrombin concentration (0 to 10 U/mL), ionic strength (0.05 to 0.3 M), pH (5.5 to 8.1), and lowering albumin concentration (100 to 0 mg/mL) resulted in decreased clot turbidity and increased clot strength using species-matched bovine and human fibrinogen and thrombin. Whereas, increasing fibrinogen concentration (1 to 5 mg/mL) resulted in increased clot turbidity and increased clot strength for both species-matched and cross-species fibrinogen and thrombin. Clotting times with both techniques followed a similar trend and were observed to be unchanged when varying albumin concentration, elongated with increasing fibrinogen, and shortened with increasing pH, ionic strength, and thrombin. TEG and turbidimetry track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV + patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin + endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

In an era where ultra-high antibody concentrations, high viscosities, low volumes, auto-injectors and long storage requirements are already complex problems with the current unconjugated monoclonal antibodies on the market, the formulation demands for antibody-drug conjugates (ADCs) are significant. Antibodies have historically been administered at relatively low concentrations through intravenous (IV) infusion due to their large size and the inability to formulate for oral delivery. Due to the high demands associated with IV infusion and the development of novel antibody targets and unique antibody conjugates, more accessible routes of administration such as intramuscular and subcutaneous are being explored. This review will summarize various site-specific and non-site-specific antibody conjugation techniques in the context of ADCs and the demands of formulation for high concentration clinical implementation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Antibody Therapeutics.)

Stimulants cause hyperthermia, in part, by increasing heat generation through exercise. Stimulants also delay the onset of fatigue and exhaustion allowing animals to exercise longer. If used in a warm environment, this combination (increased exercise and decreased fatigue) can cause heat stroke. The dorsomedial hypothalamus (DMH) is involved in mediating locomotion from stimulants. Furthermore, inhibiting the DMH decreases locomotion and prevents hyperthermia in rats given stimulants in a warm environment. Whether the DMH is involved in mediating exercise-induced fatigue and exhaustion is not known. We hypothesized that disinhibiting neurons in the dorsomedial hypothalamus (DMH) would delay the onset of fatigue and exhaustion in animals exercising in a warm environment. To test this hypothesis, we used automated video tracking software to measure fatigue and exhaustion. In rats, using wearable mini-pumps, we demonstrated that disinhibiting the DMH, via bicuculline perfusion (5 µM), increased the duration of exercise in a warm environment as compared to control animals (25 ± 3 min vs 15 ± 2 min). Bicuculline-perfused animals also had higher temperatures at exhaustion (41.4 ± 0.2 °C vs 40.0 ± 0.4 °C). Disinhibiting neurons in the DMH also increased the time to fatigue. Our data show that the same region of the hypothalamus that is involved in mediating locomotion to stimulants, is also involved in controlling exhaustion and fatigue. These findings have implications for understanding the cause and treatment of stimulant-induced-hyperthermia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/μM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms.

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function., (Published by Elsevier Masson SAS.)

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas.

Background: We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE)., Methods: Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405)., Results: Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT., Conclusion: The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.

Introduction: The targeted delivery of therapeutic agents greatly increases their effectiveness while simultaneously reducing negative side effects. In the past, targeting of therapeutics has been accomplished with nucleic acids, peptides/proteins, and conventional antibodies. A promising alternative to the conventional antibodies often used in therapeutic targeting are significantly smaller-sized antibody fragments known as single-domain antibodies (sdAbs)., Areas Covered: Recent advances in the utility of sdAbs for targeting of therapeutic agents along with relevant examples from the literature are discussed. Their advantages when compared to other targeting strategies as well as their challenges and limitations is also covered., Expert Opinion: The development of sdAb-based targeted therapeutics will likely continue. The identification of novel protein modification techniques will provide more options for sdAb modification (conjugation, immobilization, functionalization), allowing a wider array of therapeutic agents to be successfully targeted and delivered using sdAbs. This will also spur the selection of sdAbs with specificity for other targets having relevance towards therapeutics.

