text:
Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e. intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.
19 Related JoVE Articles!
Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections
Institutions: Universidad de la República, Trinity College Dublin.
Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages.
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae
inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.
Medicine, Issue 90, Sublingual immunotherapy, Pneumonia, Streptococcus pneumoniae, Lungs, Flagellin, TLR5, NLRC4
Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines
Institutions: U.S. Army Medical Research Institute of Infectious Diseases.
The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3
. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6
. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8
. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10
. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7
. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6
. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo
culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo
studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13
. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3
. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.
Infectious Diseases, Issue 66, Immunology, nasal vaccination, nasopharyngeal-associated lymphoreticular tissue, mouse, antibody, mucosal immunity, NALT ablation, NALT culture, NALT-deficient mice
Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice
Institutions: Stanford University School of Medicine, Stanford University School of Medicine.
Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo
approaches over in vitro
methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the ‘mast cell knock-in’
approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo
. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.
Immunology, Issue 99, c-kit, stem cell factor, FcεRI, immunoglobulin E, mouse model, adoptive transfer, immunology, allergy
Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care
Institutions: Duke University, Duke University, Duke University.
Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo
to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.
Immunology, Issue 96, Tumor immunotherapy, glioblastoma, chimeric antigen receptor, adoptive transfer, temozolomide, radiotherapy
Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination
Institutions: University of Michigan, University of Michigan, University of Michigan.
Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.
Immunology, Issue 98, nanoparticle, vaccine, biomaterial, subunit antigen, adjuvant, cytotoxic CD8+ T lymphocyte, whole animal imaging, tetramer staining, and lymph node
Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity
Institutions: Heinrich Pette Institute, Leibniz Institute for Experimental Virology.
Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies.
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo
, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium
, and hemorrhagic fever viruses such as Ebola virus.
Immunology, Issue 100, Mouse models, vaccines, immunity, dendritic cells, influenza, T cells
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors
Institutions: The University of Texas MD Anderson Cancer Center, The University of Texas Graduate School of Biomedical Sciences.
This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer's patch (PP). Common dendritic cell progenitors (CDPs, lin-
) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/-
mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+
and recipients being CD45.2+
. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/-
mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.
Immunology, Issue 85, hematopoiesis, dendritic cells, Peyer's patch, cytokines, adoptive transfer
Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via
functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v
.) or intracerebroventricular (i.c.v.
) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.
) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo
Here we describe the methods that we have developed for the i.v
. and i.c.v.
delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats
Institutions: University of California, Irvine (UCI).
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.
Cell Biology, Issue 6, Immunology, Immune Response, Inflammation, lymphocyte, inflammatory reaction, skin test, video protocol
Intra-lymph Node Injection of Biodegradable Polymer Particles
Institutions: University of Maryland, College Park.
Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.
size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.
Bioengineering, Issue 83, biomaterial, immunology, microparticle, nanoparticle, vaccine, adjuvant, lymph node, targeting, polymer
Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Institutions: The University of Texas at Austin, The University of Texas at Austin, The University of Texas at Austin.
Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.
Chemistry, Issue 79, Membrane Lipids, Toll-Like Receptors, Endotoxins, Glycolipids, Lipopolysaccharides, Lipid A, Microbiology, Lipids, lipid A, Bligh-Dyer, thin layer chromatography (TLC), lipopolysaccharide, mass spectrometry, Collision Induced Dissociation (CID), Photodissociation (PD)
Isolation of Lymphocytes from Mouse Genital Tract Mucosa
Institutions: University of California, Los Angeles , California NanoSystems.
Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1
. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2
, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.
Immunology, Issue 67, Mucosal immunity, sexually transmitted diseases, genital tract lymphocytes, lymphocyte isolation, flow cytometry, FACS
Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
Institutions: McMaster University, Hamilton, University of Toronto.
Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2
. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4
. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5
. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7
. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.
Immunology, Issue 65, Medicine, Physiology, Dendritic Cells, allergic airway inflammation, ovalbumin, lymph nodes, lungs, dendritic cell maturation, dendritic cell migration, mediastinal lymph nodes
Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells
Institutions: RIKEN Research Center for Allergy and Immunology.
The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2
. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4
. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli
and Salmonella enterica
serovar Typhimurium (S.
Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5
. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7
. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.
Neuroscience, Issue 58, M cell, Peyer's patch, bacteria, immunosurveillance, confocal microscopy, Glycoprotein 2
Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
Institutions: Health canada, The State Food and Drug Administration, Beijing, University of Ottawa, King Abdulaziz University, Public Health Agency of Canada.
Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2
. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3
. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4
, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5
. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7
. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6
, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7
. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8
. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used.
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.
Immunology, Issue 50, Virology, influenza, hemagglutinin, neuraminidase, quantification, universal antibody
Culture of myeloid dendritic cells from bone marrow precursors
Institutions: McMaster University, McMaster University, University of Waterloo.
Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors.
Immunology, Issue 17, dendritic cells, GM-CSF, culture, bone marrow
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research
Establishment and Characterization of UTI and CAUTI in a Mouse Model
Institutions: Washington University School of Medicine.
Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli
(UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis
. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis
bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.
Medicine, Issue 100, Escherichia coli, UPEC, Enterococcus faecalis, uropathogenic, catheter, urinary tract infection, IBC, chronic cystitis

