Principal Site Research Articles

Pre-clinical Imaging of Invasive Candidiasis Using ImmunoPET/MR.

The human commensal yeast Candida is the fourth most common cause of hospital-acquired bloodstream infections, with Candida albicans accounting for the majority of the >400,000 life-threatening infections annually. Diagnosis of invasive candidiasis (IC), a disease encompassing candidemia (blood-borne yeast infection) and deep-seated organ infections, is a major challenge since clinical manifestations of the disease are indistinguishable from viral, bacterial and other fungal diseases, and diagnostic tests for biomarkers in the bloodstream such as PCR, ELISA, and pan-fungal β-D-glucan lack either standardization, sensitivity, or specificity. Blood culture remains the gold standard for diagnosis, but test sensitivity is poor and turn-around time slow. Furthermore, cultures can only be obtained when the yeast resides in the bloodstream, with samples recovered from hematogenous infections often yielding negative results. Consequently, there is a pressing need for a diagnostic test that allows the identification of metastatic foci in deep-seated Candida infections, without the need for invasive biopsy. Here, we report the development of a highly specific mouse IgG3 monoclonal antibody (MC3) that binds to a putative β-1,2-mannan epitope present in high molecular weight mannoproteins and phospholipomannans on the surface of yeast and hyphal morphotypes of C. albicans, and its use as a [64Cu]NODAGA-labeled tracer for whole-body pre-clinical imaging of deep-seated C. albicans infections using antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI). When used in a mouse intravenous (i.v.) challenge model that faithfully mimics disseminated C. albicans infections in humans, the [64Cu]NODAGA-MC3 tracer accurately detects infections of the kidney, the principal site of blood-borne candidiasis in this model. Using a strain of the emerging human pathogen Candida auris that reacts with MC3 in vitro, but which is non-infective in i.v. challenged mice, we demonstrate the accuracy of the tracer in diagnosing invasive infections in vivo. This pre-clinical study demonstrates the principle of using antibody-guided molecular imaging for detection of deep organ infections in IC, without the need for invasive tissue biopsy.

Dangerous drugs, dangerous mothers: Gender, responsibility and the problematisation of parental substance use

If, as many would have it, the ‘drugs problem’ is among the more perilous and uncompromising challenges of our times, parental substance misuse represents one of its most insidious expressions. The past 15 years has seen the ‘hidden harms’ experienced by the children of drug users emerge as a principal concern for national policy actors and local service provision. However, there has been relatively little critique of the assumptions and epistemological foundations underscoring this policy shift, or of the preoccupation with the ‘family’ in drug policy in general. Through examination of seminal policy documents relating to parental substance misuse, and using Carol Bacchi’s ‘What’s the Problem Represented to Be?’ (WPR) approach, this article attends more closely to the formulation of parental drug use as a significant policy problem, and to the family as a principal site for the constitution of drug harms.

Emotions and the Body in Early Modern Medicine

Drawing on Latin treatises, letters, and autobiographical writings, this article outlines the changes in the—thoroughly somatic—learned medical understanding of the emotions (or “affectus/passiones animi”) between 1500 and 1800 and their impact on lay experience. The mixture of the four natural humors explained individuals’ different propensity to certain emotions. The emotions as such, however, were described primarily as movements of the spirits and the blood towards or away from external objects. The term “e(s)motion” emerged. The final part highlights the 18th-century shift from spirits and blood to the nerves as the principal site of the emotions. Physicians and laypersons alike now associated the emotions closely with the peculiar nervous sensibility and irritability of individuals and groups.

HLA-DQ Specific Alloantibodies Modify Allogenic Endothelial Cell Immunogenicity and Impair the Expansion of Anti-Inflammatory FoxP3 High Regulatory T Lymphocytes