All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Nanoparticle-based therapeutics are poised to play a critical role in treating disease. These complex multifunctional drug delivery vehicles provide for the passive and active targeted delivery of numerous small molecule, peptide and protein-derived pharmaceuticals. This article will first discuss some of the current state of the art nanoparticle classes (dendrimers, lipid-based, polymeric and inorganic), highlighting benefits/drawbacks associated with their implementation. We will then discuss an emerging class of nanoparticle therapeutics, bacterial outer membrane vesicles, that can provide many of the nanoparticle benefits while simplifying assembly. Through molecular biology techniques; outer membrane vesicle hijacking potentially allows for stringent control over nanoparticle production allowing for targeted protein packaged nanoparticles to be fully synthesized by bacteria.

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides., (© 2015 Wiley Periodicals, Inc.)

The potent fibrinolytic enzyme, plasmin has numerous clinical applications for recannulizing vessels obstructed by thrombus. Despite its diminutive size, 91 kDa, success in the recombinant expression of this serine protease has been limited. For this reason, a truncated non-glycosylated plasmin variant was developed capable of being expressed and purified from E. coli. This mutated plasmin, known as δ-plasmin, eliminates four of the five kringle domains present on native plasmin, retaining only kringle 1 fused directly to the unmodified catalytic domain of plasmin. This study demonstrates that δ-plasmin exhibits similar kinetic characteristics to full length plasmin despite its heavily mutated form; KM = 268.78 ± 19.12, 324.90 ± 8.43 μM and Kcat = 770.48 ± 41.73, 778.21 ± 1.51 1/min for plasmin and δ-plasmin, respectively. A comparative analysis was also carried out to investigate the inhibitory effects of a range of benzamidine based small molecule inhibitors: benzamidine, p-aminobenzamidine, 4-carboxybenzamidine, 4-aminomethyl benzamidine, and pentamidine. All of the small molecule inhibitors, with the exception of unmodified benzamidine, demonstrated comparable competitive inhibition constants (Ki) for both plasmin and δ-plasmin ranging from Ki < 4 μM for pentamidine to Ki > 1000 μM in the case of aminomethyl benzamidine. This result further supports that δ-plasmin retains much of the same functionality as native plasmin despite its greatly reduced size and complexity. This study serves the purpose of demonstrating the tunable inhibition of plasmin and δ-plasmin with potential applications for the improved clinical delivery of δ-plasmin to treat various thrombi., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pulmonary embolism (PE) is a leading cause of sudden cardiac death, and a model is needed for testing potential treatments. In developing a model, we compared the hemodynamic effects of isoflurane and α-chloralose in an acute swine model of PE because the choice of anesthesia will likely affect the cardiovascular responses of an animal to PE. At baseline, swine that received α-chloralose (n = 6) had a lower heart rate and cardiac output and higher SpO2, end-tidal CO2, and mean arterial pressure than did those given isoflurane (n = 9). After PE induction, swine given α-chloralose compared with isoflurane exhibited a lower heart rate (63 ± 10 compared with 116 ± 15 bpm) and peripheral arterial pressure (52 ± 12 compared with 61 ± 12 mm Hg); higher SpO2 (98% ± 3% compared with 95% ± 1%), end-tidal CO2 (35 ± 4 compared with 32 ± 5), and systolic blood pressure (121 ± 8 compared with 104 ± 20 mm Hg); and equivalent right ventricular:left ventricular ratios (1.32 ± 0.50 compared with 1.23 ± 0.19) and troponin I mean values (0.09 ± 0.07 ng/mL compared with 0.09 ± 0.06 ng/mL). Isoflurane was associated with widely variable fibrinogen and activated partial thromboplastin time. Intraexperiment mortality was 0 of 6 animals for α-chloralose and 2 of 9 swine for isoflurane. All swine anesthetized with α-chloralose survived with sustained pulmonary hypertension, RV-dilation-associated cardiac injury without the confounding vasodilatory or coagulatory effects of isoflurane. These data demonstrate the physiologic advantages of α-chloralose over isoflurane for anesthesia in a swine model of severe submassive PE.