domain: medical


text:
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Be informed daily with these free e-newsletters
November 13, 2016
Michigan soybean farmers have their checkoff interests represented at the state level by the Michigan Soybean Promotion Committee (MSPC) and by the United Soybean Board (USB) at the national level. New appointments were made recently to both boards.
At the state level, MSPC appointments are made by Governor Snyder and there are seven farmer directors who serve on this board.
George Zmitko of Owosso represented District 6 (Clinton, Ionia, Genesee and Shiawassee counties) and recently served tenure on the board. George stated, “I appreciate having had the opportunity to serve Michigan’s soybean farmers on the MSPC as a director in both the secretary and treasurer positions.”
Michigan farmers appointed to state and national soybean boards
The Governor named R. Alan Moore of Bannister as Zmitko’s successor in District 6. Moore has farmed for over 45 years. He previously served on the MSPC board and has also served on USB, North Central Soybean Research Program (NCSRP) board and National Biodiesel Board (NBB). Moore says, “It is my goal to help others in farming and to share my knowledge with my fellow farmers. I believe we can grow well together if we share our knowledge. I look forward to serving again on the MSPC.”
In District 7, Governor Snyder reappointed Steve Koeman of Hamilton for a second three-year term. Steve has farmed for 25 years and raises soybeans, corn and hogs. Steve says, “I’ve enjoyed serving the first term on the MSPC and look forward to what this next term will bring. I like promoting soybean uses and exports for Michigan’s farmers.”
At the national level, USB appointments are made by the United States Department of Agriculture – Agricultural Marketing Service (USDA-AMS) Agriculture Secretary, Tom Vilsack. Michigan soybean farmers are represented on USB with three board seats.
David Williams of Elsie and James Domagalski of Columbus were recently reappointed to the board. Both will serve an additional three-year term through December 2019.
Williams has been farming for 26 years and grows soybeans, corn and soft red winter wheat. Additionally, he is the current president of the Michigan Soybean Association.
Domagalski has farmed for over 30 years and raises corn, soybeans and wheat. Beyond his service on USB, he has also served on the MSPC, NBB and United States Soybean Export Council (USSEC).
Williams and Domagalski serve alongside Herb Miller of Niles on the board.
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domain: news


text:
There are several babies on the way in our family, so I’ve started making some baby quilts. I tried several new techniques on this one. I pieced the triangles on the serger instead of the sewing machine. It went together quickly and should add to the durability of the quilt. Then I quilted using special rulers and a ruler foot, with two different sewing machines. There’s quite a learning curve to using the rulers!
When it came time to bind the quilt, I could not find the binding I had originally cut. So I found coordinating solid fabrics and tried the faux piping method of binding. I think it looks much better than what I had planned!
The center block, a found block from an unknown quilter, was provided as part of a challenge. The fan blocks came from my Grandmother’s leftovers. She was well known for her prolific quiltmaking. I felt a satisfying connection to my Grandmother and other quilters from the past as I constructed this quilt from vintage fabrics and trims.
What I thought would be easy enough–making one block a day in the Splendid Sampler sew-along–lasted one day. Until today! This pattern called for an embroidered motif in the center. I had a piece of tatted lace that my sister-in-love Marie had made some time ago. Unfortunately it had gotten stained and set aside, waiting to be useful. It was the perfect size and shape for this block! I used sharpie markers and alcohol to dye it. I like the result!
We presented the finished quilt to Birthright at our March quilt guild meeting. It will be auctioned for a fundraiser!
So, instead of getting any of the old projects finished, I’ve quilted a project for a friend and she is pleased. A group of us got together and made a quilt top to donate to Birthright. It’s a really cute and bright hugs and kisses quilt. I’ll get a picture when it’s finished.
Not what I had planned but satisfying and good things are done!
In one of my quilt guilds, the president has challenged us to complete unfinished projects this year. She asked us to submit a list of projects. Then, as we finish projects, we bring them for show and tell at the monthly meeting. She has a point system with prizes at the end of the year. I’m not looking for prizes but I consider this a good way to make progress on projects I’d like to see completed!
So far I have started and completed a new purse for myself–not an unfinished project from the list, but a satisfying accomplishment. I used ByAnnie’s Soft and Stable to give it body and stability. I also upcycled some parts from old purses. I’m pleased with the results.
This morning I started another new project. This is a year-long project called Splendid Sampler. Over 80 quilt designers have compiled 100 six inch blocks that will be released one at a time twice a week. This sounds like something I can keep up with. The first block is a heart, perfect for Valentine’s Day.At least I am on track for today!
As for the unfinished projects? Maybe later….

domain: hobby


text:
Julia & Eric Roberts Approached To Be Stuttering Spokespeople
A recent newsletter reads, "The Roberts siblings stuttered as children and ironically give credence to the factor the heredity/genetic link in the stuttering equation. They have no doubt inspired young people with the problem."
Representative Jane Fraser tells the New York Post, "It would be marvelous to have them participate."
Heroes actor Eric recently revealed he still suffers occasional stuttering relapses, most notably on a late night talk show. He said, "I got on a stuttering jag and the audience got uncomfortable and started laughing. I stayed relaxed, but I didn't have the presence of mind, until afterwards, to acknowledge the stutter and put the audience at ease."
Other stuttering celebrities, named by the SFA, include Bruce Willis, Carly Simon, Harvey Keitel, Jane Seymour and Tiger Woods.
-Julia Roberts Pictures
(This news article provided by World Entertainment News Network)
First Look: Check Out Rob Lowe As JFK In 'Killing Kennedy' Miley Cyrus Throws One 'Crazy' Party In Latest Video, 'We Can't Stop'

