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There is a good amount of original music that can be described as "roots" or "folk rock" in this region. Although the Twin Cities based band, Swallows, do achieve these genres in their music, the delivery and mix of elements in their fifth album "Witching & Divining" sets them uniquely apart.
The album begins with the bluesy song "Long Long Shadow." It could be compared to 4onthefloor except not so hard rocking and a bit more mysterious. It seems that every musician in the band is a multi-instrumentalist and this full array of instruments that include cello, keys/organ, guitars, vocal harmonies and a very thick and diverse arrangement of percussion gives the band a very full and unique sound. As far as how the instruments come together on the album, it is done well and tastefully.
The band also describes itself as tribal folk and this in the next track "The Winnowing." What also shows is an incredible amount of dynamics from one song to another. Although the band can certainly rock out with a feel of 70s psychedelic rock at times or bluesy country at other times, songs like this one are very subtle from ambient noises from the guitar, soft moans of the cello and the very laid back percussion that brings in things like chimes, sandpaper and other unconventional percussive instruments. Perhaps the least subtle thing on this track and a lot of the other songs on the album are band leader Jeff Crandall's rough and gritty Tom Waits like voice. Even the melodies and delivery carry that alternative bluesy Waits feel to them.
One of the more "roots" oriented songs would be the steady paced "Brigid's Cross" which brings in some of the more traditional instruments one would associate with that style of music. Mandolin and harmonica always help for creating that type of feel and this song is no exception. This and several of the other songs on the album features cello player, Toni Tinetti, harmonizing with Crandall as female backing vocals. This backing vocals really makes for an a nice impact for songs like "Brigid Cross," but also creates something smooth and beautiful with laid back and ambient folk songs like "High Water." Other members include Aaron Kerr (bass/cello/vocals), Tyson Allison (guitar/keys/bass/percussion), Mike Nordby (mandolin/percussion/vocals) and Justin DeLeon (drums.)
When comparing songs like "High Water" to more some of the more rock oriented songs like "Seismic," it's interesting to see all of the sides of this band. An album should always be an experience that takes the listener through a journey throughout the album and "Witching & Divining" definitely steps up in this area. The band is not stuck to any one particular sound or genre, yet the songs fit together by creating a certain mood within the album. In the end it doesn't really matter what specific genre Swallows falls under, calling it "alternative" is your best bet. What's happening here is a solid album that roams around in a realm of moody grittiness but shines through here and there with subtle beauty and mystery. Even looking at the album's cover portrays the feel of this album well.
Swallows will be playing at Beaner's Central in West Duluth on Saturday, February 2 along with established songwriter, Kary Vernon from the Twin Cities and the piano oriented songs of Mary Bue from Duluth. Crandall mentioned that this show will be more acoustic but hopes to return again to the Twin Ports with an electric set.


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At the Design and Computation Group, we strive for engaging with specialists in the subjects we take an interest in, allowing us to keep up to date on such subjects, presenting research we have been working on, and maintaining contact with good old friends as well as making new ones.
To access the certificates click here.
O desenvolvimento das teorias da informação ao longo do século XX é marco de início de uma mudança de paradigma da nossa forma de ver o mundo. Isso implica em reformulação dos modelos explicativos. Envolvido por esse pensamento, fazemos um breve levantamento da evolução dos modelos sobre o urbano, que também tem absorvido vários princípios do paradigma da informação. Compreender a cidade e formalizá-la como informação e suas inter-relações tem trazido à tona a percepção de redes e suas complexidades, ajudando a formar uma visão pelo menos um pouco mais completa.