Based on the importance of behavior and life satisfaction for the health of young people and the considerable regional differences that exist in mainland Portugal, an investigation was conducted. It sought to verify differences in levels of the practice of sport, tobacco and alcohol consumption and life satisfaction among students of the 3rd cycle of basic education living in different inland/coastal and rural/urban areas. The sample consisted of 5624 adolescents, aged between 12 and 17 years. The instrument used for data collection was the "Inventory of Health-Related Behavior of Adolescents" and a translated version of "The Satisfaction With Life Scale". The results revealed that there are higher levels of smoking among young people living in urban and inland areas, higher levels of alcohol consumption among young people living in rural and inland areas and higher levels of life satisfaction among adolescents living in urban and coastal areas. Levels of sport were identical in all geographical areas. It was also found that there were significant differences in all variables of the analysis.

Carfilzomib, a recently FDA-approved proteasome inhibitor, has remarkable anti-myeloma (MM) activity. However, its effectiveness is limited by associated severe side-effects, short circulation half-life, and limited solubility. Here, we report the engineering of liposomal carfilzomib nanoparticles to overcome these problems and enhance the therapeutic efficacy of carfilzomib by increasing tumoral drug accumulation while decreasing systemic toxicity. In our design, carfilzomib was loaded into the bilayer of liposomes to yield stable and reproducible liposomal nanoparticles. Liposomal carfilzomib nanoparticles were efficiently taken up by MM cells, demonstrated proteasome inhibition, induced apoptosis, and exhibited enhanced cytotoxicity against MM cells. In vivo, liposomal carfilzomib demonstrated significant tumor growth inhibition and dramatically reduced overall systemic toxicity compared to free carfilzomib. Finally, liposomal carfilzomib demonstrated enhanced synergy in combination with doxorubicin. Taken together, this study establishes the successful synthesis of liposomal carfilzomib nanoparticles that demonstrates improved therapeutic index and the potential to improve patient outcome in MM., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.

The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBS(Biotin)) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1J/cm(2) yielded an average conjugation efficiency of ≈ 1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBS(Biotin) method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: ε-NH3(+) surface conjugation, NHS-Biotin, and direct physical adsorption. Taken together, the UV-NBS(Biotin) method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Hysterectomy dates back to 120BC and is the second most commonly performed gynecological surgery in the world. Cosmetic demands and the necessity of rapid return to work have contributed to the minimally invasive laparoscopic approach for hysterectomy. The majority of reports describe the use of three or four incisions to perform the surgery (two or three for manipulation and one for optics)., Methods: This work describes our experience with using only two ports for 11 patients who underwent video-laparoscopic hysterectomy surgery. One port was used for the optical system, and the second was used for manipulation. Early and late surgery complications, as well as the time to return to work and daily activities, were assessed., Results: The mean age of the patients was 41.4 years old (range 16 to 52 years) and the mean uterine weight was 133.54 g, ranging from 35 g and 291 g. The operative time ranged from 30 to 60 minutes (average 46.4 minutes) and the hospital stay ranged between 24 and 48 hrs. No intraoperative complications occurred, and no early or late postoperative complications were recorded. Patients reported minimal pain during the first 24-48 hrs in the hospital. Patients returned to their daily activities within seven days after surgery. Clinical care follow-up continued until the 40th postoperative day., Conclusion: The laparoscopic hysterectomy technique with a single port for manipulation is a feasible procedure when the uterine weight is not greater than 400 mg with little postoperative pain. The patients had an early return-to-work and daily activities and a better cosmetic outcome. These preliminary data led us to make the one-operative port laparoscopic hysterectomy the procedure of choice for patients with a low uterine weight.

Liposome Extruder Purification (LEP) allows for the rapid purification of diverse liposome formulations using the same extrusion apparatus employed during liposome formation. The LEP process provides a means for purifying functionalized liposomes from non-conjugated drug or protein contaminants with >93% liposome recovery and >93% contaminant removal in a single step.

The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 μM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

This study describes the design of a well-defined homotetravalent synthetic allergen (HTA) system to investigate the effect of hapten-IgE interactions on mast cell degranulation. A library of DNP variants with varying affinities for IgE(DNP) was generated (K(d) from 8.1 nM to 9.2 μM), and 8 HTAs spanning this range were synthesized via conjugation of each DNP variant to the tetravalent scaffold. HTAs with hapten K(d) < 235 nM stimulated degranulation following a bell-shaped dose response curve with maximum response occurring near the hapten K(d). HTAs with hapten K(d) ≥ 235 nM failed to stimulate degranulation. To mimic physiological conditions, the percent of allergen specific IgE on cell surface was varied, and maximum degranulation occurred at 25% IgE(DNP). These results demonstrated that moderate hapten-IgE affinities are sufficient to trigger mast cell degranulation. Moreover, this study established the HTA design as a well-defined, controllable, and physiologically relevant experimental system to elucidate the mast cell degranulation mechanism.