domain: celebrity


text:
Hire Dinner Party Catering in Bourton-on-the-Water
Browse Dinner Party Caterers
Recent review: Thank you so much for creating such a delicious meal - it was perfect and everyone thoroughly enjoyed the food. The kitchen was left immaculate too and the service was fantastic. Really really loved it!
Recent review: "Absolutely amazing! Fantastic service and product provided by Jason and team. Thank you, we will definitely be using you again."
About: We provide delicious vegetarian (and some vegan) Indian Street Food dishes. Myself & my wife set up this business in July 2018, as we felt my mum's Indian dishes need to be available to the public. Our food is unique, tasty and fun! The recipes are all family owned making it truly authentic. We aim
About: We are a catering company based in the Cotswolds specialising in Spanish paella, tapas and Catalan specialities. As the chef/owner and native of Barcelona, I am passionate about bringing the food of my youth to this country. We combine genuine Spanish ingredients with fresh local produce to create a
About: McBaile is a Catering and Event Management Company for discerning clients who require the highest standard of Catering and Event Planning Services, coupled with a discreet and friendly service. Our clients have discovered the benefits of dealing with a Catering Company run by professionals and th
About: Rubols Fusion Kitchen was set up by cousins Ruby and Holly, who have a passion for food and travel. They have combined their love of Middle Eastern and Mexican food to create their own unique dishes, such as the 'Shawarrito' (a cross between a Shawarma wrap and a Burrito). They serve the 'Shawarri
Recent review: "Dear Philippa, I would like to thank you and your superb team so much for the excellent food that Relish supplied at the Blenheim Horse Trials last week. Your mother told me that you did the catering at Badminton, and your food was definitely the best that we have ever had. The standard over
About: Clean Plate Catering Company offers different food styles tailored for various occasions at venues to suit our client's requirement. So whether you require a popup restaurant date for 10 guests tasting amazing food or a social gathering hot buffet for 200 persons we are the catering company to use
Recent review: To say we were impressed is an understatement, we are all well travelled and have eaten in amazing places all over the world so to have such amazing food served in my home was an amazing experience. 15 ladies & he pleased all, everyones tastes catered for we had vegans / veggies / pescatarians a c
Recent review: I decided to try and find someone to cater for my BBQ, I had a lot of friends attending and I felt I would not have the time to spend with them if doing the cooking myself. Mark and Alice arrived early to set up the gazebo's and BBQ which all looked so impressive. I had never met them before but you
Recent review: "Too Perfection really are Perfection. We hired them for the day after our wedding where we hosted a garden party for around 50 people. Nick was brilliant in the build up and on the day. The food and the service was fantastic and all our guests loved every bite. Everyone of them commented on how goo
Recent review: Nicholas was fantastic and I couldn't recommend him more! I bought my father a cooking lesson with him for his birthday and Nicholas made him feel so comfortable. He came prepared with great courses that had been discussed beforehand, put much time and thought into the preparation and bought produce
Recent review: Jacci was very helpful before the event. We are newbies to the catering scene, but Jacci sorted us out very well. The food she provided was very good, and our guests thoroughly enjoyed coming back for seconds. Her team knew what they were doing, and were unobtrusive when required. We would certainly
Recent review: Thank you Sue for all the food. It was delicious and a great addition to my Hen weekend. The food, van and service were great. Gluten free and vegetarians were also well catered for.
Recent review: An outstanding service provided by Luke, who served us a beautiful meal and couldn’t do enough to show us a fabulous evening. I would highly recommend and will be using them again!
About: Home cooking at it's best. We do amazing all vegetarian cooking on the day in front of you. We specialise in home made samosas, onion bhaji, curries & rice and authentic snacks. This is what I grew up with and wish to share with you. Currently I am trading at Gloucester King's square - opposite Ic
Recent review: "Thanks for making a great day even greater Every one loved your Hog and still there was much left over Thanks for being so efficient and so well organised"
Recent review: We hired Pink Pineapple Catering for a work summer social. They provided us enough food for 40 people, with the option of increasing this if more people showed up on the day. The food was Fantastic and their customer service was professional. The next day numerous people in the office came up to me
About: Full event planners, catering and hire. Exclusive Occasions Balloons & Banners for all occasions
Recent review: Claire and Umberto were brilliant! From booking to the event the communication was excellent. Claire was always happy to help with the organising of the event. On the evening, Claire and Umberto arrived and were friendly and it was a lovely relaxed atmosphere. They cooked and served the food, it was

domain: food


We have problem with our macchiatobin, the vanilla kernel not work:
[ 58.364030] mvpp2 f2000000.ethernet eth0: could not attach PHY (-19)
[ 58.381763] mvpp2 f2000000.ethernet eth0: could not attach PHY (-19)
[ 67.443146] mvpp2 f4000000.ethernet eth1: could not attach PHY (-19)
[ 67.460872] mvpp2 f4000000.ethernet eth1: could not attach PHY (-19)
How fix this?
up, some bugs in kernel 4.19.16
[ 5.401915] mvpp2 f10f0000.ethernet eth0: Using hardware mac address 00:00:00:00:00:04
[ 17.784700] mvpp2 f10f0000.ethernet eth0: could not attach PHY (-22)
[ 78.929793] mvpp2 f10f0000.ethernet eth0: could not attach PHY (-22)
dst file: https://pastebin.com/5gSkKwYK
.config file: https://pastebin.com/Dw7abMz9
last kernel with work mvpp2 ethernet is 4.18.20, but not work in 4.19.1
i replace folder drivers/net/ethernet/marvell from kernel 4.18.20 to 4.19.1 and ethernet is working
I'd like to add to this old issue, that it might be related to the Single-Shot board and an issue discussed on the official Macchiato forums (http://macchiatobin.net/forums/topic/kernel-vanilla/page/4/).
The issue that causes these messages might be related to the armada kernel driver trying to access RJ45 specific instructions that won't be available (due to the Single-Shot board missing these physical ports).
The official wiki on Macchiato Bin (http://wiki.macchiatobin.net/tiki-index.php?page=Build+from+source+-+Kernel) mentions this briefly, and if anything this is more a shortcoming in the driver itself that it can not handle both setups.
To verify that this is the issue, you can download the single-shot armada driver provided by macchiatobin/solidrun or - if you run Arch Linux ARM - this driver is pre-shipped and can be found at /boot/dtbs/marvel/armada-8040-mcbin-singleshot.dtb, which has to be re-named to /boot/dtbs/marvel/armada-8040-mcbin.dtb
After a reboot, link should come up and iproute2 commands should work as expected.
Hi, updated kernel + vanilla updated uboot + single dts have resolved issue


