Microbial-immune cross-talk and regulation of the immune system

We are all born germ-free. Following birth we enter into a lifelong relationship with microbes residing on our body's surfaces. The lower intestine is home to the highest microbial density in our body, which is also the highest microbial density known on Earth (up to 10(12) /g of luminal contents). With our indigenous microbial cells outnumbering our human cells by an order of magnitude our body is more microbial than human. Numerous immune adaptations confine these microbes within the mucosa, enabling most of us to live in peaceful homeostasis with our intestinal symbionts. Intestinal epithelial cells not only form a physical barrier between the bacteria-laden lumen and the rest of the body but also function as multi-tasking immune cells that sense the prevailing microbial (apical) and immune (basolateral) milieus, instruct the underlying immune cells, and adapt functionally. In the constant effort to ensure intestinal homeostasis, the immune system becomes educated to respond appropriately and in turn immune status can shape the microbial consortia. Here we review how the dynamic immune-microbial dialogue underlies maturation and regulation of the immune system and discuss recent findings on the impact of diet on both microbial ecology and immune function.

Potential role of CYP2D6 in the central nervous system

1. Cytochrome P450 2D6 (CYP2D6) is a pivotal enzyme responsible for a major drug oxidation polymorphism in human populations. Distribution of CYP2D6 in brain and its role in serotonin metabolism suggest that CYP2D6 may have a function in the central nervous system. 2. To establish an efficient and accurate platform for the study of CYP2D6 in vivo, a human CYP2D6 (Tg-2D6) model was generated by transgenesis in wild-type (WT) C57BL/6 mice using a P1 phage artificial chromosome clone containing the complete human CYP2D locus, including the CYP2D6 gene and 5'- and 3'-flanking sequences. 3. Human CYP2D6 was expressed not only in the liver but also in the brain. The abundance of serotonin and 5-hydroxyindoleacetic acid in brain of Tg-2D6 is higher than in WT mice, either basal levels or after harmaline induction. Metabolomics of brain homogenate and cerebrospinal fluid revealed a significant up-regulation of L-carnitine, acetyl-L-carnitine, pantothenic acid, 2'-deoxycytidine diphosphate (dCDP), anandamide, N-acetylglucosaminylamine and a down-regulation of stearoyl-L-carnitine in Tg-2D6 mice compared with WT mice. Anxiety tests indicate Tg-2D6 mice have a higher capability to adapt to anxiety. 4. Overall, these findings indicate that the Tg-2D6 mouse model may serve as a valuable in vivo tool to determine CYP2D6-involved neurophysiological metabolism and function.

Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

No evidence for a local renin-angiotensin system in liver mitochondria

The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.

Asymptotic freedom, dimensional transmutation, and an infrared conformal fixed point for the δ-function potential in one-dimensional relativistic quantum mechanics

We consider the Schrödinger equation for a relativistic point particle in an external one-dimensional δ-function potential. Using dimensional regularization, we investigate both bound and scattering states, and we obtain results that are consistent with the abstract mathematical theory of self-adjoint extensions of the pseudodifferential operator H=p2+m2−−−−−−−√. Interestingly, this relatively simple system is asymptotically free. In the massless limit, it undergoes dimensional transmutation and it possesses an infrared conformal fixed point. Thus it can be used to illustrate nontrivial concepts of quantum field theory in the simpler framework of relativistic quantum mechanics.

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Solution of the relativistic Schrödinger equation for the δ ′ -Function potential in one dimension using cutoff regularization

We study the relativistic version of the Schrödinger equation for a point particle in one dimension with the potential of the first derivative of the delta function. The momentum cutoff regularization is used to study the bound state and scattering states. The initial calculations show that the reciprocal of the bare coupling constant is ultraviolet divergent, and the resultant expression cannot be renormalized in the usual sense, where the divergent terms can just be omitted. Therefore, a general procedure has been developed to derive different physical properties of the system. The procedure is used first in the nonrelativistic case for the purpose of clarification and comparisons. For the relativistic case, the results show that this system behaves exactly like the delta function potential, which means that this system also shares features with quantum filed theories, like being asymptotically free. In addition, in the massless limit, it undergoes dimensional transmutation, and it possesses an infrared conformal fixed point. The comparison of the solution with the relativistic delta function potential solution shows evidence of universality.

Investigation of hemodynamics in an in vitro system simulating left ventricular support through the right subclavian artery using 4-dimensional flow magnetic resonance imaging.