Aluno de doutorado no PROURB/UFRJ e pesquisador no LAMO, com estudos sobre o urbano a partir da teoria da complexidade. Mestrado em Arquitetura, Tecnologia e Cidade pela UNICAMP, sobre Arquitetura Performativa. Desenvolve pesquisa na área de Teoria da informação aplicada para arquitetura, nos temas: materialização digital, algoritmo de arquitetura, teoria geral dos sistemas (TGS), tendo ênfase no estudo de emergência de complexidade. Graduado em Arquitetura e Urbanismo pelo UFC (Universidade Federal do Ceará) em 2011. Graduado no Curso Superior de Tecnologia Em Mecatrônica pelo Centro Federal de Educação Tecnológica do Ceará (2005).
Esta apresentação foca em projetos especulativos que produzem caricaturas de tendências arquitetônicas contemporâneas. Estratégias como sampling e remixing por meio de modelagem computacional são utilizadas para produzir uma diferente relação com a história da arquitetura.
The lecture took place on April 06 at 10h30, in room 5.0.10 (Pav. 5, Faculdade de Arquitetura, Universidade de Lisboa).
Victor Sardenberg é pesquisador associado no dMA – Digital Methods in Architecture na Leibniz Universität Hannover. É Mestre pela Städelschule, em Frankfurt, e bacharel pelo Mackenzie, em São Paulo.
Os exercício de kit-de-partes pertencem a uma tradição didática onde a composição arquitetônica é limitada pelo uso de uma coleção específica de elementos e por regras claras de combinação. Um kit-de-partes pode ser entendido como uma gramática arquitetônica e, portanto, é passível de codificação.
O Supergrid é um algoritmo que explora o potencial computacional implícito na sintaxe do kit-de-partes para compor um grande catálogo na forma de um extenso modelo físico produzido digitalmente visando explicitar e explorar o rico potencial contido na coleção de componentes elementares da arquitetura cotidiana.
The lecture will take place on March 28 at 14h00, in room 5.0.13 (Pav. 5, Faculdade de Arquitetura, Universidade de Lisboa).
After Prof. Engel's presentation, the lecture will continue with presentations of the following DCG PhD candidates, followed by a Q&A session:
14h30 – Rui de Klerk – Semantics in design generation using ontologies to control shape generation
15h00 – Sara Garcia – A Grammar-based Tool for Multipurpose Chair Design
15h30 – Daniel Mateus – Energy-based Design: Digital Design of Building Skins
Pedro Engel é arquiteto, professor do sector de Estudo da Forma na Faculdade de Arquitetura e Urbanismo da Universidade Federal do Rio de Janeiro. Sua atuação concentrada no ensino de fundamentos do projeto pauta seus diversos interesses de pesquisas: métodos didáticos, processos de concepção, meios de representação e sistemas computacionais de geração da forma. Junto ao Laboratório de Modelos da FAU-UFRJ – LAMO – vem desenvolvendo como designer projetos que exploram métodos de fabricação digital e modelagem numa interface entre arte, arquitetura e pesquisa acadêmica.
Watch this lecture (live streaming or stored video) in Youtube
Professors Henri Achten (Czech Technical University in Prague), Pieter Vermaas (Technical University Delft) and José Pinto Duarte (FA/ULisboa + Penn State University) will be visiting us next week.
To celebrate this we are preparing a special edition of DCG lectures for next thursday (March 10th), to show some of the work in progess within the DCG group and talk about ongoing researches.
Henri Achten
Sara Eloy + Pieter Vermaas – "The Quality of Designs by Shape Grammar Systems and Architects: A Comparative Test on Refurbishing Lisbon's Rabo-de-Bacalhau Apartments"
Eduardo Castro e Costa – "Mass Personalization of Ceramic Tableware"
Sara Garcia – "Classifications of Shape Grammars"
Watch the lectures in Youtube: part 1 || part 2 || part 3
This month there will be two new researchers arriving at our group: Sorour Ebadati and Rusne Sileryte.
In order to get to know each other and each other's work we will have a meeting where we present our work.
Thursday, 11th February 2016, starting at 9h30 at the School of Architecture (UL), Post-Graduation Pavilion room 5.0.12.