text:
INDIANAPOLIS — The shot that Michigan basketball will forever remember was born out of desperation, a dwindling shot clock and a vanishing lead, from a season that began with Finals aspirations. Four but that looked like something much worse in the months leading up to March.
The ball belonged to Eli Brooks, the grad student with more games in a blue-and-corn uniform than anyone in the program’s history. He returned for a fifth season to experience a moment like this, a chance to do better than Wolverines a year ago by reaching the Elite Eight. Losing in the round of 16 was not part of his plan.
Brooks veered downhill to the right side of the arc and threw the kind of looping, arcing hook shot that would have made Kareem Abdul-Jabbar proud. He floated through the Indianapolis air as a waiting crowd at Gainbridge Fieldhouse held their collective breath. Then he gently splashed the net with the most perfect of rustles.
His incredible shot extended a rocking season and gave No. 11 seed Michigan an insurmountable lead with 53 seconds left against third-seeded Tennessee. This beleaguered group of Wolverines – a group that was shut down due to COVID-19, played several games without their best player and whose coach was suspended for a horrific incident on national television – had pulled off a superb upset, 76-68, Saturday to reach the Sweet 16.
RECRUITMENT BATTLE:Michigan fight for Jett Howard adds subplot to game against Tennessee
“I just try to live in the moment,” Brooks said, “and cherish the time that we have with the people that are in my life right now in Michigan. Just enjoy the process of reliving this tournament and have that chance to get our last goal.”
That final goal – to win a national championship – lives on thanks to the stellar performances of Brooks, who scored 23 points in one of the defining moments of his illustrious career, and Dickinson, who played flawlessly to score a game-high. team of 27 points and grabbing 11 rebounds in one night, the Wolverines needed them all. Super sub Terrance Williams II scored six of his nine points in the final five minutes to spark the latest comeback after a 60-54 deficit.
Michigan held the Volunteers to 2 for 18 shots from beyond the arc and didn’t allow a 3-pointer in the second half. Brooks balanced his majestic offensive display with near-relentless harassment of sniper Santiago Vescovi, UT’s second-leading scorer, who finished with his lowest point total (five) since Jan. 29.
“That was the main focus,” Brooks said. “Keep them away from the line and avoid fake shots. We did that. That was the biggest key to winning the game.”
The return of point guard DeVante’ Jones after a concussion sidelined him against Colorado State gave UM an emotional boost, and fans cheered as he spoke for the pre-match warm-ups. But Jones’ presence was fleeting; he only played 12 minutes before spending the second half in the locker room after falling ill with a head injury, according to a team spokesperson.
For most of the first half, Juwan Howard’s side bit on the mouthpiece and turned to the SEC Tournament champion Volunteers. Wave after wave of pressure from Tennessee’s vaunted defense couldn’t faze the Wolverines, who battled a series of early turnovers as they adapted their offense to combat the relentless peddling of five orange blurs fortifying that end of the floor.
A tight defense against Jones, Brooks and freshman point guard Frankie Collins repeatedly pushed Michigan’s starting position near midfield. The lobbed entry passes to Dickinson or the drive-and-kick 3-pointers for Caleb Houstan that have become staples of Howard’s system this season were suddenly blocked. Tennessee came in forcing opponents to commit 15.6 turnovers per game, and UM lost the ball nine times in the first half.
Instead, the Wolverines ran pick-and-pops with Dickinson for a pair of 3s early; or found Moussa Diabate (13 points, 6 rebounds) on a series of rim cuts for close range finishes; or instructed guards to use dribble transfers that minimized Tennessee’s ability to break through passing lanes. When Dickinson swept a free-throw line jumper with 2:29 remaining in the half — an unusual type of shot from an unusual spot for the team’s top scorer — Michigan climbed ahead, 32 -31, to annoy the largely pro-Volunteers crowd.
Howard’s group even recovered from a calamitous streak of four straight turnovers to finish the half by hitting eight of their first 12 shots after the break, tying the game twice and taking a one-point lead. three times. Michigan fans roared as Dickinson blocked a layup attempt, altered another shot after a Volunteers offensive rebound, then dove to the floor in pursuit of a loose ball, ultimately producing a tie.
“We turned the ball over, things weren’t going our way,” Williams said. “But being the most connected team is what Coach Howard talks about, and we were the most connected team. We didn’t part ways even though we made mistakes.”
Portions of the second half were breathless in a game with six draws and 12 lead changes. Every layup from Tennessee freshman dynamo Kennedy Chandler (19 points, nine assists), every midrange jumper from James (13 points, six rebounds) was answered by the Wolverines, who wove their performance together the most daring of the season on the biggest stage. against an opponent favored by seven.
They found an answer for every burst. When the Volunteers built a six-point lead with 8:27 remaining, Williams soared for back-to-back brilliant tricks on a missed 3-pointer by Collins and a layup gone wrong from Dickinson. When the game was tied with 3:21 remaining, Brooks charged down the right side for a layup that produced a traditional three-point play to propel UM ahead, 65-62.
The pad proved large enough to keep Tennessee at bay. Howard’s team closed out the game shooting 8-for-8 from the free throw line to send Michigan to a fifth straight Sweet 16, where anything and everything seems possible given what this group has accomplished in the past. course of the last three days.
As the clock expired, Dickinson tossed the ball into the stands to cap off an extraordinary evening. This incredible, unpredictable, Hollywood-could-not-script-it season continues.
Who knows where it will end.
“Nobody believed in us,” Dickinson said. “Everyone thought we shouldn’t be in the tournament. And now people who hated us are going home to watch us next week.”


text:
thank you for the feedback, Really am looking forward to the full release of this vn. it is really unique and stunning from what i have seen.
Thanks, I'm really glad you like it so far. Full release will still take a while, but don't worry, there's more to come, eventually. :3
<3 looking forward to it mate.