OBJECTIVES Left ventricular assist devices are an important treatment option for patients with heart failure alter the hemodynamics in the heart and great vessels. Because in vivo magnetic resonance studies of patients with ventricular assist devices are not possible, in vitro models represent an important tool to investigate flow alterations caused by these systems. By using an in vitro magnetic resonance-compatible model that mimics physiologic conditions as close as possible, this work investigated the flow characteristics using 4-dimensional flow-sensitive magnetic resonance imaging of a left ventricular assist device with outflow via the right subclavian artery as commonly used in cardiothoracic surgery in the recent past. METHODS An in vitro model was developed consisting of an aorta with its supra-aortic branches connected to a left ventricular assist device simulating the pulsatile flow of the native failing heart. A second left ventricular assist device supplied the aorta with continuous flow via the right subclavian artery. Four-dimensional flow-sensitive magnetic resonance imaging was performed for different flow rates of the left ventricular assist device simulating the native heart and the left ventricular assist device providing the continuous flow. Flow characteristics were qualitatively and quantitatively evaluated in the entire vessel system. RESULTS Flow characteristics inside the aorta and its upper branching vessels revealed that the right subclavian artery and the right carotid artery were solely supported by the continuous-flow left ventricular assist device for all flow rates. The flow rates in the brain-supplying arteries are only marginally affected by different operating conditions. The qualitative analysis revealed only minor effects on the flow characteristics, such as weakly pronounced vortex flow caused by the retrograde flow via the brachiocephalic artery. CONCLUSIONS The results indicate that, despite the massive alterations in natural hemodynamics due to the retrograde flow via the right subclavian and brachiocephalic arteries, there are no drastic consequences on the flow in the brain-feeding arteries and the flow characteristics in the ascending and descending aortas. It may be beneficial to adjust the operating condition of the left ventricular assist device to the residual function of the failing heart.

A novel organ-slice culturing system to simulate meniscal repair: Proof of concept using a synovium-based pool of meniscoprogenitor cells.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait and obesity. Sixty per cent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-ß1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. This article is protected by copyright. All rights reserved.

A novel colourimetric technique to assess chewing function using two-coloured specimens: Validation and application

OBJECTIVES Chewing efficiency may be evaluated using cohesive specimen, especially in elderly or dysphagic patients. The aim of this study was to evaluate three two-coloured chewing gums for a colour-mixing ability test and to validate a new purpose built software (ViewGum©). METHODS Dentate participants (dentate-group) and edentulous patients with mandibular two-implant overdentures (IOD-group) were recruited. First, the dentate-group chewed three different types of two-coloured gum (gum1-gum3) for 5, 10, 20, 30 and 50 chewing cycles. Subsequently the number of chewing cycles with the highest intra- and inter-rater agreement was determined visually by applying a scale (SA) and opto-electronically (ViewGum©, Bland-Altman analysis). The ViewGum© software determines semi-automatically the variance of hue (VOH); inadequate mixing presents with larger VOH than complete mixing. Secondly, the dentate-group and the IOD-group were compared. RESULTS The dentate-group comprised 20 participants (10 female, 30.3±6.7 years); the IOD-group 15 participants (10 female, 74.6±8.3 years). Intra-rater and inter-rater agreement (SA) was very high at 20 chewing cycles (95.00-98.75%). Gums 1-3 showed different colour-mixing characteristics as a function of chewing cycles, gum1 showed a logarithmic association; gum2 and gum3 demonstrated more linear behaviours. However, the number of chewing cycles could be predicted in all specimens from VOH (all p<0.0001, mixed linear regression models). Both analyses proved discriminative to the dental state. CONCLUSION ViewGum© proved to be a reliable and discriminative tool to opto-electronically assess chewing efficiency, given an elastic specimen is chewed for 20 cycles and could be recommended for the evaluation of chewing efficiency in a clinical and research setting. CLINICAL SIGNIFICANCE Chewing is a complex function of the oro-facial structures and the central nervous system. The application of the proposed assessments of the chewing function in geriatrics or special care dentistry could help visualising oro-functional or dental comorbidities in dysphagic patients or those suffering from protein-energy malnutrition.

An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle

BACKGROUND: Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work. RESULTS: NanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo. CONCLUSIONS: PbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate requirements of NanoLuc are different from those of firefly luciferase, dual bioluminescence imaging for the simultaneous characterization of two lines, or two separate biological processes, is possible, as demonstrated in this work.