The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a K(d) ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBS(IBA)). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBS(IBA) column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBS(IBA) column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBS(IBA) column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBS(IBA) platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.

The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a K(d) ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm(2) yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, ε-NH(3)(+) conjugation and physical adsorption, demonstrated that the UV-NBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.

In the continuing search for effective cancer treatments, we report the rational engineering of a multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-adhesion functionalities. Very late antigen-4 (VLA-4) mediated adhesion of multiple myeloma (MM) cells to bone marrow stroma confers MM cells with cell-adhesion-mediated drug resistance (CAM-DR). In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin (Dox) conjugates, simultaneously, to selectively target MM cells and to overcome CAM-DR. Dox was conjugated to the nanoparticles through an acid-sensitive hydrazone bond. VLA-4-antagonist peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities. The nanoparticles were efficiently internalized by MM cells and induced cytotoxicity. Mechanistic studies revealed that nanoparticles induced DNA double-strand breaks and apoptosis in MM cells. Importantly, multifunctional nanoparticles overcame CAM-DR, and were more efficacious than Dox when MM cells were cultured on fibronectin-coated plates. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors with ∼10 fold more drug accumulation and demonstrated dramatic tumor growth inhibition with a reduced overall systemic toxicity. Altogether, we demonstrate the disease driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM.

Cisplatin is a first line chemotherapy for most types of cancer. However, its use is dose-limited due to severe nephrotoxicity. Here we report the rational engineering of a novel nanoplatinate inspired by the mechanisms underlying cisplatin bioactivation. We engineered a novel polymer, glucosamine-functionalized polyisobutylene-maleic acid, where platinum (Pt) can be complexed to the monomeric units using a monocarboxylato and an O --> Pt coordinate bond. We show that at a unique platinum to polymer ratio, this complex self-assembles into a nanoparticle, which releases cisplatin in a pH-dependent manner. The nanoparticles are rapidly internalized into the endolysosomal compartment of cancer cells, and exhibit an IC50 (4.25 +/- 0.16 microM) comparable to that of free cisplatin (3.87 +/- 0.37 microM), and superior to carboplatin (14.75 +/- 0.38 microM). The nanoparticles exhibited significantly improved antitumor efficacy in terms of tumor growth delay in breast and lung cancers and tumor regression in a K-ras(LSL/+)/Pten(fl/fl) ovarian cancer model. Furthermore, the nanoparticle treatment resulted in reduced systemic and nephrotoxicity, validated by decreased biodistribution of platinum to the kidney as quantified using inductively coupled plasma spectroscopy. Given the universal need for a better platinate, we anticipate this coupling of nanotechnology and structure-activity relationship to rationally reengineer cisplatin could have a major impact globally in the clinical treatment of cancer.

Unlabelled: Sensorineural hearing loss (SNHL) related to chronic suppurative otitis media (CSOM) was studied to clarify the involvement of cholesteatomas in this context., Aim: to evaluate SNHL related to CSOM and its association with cholesteatomas, disease duration and patients ages., Methods: Retrospective analysis of 115 patients with CSOM with and without cholesteatoma submitted to surgical treatment. Inclusion criteria were active unilateral disease, normal contralateral ear and age below 60 years., Results: The average age was 26.3 years, 58 males and 57 females. The duration of ear disease was, in average, 12.4 years. The average threshold of hearing was 40 dB in CSOM ear and 22 dB in the normal contralateral ear (P=0.002). CSOM with cholesteatoma occurred in 78 of 115 cases. In the abnormal ear, SNHL was seen in 15 cases, being 6 cases of profound loss, that correlated with adjusted-age (P=0.003) and absence of cholesteatoma (P=0.01), but not with disease duration (P=0.458)., Conclusion: SNHL occurred in 13% of the patients with CSOM, and was correlated with older age, but not with the presence of cholesteatoma or longer duration of ear disease.