domain: artistic


Location: San Dimas, California
Completion Date: 2003 / 2015
JVC Architects designed a new 1,000-seat sanctuary, multipurpose hall and office facilities for this 1960’s-built church campus. For the 26,595 square-foot project, JVC followed the requirements of the city of San Dimas, California, to incorporate early California architctural styles. Green design principles were also integrated, including an erosion and sedimentation control plan to control erosion and reduce its negative impacts on water and air quality, a storm water management system that limits pollution of natural water floors, a tobacco smoke control system that limits exposure of building occupants and systems. JVC also reduced the occurence of light pollution to reduce reliance on electricity.


text:
Boil the lentils following pack instructions, drain, rinse well, then drain thoroughly.
Meanwhile, mix the citrus juices, vinegar and a pinch of salt in a salad bowl, then toss in the onion rings – after a few mins they will soften and turn pink. Whisk together the oil, cumin, garlic and chutney, then toss into the onions with the cooled lentils, coriander, tomatoes, spinach and plenty of seasoning.
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text:
A Cambridgeshire woman whose wallet was stolen 18 years ago has had it returned.
It still contained her bank cards, a lucky shamrock and her prized picture of Eric Cantona.
Joanne Bevan was astonished when the wallet, stolen in 2000, was returned to her Mum's house.
Joanne, who was 21 in 2000, said having the wallet returned took her back to that time.
It was taken from the hotel she was working at in St Ives, Cambridgeshire.
On August 11 the wet and muddy purse was posted through the police station letter box.
Using the address on her provisional licence they were able to return it to her Mum's house.

domain: law


Nyesom Wike, the governor of Rivers State, has expressed worry about the Federal Government’s indifference to the plight of internally displaced persons (IDPs) in Benue State and the resulting humanitarian disaster.
He also denounced the state’s murderous herdsmen’s operations, which resulted in the eviction of people from their ancestral homes.
Wike, who visited his Benue counterpart, Samuel Ortom, in Makurdi over the weekend, said the state’s predicament merited the same level of attention from the federal government as the North East and other regions of the country.
READ ALSO: Buhari Is Part Of Nigeria’s Problem – Wike
He said: “Even if the Federal Government does not like the Benue governor, they should support him because the people of the state voted for the President. The Federal Government should take care of the IDPs in Benue as they do in the North East and other states.”
He stated he would stick by Ortom because he despised the injustice being meted out to the state as a result of his noble stand in defense of his people.


text:
Search for positions by entering key words in search box below. Use left side navigation to filter by Region, Job Category, Job Type or Salary.
TEST DATE: THURSDAY, JUNE 14, 2018 AT 2:00 P.M.
THE POSITION: The City of Los Banos is seeking an individual to fill the position of Utilities Maintenance I in the Public Works Department. This person performs a variety of unskilled or semi-skilled maintenance duties, and operates a variety of equipment in the construction, operation, repair, maintenance, and replacement of City water, sewer and storm drainage facilities and systems; and other duties as needed.
Under the general policy and program direction of the Library Commission, manage and direct the Library’s operations to assure a balanced countywide library system that includes a comprehensive program of library services.
The County of Yuba is currently recruiting Extra Help Cooks in the Probation Department. Under close supervision, prepares and provides institutional meals following federal and state nutritional guidelines, and performs other duties as assigned. This classification is assigned to the County Juvenile Hall & Camp, a division within the Probation Department, or the County Jail division in the Sheriff’s department. This position performs a full range of tasks associated with the preparation and distribution of meals to juvenile detainees and adult inmates.

domain: library


The objective of this study is to examine the effect of the geometrical modification of the land on the overall film cooling effectiveness on the cutback region of a turbine blade model. A room-temperature experiment was conducted, in which nitrogen serves as the cooling stream, and the mainstream flow is air. The adiabatic film cooling effectiveness was mapped employing the pressure-sensitive paint (PSP) technique. Data was acquired at five different blowing ratios (from 0.45 to 1.65) for both the baseline and the modified model. Detailed film cooling effectiveness from PSP measurements in correlation with the flow map in streamwise and spanwise planes from particle image velocimetry (PIV) measurements was performed, characterizing the effect of rounding the edges of the lands. The results show that the rounded edges enable the coolant flow to reach the top surface of the land area more readily, especially at low blowing ratios. Superior coolant coverage on the land surface observed in the PSP measurements are well correlated with the PIV measurements. At the high blowing ratio of 1.65, the round edge of the lands helps regulate the mixing between the coolant and mainstream flows, therefore the film cooling effectiveness in the slot region is also improved.