Biodiversity and Ecosystem Function in the Gulf of Maine: Pattern and Role of Zooplankton and Pelagic Nekton

This paper forms part of a broader overview of biodiversity of marine life in the Gulf of Maine area (GoMA), facilitated by the GoMA Census of Marine Life program. It synthesizes current data on species diversity of zooplankton and pelagic nekton, including compilation of observed species and descriptions of seasonal, regional and cross-shelf diversity patterns. Zooplankton diversity in the GoMA is characterized by spatial differences in community composition among the neritic environment, the coastal shelf, and deep offshore waters. Copepod diversity increased with depth on the Scotian Shelf. On the coastal shelf of the western Gulf of Maine, the number of higher-level taxonomic groups declined with distance from shore, reflecting more nearshore meroplankton. Copepod diversity increased in late summer, and interdecadal diversity shifts were observed, including a period of higher diversity in the 1990s. Changes in species diversity were greatest on interannual scales, intermediate on seasonal scales, and smallest across regions, in contrast to abundance patterns, suggesting that zooplankton diversity may be a more sensitive indicator of ecosystem response to interannual climate variation than zooplankton abundance. Local factors such as bathymetry, proximity of the coast, and advection probably drive zooplankton and pelagic nekton diversity patterns in the GoMA, while ocean-basin-scale diversity patterns probably contribute to the increase in diversity at the Scotian Shelf break, a zone of mixing between the cold-temperate community of the shelf and the warm-water community offshore. Pressing research needs include establishment of a comprehensive system for observing change in zooplankton and pelagic nekton diversity, enhanced observations of "underknown'' but important functional components of the ecosystem, population and metapopulation studies, and development of analytical modeling tools to enhance understanding of diversity patterns and drivers. Ultimately, sustained observations and modeling analysis of biodiversity must be effectively communicated to managers and incorporated into ecosystem approaches for management of GoMA living marine resources.

Digital Vertical Aerial Camera System for High-resolution Site Inspections in Conservation Easment Monitoring

A three-level satellite to ground monitoring scheme for conservation easement monitoring has been implemented in which high-resolution imagery serves as an intermediate step for inspecting high priority sites. A digital vertical aerial camera system was developed to fulfill the need for an economical source of imagery for this intermediate step. A method for attaching the camera system to small aircraft was designed, and the camera system was calibrated and tested. To ensure that the images obtained were of suitable quality for use in Level 2 inspections, rectified imagery was required to provide positional accuracy of 5 meters or less to be comparable to current commercially available high-resolution satellite imagery. Focal length calibration was performed to discover the infinity focal length at two lens settings (24mm and 35mm) with a precision of O.1mm. Known focal length is required for creation of navigation points representing locations to be photographed (waypoints). Photographing an object of known size at distances on a test range allowed estimates of focal lengths of 25.lmm and 35.4mm for the 24mm and 35mm lens settings, respectively. Constants required for distortion removal procedures were obtained using analytical plumb-line calibration procedures for both lens settings, with mild distortion at the 24mm setting and virtually no distortion found at the 35mm setting. The system was designed to operate in a series of stages: mission planning, mission execution, and post-mission processing. During mission planning, waypoints were created using custom tools in geographic information system (GIs) software. During mission execution, the camera is connected to a laptop computer with a global positioning system (GPS) receiver attached. Customized mobile GIs software accepts position information from the GPS receiver, provides information for navigation, and automatically triggers the camera upon reaching the desired location. Post-mission processing (rectification) of imagery for removal of lens distortion effects, correction of imagery for horizontal displacement due to terrain variations (relief displacement), and relating the images to ground coordinates were performed with no more than a second-order polynomial warping function. Accuracy testing was performed to verify the positional accuracy capabilities of the system in an ideal-case scenario as well as a real-world case. Using many welldistributed and highly accurate control points on flat terrain, the rectified images yielded median positional accuracy of 0.3 meters. Imagery captured over commercial forestland with varying terrain in eastern Maine, rectified to digital orthophoto quadrangles, yielded median positional accuracies of 2.3 meters with accuracies of 3.1 meters or better in 75 percent of measurements made. These accuracies were well within performance requirements. The images from the digital camera system are of high quality, displaying significant detail at common flying heights. At common flying heights the ground resolution of the camera system ranges between 0.07 meters and 0.67 meters per pixel, satisfying the requirement that imagery be of comparable resolution to current highresolution satellite imagery. Due to the high resolution of the imagery, the positional accuracy attainable, and the convenience with which it is operated, the digital aerial camera system developed is a potentially cost-effective solution for use in the intermediate step of a satellite to ground conservation easement monitoring scheme.