09.30 – Sorour Ebadati: The role of Iranian-Islamic architecture and urbanism in their Landscape with the aim of identity of contemporary urban space
10.00 – Fernando Lima: Algorithmic approach towards Transit Oriented Development neighborhoods: (para)metric tools for evaluating and proposing rapid transit based districts
10.30 – Coffee break
11.00 – Débora Verniz: Understanding the urban structure of informal settlements: combining techniques of 3D scan and shape grammar
11.30 – Francesco Orsi + Stefano Fiorito: Torino project
12.00 – Discussion on the presented topics
12.30 – Lunch time
13.30 – David Vale: Measuring the built environment for travel behaviour research
14.00 – Rusne Sileryte: Digital Methods for the Built Environment
14.30 – Ljiljana Cavic: Convex and Solid Voids
15.00 – João Ventura: DM4SQUARE – Data mining for public open space classification and urban potential assessment
15.30 – Discussion on the presented topics
Watch this lecture (live streaming or stored video) in Youtube
One of the initial stages of architectural design process is concerned with the generation of planar floor plans while satisfying the given topological and dimensional constraints.
Mathematically, the topological constraints given in terms of adjacency relations among rooms fall in the domain of graph theory. Therefore, in this presentation we are going to discuss some graph theoretical concepts that are relevant to architectural designs.
In addition, we will go through some of the related work and present some interested open problems in this direction.
Watch this lecture (live streaming or stored video) in Youtube
The aim of my work is enhancing the use of rehabilitation processes based on incremental strategies to transform the existing building stock. The focus of the investigation is more specifically on the conversion into housing of the large number of office buildings that have been vacant for a long period and will be unlikely reabsorbed in the market. In this way it will be possible to give simoultaneously a response to both the crescent structural vacancy rate of offices as well as the high demand of new affordable dwellings in the urban region of Milan.
The application of a shape grammar based metholodogy is investigated to enable the exploration of preliminary design solutions and their implications in terms of volumetric scenarios, functional programs and eventually costs estimations and energetic behaviour. As a result, an overall evaluation of the "adaptive capacity" of an existing building type to be reused can be carried out at the very early stages or even before the starting of the design phase.
Politecnico of Milan, Department of Architecture, Building and Construction (ABC) – Phd in Technology and Design for the Built Environment
The grammar was developed at the DCG FA/ULisboa with a grant from the Italian government and is part of my PhD being developed at the Politecnico di Milano.
Tuesday, 22nd of September 2015, starting at 10h00 at the Faculty of Architecture (UL), Post-Graduation Pavilion room 5.0.13.
This session will be dedicated to the use of shape grammars in Mass Customization of Housing, presenting:
10h00 – José Pinto Duarte on "Mass Customization of Housing – new construction"
10h45 – Sara Eloy on "Mass Customization of Housing – rehabiliation of existing types"
11h30 – José Nuno Beirão on "Mass Customization of Housing"
12H15 – Hasnyiati on "Mass Customization in Malaysia – a managerial approach"
Watch this lecture (live streaming or stored video) in Youtube
This work presents an application of Shape Grammar (SG) in automatic design of spatial trusses (ST) manufactured with tubular steel members.
SG is a powerful approach for geometric parameterization based on set of rules and recursive application over an initial shape.
This strategy offers control over a large domain of topologies from a relatively short set of parameters, which may control the main geometrical properties of the system. These characteristics (large domain, few parameters and efficient constraint) have a very important influence over the computational costs and results in usual optimization algorithms, as for example the topological optimization by Genetic Algorithms. In this context, Shape Grammar proved to be an efficient approach for improvement of structural steel products [1].
The main contributions of the present work are developments and implementation of parametric rules for a specific ST-Grammar.
A graphic user interface is provided taking advantage of OpenGL. All algorithms are implemented in a mixed Fortran/C++ language. Finally, illustrative examples of spatial trussed roof systems are shown.