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A thermal power station is a power station in which heat energy is converted to electric power.In most, a steam-driven turbine converts heat to mechanical power as an intermediate to electrical power. Water is heated, turns into steam and drives a steam turbine which drives an electrical generator.After it passes through the turbine the steam is condensed in a condenser and recycled to where
Pipe, Tube, Angle, Channel & Flat Bar Rolling Machines A+ Rating Radius rolling of angle, channel, flat bar, and T-shapes is usually done on a pyramid style 3 roll bender. Tube and pipe can also be rolled but not to the small radius that can be repeatably and accurately done on a rotary mandrel draw bender machine using dies that match the tube/pipe size. Please remember that tube is measured
Diesel engines are the power behind our biggest machines—trucks, trains, ships, and submarines. On the face of it, they're similar to ordinary gasoline (petrol) engines but they generate more power, more efficiently by working in a subtly different way. Let's take a closer look! Photo: Diesel engines (like the one in this railroad locomotive) are ideal for pulling heavy trains. This is a
Industrial Steam Boiler Manufacturers. Clayton Industries was established in October 20, 1930. We have a worldwide reputation as a leading industrial steam boiler manufacturer of industrial process steam generation products. These include fired boilers and unfired waste heat boilers. Clayton's unique controlled circulation counter-flow design offers many operational advantages and benefits
Depreciation allowance as percentage of actual cost (a) Plant and Machinery in generating stations including plant foundations :— (i) Hydro-electric. 3.4 (ii) Steam electric NHRS & Waste heat recovery Boilers/plants. 7.84 (iii) Diesel electric and Gas plant. 8.24 (b) Cooling towers and circulating water systems. 7.84
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BLEEDING AIR FROM DIESEL FUEL LINES AND FILTERS Farm Machinery Fact Sheet FM-01 By Dr. Von H. Jarrett, Extension Agricultural Engineer When you change a diesel fuel filter, run out of fuel or disturb the fuel system, air is trapped. When you try to start the engine this air acts as a lock, preventing the normal supply of fuel into the cylinder.
HKEY_LOCAL_MACHINE und HKEY_CURRENT_USER sind Registrierungsschlüssel (Keys) in der zentralen Registrierungsdatenbank (Registry) von Windows. Diese Schlüssel enthalten bestimmte Einträge und
2 ton oil boiler original in France - carolienvaneyck.be. Boiler name: 2 ton oil boiler original in France. The company currently has the leading production process equipment in the industrial boiler industry. The main equipments include: high-speed CNC plane drilling, CNC drum drilling, robot welding equipment, 3D laser cutting machine, 4-axis
Union Pacific 4-8-8-4 Big Boy Locomotive. 26 Replies . By William Pearce. For some time, locomotives of the Union Pacific Railroad (UP) had struggled to climb the Wasatch mountains between Ogden, Utah and Green River, Wyoming. This 176-mile (283-km) stretch of track started out at 4,300 ft (1,310 m) above sea level in Ogden, climbed the Wasatch Range to 7,300 ft (2,225 m) at the Aspen Tunnel
Boiler efficiency does not remain fixed and large variations from ideal values take place during the course of operation. Shifting to real time efficiency monitoring can improve the boiler efficiency significantly depending upon the boiler type and actual conditions at site. In nutshell, monitoring and maintaining the boiler efficiency for the overall operational life of the boiler is a must
Size / Capacity: 121 tons (110 metric tons) Since 1982, NS Machine has served the metal working industry by supplying machine tools and manufacturing machinery for the following industries: Sheet Metal Fabrication, Tank Head & Shell Fabrication, Tube Fabrication, Rollforming, Machining, Stamping, Coil Processing and more. Our inventory includes a wide variety of used machinery including
Unit Conversion--How to Calculate Boiler Horsepower 2016-11-17 14:13:21. HP mean Horse power and BHP mean Boiler Horse power. Our clients are usually confused with the two units. Now, let us make them clear. Boiler Horsepower - BHP. The Boiler Horsepower (BHP) is the amount of energy required to produce 34.5 pounds of steam per hour at a pressure and temperature of 0 Psig and 212 ℉, with
All dies verweist somit diese Motoren, mehr noch als früher, in den Luxusbereich. Bei Lkws gibt es nach Überschreiten der 500-PS-Grenze bei aufgeladenen Sechszylinder-Dieseln kaum noch einen Grund für die Nutzung von V8-Motoren. Diese Motoren werden in kleinen Stückzahlen weiter gebaut werden, um in Zugmaschinen für Schwertransporte und Schienenfahrzeuge sowie in Boote eingebaut zu werden
Sentinel Waggon Works Ltd was a British company based in They began producing steam road vehicles in 1905 and in 1906 introduced a 5 ton vertical-boiler wagon, which featured a 2-cylinder undertype engine and chain drive. Around 1915 Alley & McLellan moved the steam wagon production to a new factory in England and it continued under a separate company (see below)., and in 1918 the company
Founded in 1973, the Machine Moving & Engineering Group operates nationally and we specialise in the relocation and installation of all forms of machinery and equipment. With over 45 years of experience in our field within South Africa and Africa, we can provide you with unparalleled service excellence and professionalism.. We offer a fully comprehensive turnkey package, incorporating:
BMW X4 M40i: Kraftstoffverbrauch in l/100 km (kombiniert): 8,1-7,8; CO2-Emissionen in g/km (kombiniert): 185-177 . BMW X4 M40d: Kraftstoffverbrauch in l/100 km (kombiniert): 5,9-5,6; CO2-Emissionen in g/km (kombiniert): 156-147 . Die offiziellen Angaben zu Kraftstoffverbrauch, CO2-Emissionen und Stromverbrauch wurden nach dem vorgeschriebenen Messverfahren VO (EU) 715/2007 in der jeweils
THE INDIAN BOILERS ACT, 1923 (Act No. 5 of 1923)* (23rd February, 1923) An Act to consolidate and amend the law relating to steam-boilers . Whereas it is expedient to consolidate and amend the law relating to steam-boilers; It is hereby enacted as follows: 1. Short title, extent and commencement (1) This Act may be called the Indian Boilers Act, 1923. (2) It extends to the whole of India
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Der neue S8, der im Herbst bestellbar ist und gegen Jahresende - und damit zwei Jahre nach dem normalen A 8 der aktuellen Generation - auf den Markt kommt, darf Super Plus statt Diesel verbrennen.
Ford Fiesta 8 ST 1,5 Turbo: Ford Focus 1 RS: Ford Focus 2 RS 2009 : Ford Focus 2 ST 225: Ford Focus 3 RS 2016: Ford Focus 3 ST 250/TDCi: Ford Focus 4 ST 280/TDCi: Ford Focus ST 170: Mondeo ST 200/220: Neue Produkte im Juni: Powerflex Drehmomentstütze Ford Fiesta 8 ST 1,5 / Sport 147,90EUR (inkl. 19 % UST exkl.Versandkosten) Powerflex Buchse Einsatz Drehmomentstütze gr. Fiesta 8 ST /Sport 29
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Verbrauch: 3.8 - 7.0 l/100 km Im Unterschied zu den Benzinern ist bei den Diesel-Aggregaten ein Sechsganggetriebe serienmäßig mit an Bord. Beim Allradantrieb kann der Fahrer zwischen den drei Modi 2WD, 4WD und Â‚gesperrtÂ' wählen. Unterschiede des Peugeot 4008 zum Mitsubishi ASX. Um die Vorlage von Mitsubishi französisch wirken zu lassen, wurde der Japaner mit neuer Front und
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Steam, Boiler, and Blowdown Pressure are the same. Combustion Efficiency is the % of fuel energy that is directly added to the feedwater and not otherwise lost or used. Blowdown Rate is the % of incoming feedwater mass flow rate that leaves the boiler as a saturated liquid at boiler pressure.
Boiler horsepower (BHP) is a measure of boiler energy output. One boiler horsepower (from and at 212 °F): † BHP = 34.5 lbs/hr † BHP = 33,472 Btu/hr † BHP = 9.8 kW † BHP should not be confused with other horsepower measurements. Boiler Trim: Accessories added to the boiler, such as auxiliary low water cutoff, special tappings, special gas
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1.1* Scope. This code applies to the following: A.1.1 Technological advances in recent years and, in particular, the pervasiveness of microprocessor-based hardware make it even more important that only highly qualified individuals be employed in applying the requirements of this code to operating systems. Each type of hardware has its own unique features and operational modes.
Machinists and tool and die makers set up and operate machine tools to produce precision metal parts, instruments, and tools. Work Environment. Machinists and tool and die makers work in machine shops, toolrooms, and factories. Although many work full time during regular business hours, overtime may be common, as is evening and weekend work.
Fulton is a global manufacturer of steam, hot water and thermal fluid (hot oil) heat transfer products. Fulton specializes in complete industrial/commercial boiler and heater systems.
Automatic Diesel Boiler In Punjab - luxushaeppchen.de. diesel fired industrial boiler - ricoma-stickmaschinen.de. diesel fired steam boiler manufacturer in europe . Steam locomotive - Wikipedia. 2019-3-24 · A steam locomotive is a type of railway locomotive that produces its pulling power through a steam engine.These locomotives are fueled by burning combustible material usually coal, wood
A boiler is defined as "a closed vessel in which water or other liquid is heated, steam or vapor is generated, steam is superheated, or any combination thereof, under pressure or vacuum, for use external to itself, by the direct application of energy from the combustion of fuels, from electricity or nuclear energy."