Recent clinical studies have highlighted the prevalence of de novo circulating Donor-Specific Antibodies (DSA) against HLA-DQ following solid organ transplantation. The emergence of HLA-DQ DSA has been associated with poor graft outcome. The allograft microvascular endothelium is known to upregulate HLA-DQ expression during rejection. The allograft endothelium is directly exposed to circulating anti-donor antibodies and as a result, becomes a principal site of DSA binding and thereby the target of antibody-mediated damage. Endothelial activation is a feature of antibody-mediated rejection and is reported to arise from inflammation, DSA binding and complement activation. Activated endothelial cells modulate the recipient’s immune response as a result of their interactions with CD4+ T lymphocytes. Plasmapheresis samples were obtained from alloimmunized patients with high levels of circulating DSA. Antibodies were isolated using protein G beads, and their HLA specificity was assessed by Luminex. Antibodies were evaluated for binding to microvascular endothelial cells, and a time course of binding was established by flow cytometry. Patient antibodies specific for relevant HLA-DQ alleles were used to assess potential signal transduction from antibody-binding to endothelial cells. HLA-DQ DSA induced transient phosphorylation of Akt and S6 Kinase. This was confirmed using a monoclonal anti-HLA DQ antibody. Alloantibodies against HLA-DQ alleles not expressed by our cell model did not induce specific signalling events. Previously, we found that HLA-DR+ endothelial cells are capable of inducing alloproliferation of CD4+ T lymphocytes and selectively expanding Th17 and FoxP3high T regulatory cell (Treg) subsets (Taflin PNAS 2011, Lion Am J Transplant 2016). The presence of intragraft Th17 correlates with shorter graft survival, whilst the proportion of Treg cells has been implicated in tolerance. To assess the functional consequences of DSA binding, endothelial cells were stimulated with HLA-DQ DSA for one hour preceding co-culture with allogenic peripheral blood mononuclear cells. After seven days, CD4+ T cell subsets were analysed by intracellular cytokine staining. HLA-DQ DSA modified endothelial cell immunogenicity, resulting in a reduction in alloproliferation and in Treg expansion. Alloantibodies against HLA-DQ alleles not expressed by our cell model did not alter alloproliferation or Treg expansion. The impaired amplification of anti-inflammatory Treg cells, in combination with an unaltered expansion of the Th17 subset, leads to an overall pro-inflammatory orientation of alloreactive CD4+ T cells. This altered composition of CD4+ T cells, due to HLA-DQ binding to endothelial cells, may exacerbate allograft damage and reduce allograft tolerance. AC was supported by the Société Francophone de Transplantation (SFT).

Abstract 4582: Capturing T cell receptor clones from circulating free DNA in cancer patients with solid malignancies

Abstract Introduction Understanding the tumor microenvironment is challenging in situations where a tumor biopsy is unavailable. In these situations, liquid biopsies provide a less invasive alternative that can be collected at multiple timepoints throughout treatment. In particular, analyses of circulating free DNA (cfDNA) from plasma samples can distinguish genomic alterations from shed tumor DNA (ctDNA) within the cfDNA population. In this study, we aim to better understand the origin of cfDNA as well as its potential in understanding the intratumoral immune infiltration by developing and validating a strategy for identifying T cell receptor β (TCR β) rearrangements in the cfDNA from patients with solid malignancies. Methods We devised a target enrichment approach for high-throughput DNA sequencing that employs a hybridization capture strategy to bait germline variable (V) and joining (J) gene sequences in the TCR β loci. The target sequences are proximal to rearrangement sites flanking the CDR3, the principal site of contact for recognition specificity with foreign antigens presented by the major histocompatibility complex (MHC). We controlled for amplification bias by tagging DNA fragments with unique molecular indices (UMI). We validated our approach on dilutions of T cell lines in non-T cell lines, compared T cell levels determined from our method to those derived from flow cytometry, and compared data complexity at DNA input levels from 100 ng to 2 ng. Finally, we analyzed 27 pre-treatment plasma samples from cancer patients with solid malignanices, including quantification of total cfDNA concentrations, analyzing somatic genomic alterations using whole exome sequencing (WES), and employing our method for capture of TCR β rearrangements. Results Results indicate we are able to capture T cell rearrangements from cfDNA in cancer patient plasma. T cell rearrangements were identified in more than 25% of samples (7/27). Interestingly, the total cfDNA levels were not correlated to the number of T cell rearrangements detected, but all samples with detectable somatic alterations in the cfDNA (3/27) were also those with T cells identified. These preliminary results suggest a connection between apoptosis of lymphocytes and solid tumors that shed DNA into the bloodstream, and demonstrate potential utility as liquid biopsy for identifying tumor infiltrating lymphocytes. Citation Format: Lara McGrath, Elizabeth Maloney, Daniel Stetson, Tristan J. Lubinski, Rory Kirchner, Brian Dougherty, J. Carl Barrett. Capturing T cell receptor clones from circulating free DNA in cancer patients with solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4582.