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A casino online is a digital platform that lets players wager real money and win cash prizes without leaving the comfort of their home. They can access a much wider range of gambling games than their retail counterparts, and often offer more generous welcome bonuses and loyalty rewards as well. Some casinos also offer a full range of live dealer tables to enhance the experience.
There are many ways to gamble for real money online, but a safe and legitimate way is through regulated casino sites. These operate within the jurisdiction of their respective gaming authorities and abide by strict security standards to protect player data and money. They use 128-bit SSL encryption to prevent hacking and have independent auditors in place to test their systems and payouts on a regular basis.
Regulated casino online sites offer a wide variety of casino games, including slots, blackjack, roulette, video poker, baccarat and craps. They are available through a web browser or, in some cases, a dedicated mobile app. Many of these apps feature an attractive design and a user-friendly interface that makes them easy to navigate. Most regulated online casinos also offer a secure and fast banking option that lets you deposit and withdraw funds quickly and easily.
Physical casinos have a special atmosphere that is hard to replicate online, and their location within the city or town helps generate revenue for other local businesses such as hotels, restaurants and nightclubs. They are often part of a larger complex that also hosts events such as poker tournaments and special time-limited jackpots. They are also popular locations for TV and film productions, creating additional revenue streams.
When choosing an online casino, it is important to consider the types of games that they offer and their availability on both desktop and mobile devices. Most reputable online casinos offer a comprehensive selection of casino games from some of the best software developers in the world. Those that specialize in slot games typically offer high-quality graphics and innovative gameplay features, while those that feature table games have a strong focus on reliability and fairness. Regardless of what type of games you prefer, all top-rated casino websites have a variety of secure banking options that let you fund your account quickly and easily. They accept credit cards, e-wallets such as PayPal and VIP Preferred, and popular cryptocurrencies such as Bitcoin. They are also compatible with most major mobile operating systems.

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A veteran campaigner has vowed never to give up his battle to force council chiefs to bring illegal parkers to book.
Jack Gledhill says the staff and customers of businesses along Whitegate Drive, Blackpool, have been parking illegally for years and have not received so much as a ticket.
And the 76-year-old, of Gainsborough Road, claims bollards and railings are “ineffectual”.
For five years he has contacted Blackpool Council with numerous letters pointing out that motorists are breaking the law on a daily basis and demanding that something is done.
Mr Gledhill said: “I have pointed out the requirements of the current legislation on countless occasions, but nothing is done. The double yellow lines on the road clearly indicate that no one should be parking there, but every day the road is full of illegally parked cars. There is no sign of a traffic warden, no cars are ticketed and the decision to build bollards – one of which has already been knocked down – to stop drivers parking where they shouldn’t be parking in the first place is a waste of money. I will continue fighting against illegal parking all the time people continue to park illegally.”
Mr Gledhill says notices were recently placed on 200 illegally parked cars. However, the following day they were parked once more in their usual spots.
He said: “Doing something like that is pointless, if you are not going to follow it up by handing out parking tickets.”
Blackpool Council said it had previously contacted Mr Gledhill directly and declined to comment further.


No A/C, what will we do?
It’s easy to take modern conveniences, like a climate-controlled home, for granted. For most of us, keeping the home cool on the hottest days of the year is as easy as tapping a button or turning a dial on the thermostat. That wasn’t always the case, though. Before air conditioning was invented by Willis Carrier in 1902, and before it became a mainstream appliance about 60 years ago, people had to deal with the heat a little more creatively.
It’s simple, but true. During stifling heat waves, people often left the hot, still air of their homes to find refuge in the shade of the biggest and most sprawling nearby trees. A home with trees in the yard was about more than having nice curb appeal. It was a hot commodity, especially for the hottest days of the year.
This technique is believed to date back to the time of ancient Egyptians, who would dampen thin materials and then hang them in doorways. The dampness combined with a breeze provided at least some relief when the temperature spiked above what was tolerable. It’s been reported that on his deathbed in 1881, President James Garfield received some relief from the heat with a more advanced version of this trick: cotton sheets drenched in ice water with air blown through them. It took a lot of energy and manpower to keep this system going, though. Garfield’s caretakers burned through 500,000 pounds of ice in a two-month period trying to keep him comfortable.
Ancient Rome is known for its amazing aqueduct advances that made it possible for citizens to easily obtain water. The wealthier citizens were able to take this technology one step further by installing mini-aqueduct systems in their own homes that circulated cool water through the walls. By keeping the materials of the walls cool, the entire home was less hot despite the temperatures outside.
In the summer? Yes, actually. During the Renaissance period, it was common for men to haul blocks of snow and ice from mountainous regions and then sell it to the locals. There were no freezers, but special cooling pits were created to transport the cool materials from one spot to the next. In homes, people would use the snow and ice for drinks or to keep food from spoiling. Some even used the evaporation from the coolness to create an ancient version of the modern air conditioner.
Electric fans have been around for over 100 years, but before that people were smart about using the materials around them to stay cool manually. It still works today, of course. Fanning oneself with hand-made fans, palm fronds, or feathers creates a nice breeze on the skin, which helps your body’s natural cooling function—sweating—by helping to cool and evaporate the moisture more quickly.
If you’ve ever driven through a post-World War II neighborhood, you’ve probably noticed some similar exterior design techniques among the homes. One of the most prominent features is the shaded front porch. This was a luxury before the days of air conditioning. People would escape the trapped heat of their homes for at least a little relief outside. Porch swings, chairs and even front steps provided a cooler spot to sit and deal with the warmest weather of the year. Once in-home air conditioning went mainstream, shaded front porches became more of curb-appeal feature and less of a functional space for keeping cool.
The next time you adjust the temperature of your home with the simple push of a button, remember that there was once a time that keeping cool wasn’t so easy. Air conditioning is one of the greatest modern conveniences—and one that deserves plenty of credit for keeping people safe and comfortable when the temperature spikes. Enjoy it!
For over 45 years, Bob Jenson has been providing quality heating and air services to the San Diego community.
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I know people have been camping out by their computers, just WAITING to see what I have to say about the FX show "American Horror Story". Well, sorry to keep you waiting, I was behind a bit. The day it debuted, I forgot about it and missed the first episode. So I had to wait for all the episodes to repeat to catch up. Now that I'm caught up, I can finally talk about this show.
Oh and spoilers if you're not caught up or haven't seen any episode but plan on it later. This post will be right here waiting for you when you do catch up.
With that said...
I love the fact the teenager Tate did a Columbine before there was a Columbine. He's like a deadly hipster. Of course there's that whole thing of him not remembering doing that, which I wonder what that's all about. Does he really not remember killing those kids or is he just pretending? And what's up with the whole "he doesn't know he's a ghost thing, so we need your help to send him to the other side" thing?
Did anyone else think the bitchy old lady next door was also a ghost? I did for the longest time. Hell, I'm still convinced she is.
How come the bitchy old lady's daughter isn't running around being a ghost? Can you only be a ghost in this house? Well, that's not true cause those kids Tate shot died at school and they showed up. Maybe she didn't wanna roam around as a ghost is the only thing I got.
My favorite part has to be the maid and how she changes her look, depending on who's looking at her. When she's the hot young chick, she says all these strange things in a sexual innuendo type way, but you gotta keep in mind, she's ALSO this old lady. It's all very strange.
What THE FUCK is up with that gimp dude?
I'm starting to have a problem with how much weird shit is going on this show. I don't know how many quirks they're gonna give Jessica Lange's character but NOW she had a deformed son that was chained up in the attic? What's next, a two-headed dog? I hope it stops there otherwise the show's gonna end up being goofy for no reason.
Overall, it is a great show. Unlike "Lost" when they present something, they actually explain it right away, but leave a few small things unexplained (like gimp dude, or the baby being twins). I hope this show doesn't end up like "The Walking Dead" and slow down or get too full of itself. It's perfect the way it is and I hope it continues the path it's going down now.


Noida: The district administration team led by SDM Dadri, Alok Gupta, has taken strict action against five builders in Noida who have not paid their dues. These builders may face arrest if they do not clear their debts within 24 hours. The administration has warned them that their offices will be sealed, and their property will be confiscated if they fail to comply.
The district administration team has done munadi (the beating of drums) in front of the office of these builders.
The action taken by SDM Dadri has caused a stir among the builders. The district administration has given ample time to the builders to clear all the dues, but they haven’t listened. Now the administration has tightened its noose around these builders.
Also read: UP RERA warned the house buyers in Noida
Under the leadership of Alok Gupta, SDM Dadri, the team of Dadri Tehsil went to the office of Supertech in Noida Sector 96 and did the munadi. After that, when the crowd gathered, they announced that notices had been issued to the builder many times, but he was not paying the dues. So finally, the administration has taken this action against them.
SDM DadriAlok Gupta said that apart from sealing the offices of these defaulters, the property of the builder will also be confiscated, and the builder will also be arrested. The order of Munadi at their offices will now be run continuously by the district administration. So that the buyers coming to his office also has information about the builder and do not get trapped in their clutches.
List of these builders
2 Mahagun India
3 Rudra Palace Heights
4 Future World Green Homes
5 Space Engineer
Abhishek Kumar, the president of NEFOWA, has stated that SDM Dadri made an excellent decision. This will act as an advisory to new buyers so that they are not manipulated by these builders. He told us that SDM should also take decisions like this in favor of the buyers so that they will also get back their money, which got stuck with these buyers.
N.P. Singh, president of DDRWA, has told us that the SDM should take this action earlier. These defaulters’ builders have taken the money from the buyers but haven’t cleared the debt with the administration. He also requested that the SDM look into the problem of the buyers and take some decisions in favor of them. So that buyers will get their money back from these builders.


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Personality essay questions
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domain: psychology