Extraosseous Ewing's Sarcoma of the Maxillary Sinus: A Very Rare Entity

AbstractEwing's sarcoma is rare sarcoma of bone and soft tissue that uncommonly involve the head and neck in both bone and soft tissue locations. The term Ewing's sarcoma family of tumors comprises both Ewing's sarcoma and peripheral neuroectodermal tumor. It was in 1975, Angervall and Enzinger described the Ewing's sarcoma arising in soft tissues (extraskeletal). The principal sites of extraskeletal Ewing's sarcoma are the paravertebral region and chest wall, generally in close association with vertebrae or the ribs. These tumors may also arise in the soft tissues of the lower extremities and rarely in the pelvic region and hip regions. Superficially located cases do occur but are rare. Here, we present a case of Ewing's sarcoma involving the maxillary sinus region. A 12-year-old boy reported to our department with a swelling on the right side of middle third of the face for 15 days. Intraorally, there was only obliteration of the labial vestibule. On histopathological examination, a provisional diagnosis of malignant round cell tumor was given. Immunohistochemistry with a panel of markers was done to come to a definitive diagnosis. CD99 positivity confirmed the diagnosis of Ewing's sarcoma.

Fumigating the Hygienic Model City: Bubonic Plague and the Sulfurozador in Early-Twentieth-Century Buenos Aires.

The 1899/1900 arrival of bubonic plague in Argentina had thrown the model status of Buenos Aires as a hygienic city into crisis. Where the idea of foreign threats and imported epidemics had dominated the thinking of Argentina's sanitarians at that time, plague renewed concerns about hidden threats within the fabric of the capital's dense environment; concerns that led to new sanitary measures and unprecedented rat-campaigns supported by the large-scale application of sulphur dioxide. The article tells the story of early twentieth-century urban sanitation in Buenos Aires through the lens of a new industrial disinfection apparatus. The Aparato Marot, also known as Sulfurozador was acquired and integrated in the capital's sanitary administration by the epidemiologist José Penna in 1906 to materialise two key lessons learned from plague. First, the machine was supposed to translate the successful disinfection practices of global maritime sanitation into urban epidemic control in Argentina. Second, the machine's design enabled public health authorities to reinvigorate a traditional hygienic concern for the entirety of the city's terrain. While the Sulfurozador offered effective destruction of rats, it promised also a comprehensive - and utopian - disinfection of the whole city, freeing it from all imaginable pathogens, insects as well asrodents. In 1910, the successful introduction of the Sulfurozador encouraged Argentina's medico-political elite to introduce a new principle of 'general prophylaxis'. This article places the apparatus as a technological modernisation of traditional sanitary practices in the bacteriological age, which preserved the urban environment -'elterreno'- as a principal site of intervention. Thus, the Sulfurozador allowed the 'higienistas' to sustain a long-standing utopian vision of all-encompassing social, bodily and political hygiene into the twentieth century.

The control of the adsorption of bovine serum albumin on mercaptan-modified gold thin films investigated by SERS spectroscopy

Nanostructured gold thin films were built from deposition of colloidal gold nanoparticles on silanized glass slides, and used to study the adsorption of bovine serum albumin (BSA) after chemical treatment of gold surface with the mercaptans 2-mercaptoethanol, 3-mercaptoproprionic acid, 1,3-propanedithiol and 1-propanethiol. Surface-enhanced Raman scattering (SERS) spectroscopy was used for investigating the chemical interactions of BSA with the modified gold surfaces. In the presence of the surface modifier 2-mercaptoethanol, a promoter of hydrogen bonds, the stable interactions among BSA and gold surfaces led to high reproducibility of the SERS spectral pattern in the most monitored points of the mapped surface. The vibrational assignment endorsed the assumption that lysine residue, majority present in the molecular structure, were the principal anchor site of BSA involved in the interactions with 2-mercaptoethanol-modified gold surface.

Review: Synovial Cell Metabolism and Chronic Inflammation in Rheumatoid Arthritis.

Metabolomic studies of body fluids show that immune-mediated inflammatory diseases such as rheumatoid arthritis (RA) are associated with metabolic disruption. This is likely to reflect the increased bioenergetic and biosynthetic demands of sustained inflammation and changes in nutrient and oxygen availability in damaged tissue. The synovial membrane lining layer is the principal site of inflammation in RA. Here, the resident cells are fibroblast-like synoviocytes (FLS) and synovial tissue macrophages, which are transformed toward overproduction of enzymes that degrade cartilage and bone and cytokines that promote immune cell infiltration. Recent studies have shown metabolic changes in both FLS and macrophages from RA patients, and these may be therapeutically targetable. However, because the origins and subset-specific functions of synoviocytes are poorly understood, and the signaling modules that control metabolic deviation in RA synovial cells are yet to be explored, significant additional research is needed to translate these findings to clinical application. Furthermore, in many inflamed tissues, different cell types can forge metabolic collaborations through solute carriers in their membranes to meet a high demand for energy or biomolecules. Such relationships are likely to exist in the synovium and have not been studied. Finally, it is not yet known whether metabolic change is a consequence of disease or whether primary changes to cellular metabolism might underlie or contribute to the pathogenesis of early-stage disease. In this review article, we collate what is known about metabolism in synovial tissue cells and highlight future directions of research in this area.

An international qualitative study exploring patients’ experiences of cutaneous leishmaniasis: study set-up and protocol

IntroductionLack of investments in drug development, lack of standardisation of clinical trials and the complexity of disease presentations contribute to the current lack of effective, safe and adapted treatments for...

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.

Burnout and Scope of Practice in New Family Physicians.

Family physicians report some of the highest levels of burnout, but no published work has considered whether burnout is correlated with the broad scope of care that family physicians may provide. We examined the associations between family physician scope of practice and self-reported burnout. Secondary analysis of the 2016 National Family Medicine Graduate Survey respondents who provided outpatient continuity care (N = 1,617). We used bivariate analyses and logistic regression to compare self-report of burnout and measures of scope of practice including: inpatient medicine, obstetrics, pediatric ambulatory care, number of procedures and/or clinical content areas, and providing care outside the principal practice site. Forty-two percent of respondents reported feeling burned out from their work once a week or more. In bivariate analysis, elements of scope of practice associated with lower burnout rates included providing more procedures/clinical content areas (mean procedures/clinical areas: 7.49 vs 7.02; P = .02) and working in more settings than the principal practice site (1+ additional settings: 57.6% vs 48.4%: P = .001); specifically in the hospital (31.4% vs 24.2%; P = .002) and patient homes (3.3% vs 1.5%; P = .02). In adjusted analysis, practice characteristics significantly associated with lower odds of burnout were practicing inpatient medicine (OR = 0.70; 95% CI, 0.56-0.87; P = .0017) and obstetrics (OR = 0.64; 95% CI, 0.47-0.88; P = .0058). Early career family physicians who provide a broader scope of practice, specifically, inpatient medicine, obstetrics, or home visits, reported significantly lower rates of burnout. Our findings suggest that comprehensiveness is associated with less burnout, which is critical in the context of improving access to good quality, affordable care while maintaining physician wellness.

Oropharyngeal (OPG) vaccination activates head and neck lymphoid tissues required for immune protection against mucosal Brucella infection

Abstract Human brucellosis remains a global health problem with no human vaccines existing. Most human infections occur mucosally with eventual systemic brucellae dissemination. Current interventions primarily focus on the systemic disease, rather than considering neutralizing the infection at its source. A number of studies show that the OPG mucosa is the principal site of brucellae uptake following oral or inhalational exposures. We have devised an OPG infection method using an attenuated Brucella mutant by allowing the mice to drink (ad bibitum) from a pipette tip. BALB/c mice were fed once daily for 3 days with 108 CFUs. Individual head and neck lymph nodes (HNLNs), and spleens were examined at 2 weeks post-infection for bacterial colonization and proinflamatory cytokine responses. Elevated colonization was observed in submandibular LNs (SmLNs) and deep cervical LNs (CLNs). Modest colonization was detected in the parotid LNs (PrLNs) and spleens. Splenic and individual HNLN lymphocytes were analyzed for IFN-g, TNF-a, granzyme B, and perforin production by flow cytometry. SmLN, CLN, and PrLN IFN-g-producing CD8+ T cells significantly exceeded IFN-g-producing CD4+ T cells by 2.5-, 2.5-, and 1.5-fold, respectively. Increased numbers of CD4+ and CD8+ T cells expressing TNF-a, granzyme B, and perforin were also observed. All activated lymphocytes were predominantly of effector-memory phenotype, CD44+CD62LlowCCR7low. These studies demonstrate that ad bibitum vaccination against brucellosis results in colonization and activation of the HNLNs, mimicking natural human infection, enabling future inquiry into the immune mechanisms responsible for protection to mucosal Brucella infections. Work supported by NIH AI-125546.

A novel approach for synthesis of exfoliated biopolymeric-LDH hybrid nanocomposites via in-stiu coprecipitation with gum Arabic: Application towards REEs recovery

Delamination and exfoliation of layered double hydroxides (LDH) is an interesting way for the synthesis of novel nanocomposites. Herein, we report the synthesis of various exfoliated biopolymeric-LDH nanocomposites via in-situ coprecipitation method with gum Arabic (GA). The influence of various divalent ions on the morphological characteristics of GAnXA (n = wt% of GA and X = Mg, Ca, Ba, Sr) was explored Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), and Scanning Electron Microscopy (SEM). X-ray diffraction pattern was used for the determination of phase composition of synthesized nanocomposites. The surface functional groups of GAnXA were investigated by Fourier Transform Spectroscopy (FTIR). Furthermore, the surface charge characteristics was explored by zeta potential analysis. Specific surface area of GAnXA was determined by BET analysis. The overall adsorption of REEs decreased with an increase in ionic size of divalent ions and the noticeable difference in the morphologies of exfoliated GAnXA was observed. In a single component system, the REEs adsorption capacities followed the order: Sc > Y > Nd > Ce > Eu > La, whereas, in multicomponent system, adsorption seems to be competitive and presence of competing ions affect the overall REEs removal. Moreover, HREEs removal superseded over LREEs and the nanocomposites (GA5CA, GA5SA, GA5BA) were highly selective for Sc recovery. The post-adsorption FTIR and SEM results revealed the importance of surface hydroxyl and carboxyl functional groups enacting as the principal REE binding sites. Overall, ability to extract REEs at pH 4 (slightly acidic) presented a facile route for the recovery and separation of REEs from aqueous medium and enhances the possibility of its use in many industrial applications.

Excess iron stress reduces root tip zone growth through nitric oxide‐mediated repression of potassium homeostasis in Arabidopsis

The root tip zone is regarded as the principal action site for iron (Fe) toxicity and is more sensitive than other root zones, but the mechanism underpinning this remains largely unknown. We explored the mechanism underpinning the higher sensitivity at the Arabidopsis root tip and elucidated the role of nitric oxide (NO) using NO-related mutants and pharmacological methods. Higher Fe sensitivity of the root tip is associated with reduced potassium (K+ ) retention. NO in root tips is increased significantly above levels elsewhere in the root and is involved in the arrest of primary root tip zone growth under excess Fe, at least in part related to NO-induced K+ loss via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4) and reduced root tip zone cell viability. Moreover, ethylene can antagonize excess Fe-inhibited root growth and K+ efflux, in part by the control of root tip NO levels. We conclude that excess Fe attenuates root growth by effecting an increase in root tip zone NO, and that this attenuation is related to NO-mediated alterations in K+ homeostasis, partly via SNO1/SOS4.

Hydration Structure and Dynamics of Inhibitor-Bound HIV-1 Protease.

Water is essential in many biological processes, and the hydration structure plays a critical role in facilitating protein folding, dynamics, and ligand binding. A variety of biophysical spectroscopic techniques have been used to probe the water solvating proteins, often complemented with molecular dynamics (MD) simulations to resolve the spatial and dynamic features of the hydration shell, but comparing relative water structure is challenging. In this study 1 μs MD simulations were performed to identify and characterize hydration sites around HIV-1 protease bound to an inhibitor, darunavir (DRV). The water density, hydration site occupancy, extent and anisotropy of fluctuations, coordinated water molecules, and hydrogen bonds were characterized and compared to the properties of bulk water. The water density of the principal hydration shell was found to be higher than bulk, dependent on the topology and physiochemical identity of the biomolecular surface. The dynamics of water molecules occupying principal hydration sites was highly dependent on the number of water-water interactions and inversely correlated with hydrogen bonds to the protein-inhibitor complex. While many waters were conserved following the symmetry of homodimeric HIV protease, the asymmetry induced by DRV resulted in asymmetric lower-occupancy hydration sites at the concave surface of the active site. Key interactions between water molecules and the protease, that stabilize the protein in the inhibited form, were altered in a drug resistant variant of the protease indicating that modulation of solvent-solute interactions might play a key role in conveying drug resistance. Our analysis provides insights into the interplay between an enzyme inhibitor complex and the hydration shell and has implications in elucidating water structure in a variety of biological processes and applications including ligand binding, inhibitor design, and resistance.

Duplicated TLR5 of zebrafish functions as a heterodimeric receptor

Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the homodimeric TLR5-flagellin interaction. Here we provide evidence that zebrafish (Danio rerio) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5-flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach.

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

An <em>In Vivo</em> Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer's disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed. We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer's disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed. We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.