Juarez Franco is Phd in Civil Engineering and Master in Architecture. His research is addressed to automatic design and optimization of framed steel structures, with interest in shape grammar formalism, genetic algorithms and buckling of thin-walled structures.
Watch this lecture (live streaming or stored video) in Youtube
This presentation will share the Gonçalo Castro Henriques' recent experience at the federal university of Rio de Janeiro, presenting «LAMO – Laboratório de Modelos 3d e fabricação digital» (laboratory of 3d models and digital fabrication) and a brief resume of the lab history, approach and future goals in Latin America context.
LAMO is a young lab, developing a collaboration network with other laboratories and research centers. the presentation will highlight the contribution of 3 projects: «wiki-house/casa revista (2015)» and the international workshops «sensitive shelters/ abrigos sensíveis (2014)», and «defying gravity/desafiando a gravidade (2015)».
Watch this lecture (live streaming or stored video) in Youtube
28th of May 2015, 10h00 at the Faculty of Architecture (UL) – Doct. Pavilion classroom 5.0.
In this presentation Manuela Ruiz Montiel will talk about some techniques and experiences related to the use of computers in order to suggest starting points in the context of housing design.
These techniques are mainly based in shape grammars and machine learning methods.
So far, we have focused on habitability guidelines to generate the so-called housing
unit schemes. Currently we are also considering energy efficiency directives over the machine learning process.
Manuela Ruiz-Montiel is a PhD student with a particular interest in how computers can be used to trigger and enhance the creative process in fields where shape design plays an important role, like architecture or industrial design.
She holds a master's degree in Computer Science from the University of Málaga, Spain. Now she combines her research activity with a job as a tutor teacher in the Open University of Spain (UNED).
Watch this lecture (live streaming or stored video) in Youtube
Due to the raise in human population over the last few years, many countries over the world are facing a problem of housing shortage. In order to build so many houses in a short period of time, we need advanced & logical methods and technologies to develop new housing designs.
Krishnendra's work is concerned with the automated generation of floor plan layouts which might speed the production of design documents, reduce errors and omissions, and provide inexpensive and vivid visualizations.
Using some mathematical techniques, in this work I present a systematic pathway for the floor plan design when given the shape of required floor plan, the list of spaces, the dimensions of each space and the weighted matrix of required adjacencies between the spaces. In addition, a lot of covariants (associated with the architectural designs) will be presented, that will help in improving the quality of the solutions.
Rudi Stouffs is a researcher and educator in computational design, with interests in computational issues of description, modeling and representation for design in the areas of information exchange, collaboration, shape recognition and generation, geometric modeling, visualization and e-learning. Has led and participated in research projects in the Netherlands, Switzerland and the US, supported by scientific funding organizations.
Specialties:Digital design & manufacturing, spatial grammars
My name is Nina Verkerk, and study Architecture at TU Delft (the Netherlands) and studied a year of Applied Mathematics (also TU Delft). Since secondary school, I have an interest in both, creative design and the systematics behind mathematics. In the master graduation studio Explorelab (TU Delft) (http://www.explorelab.nl/) it is possible to develop your fascination or passion. I choose to work on shape grammar as a combination of my both interest. First I had to research my topic, and now I need to design a building using that research.
Because there are no teachers at TU Delft who have a lot of knowledge or experience with shape grammar, figuring out the system behind shape grammar was part of my research. Because most traditional papers are very specific for one building style, I decided to try to develop my knowledge on more general level: My study gives a general understanding of the formal terms, a generic approach of rule systems, and a possible application in the architectural design process. The goal of this project is to make the theorem of shape grammar easier to understand and more accessible in the design process.
(for more information click here)
But for my graduation I also need to design a building (I choose to design a secondary school), with the knowledge I found during my research. And that is where I'm working at now. I hope that you (and your colleagues) can help me to develop my design with insights I did not find myself. I'm also very interested in the research you (and your colleagues) are doing, and really would like to learn about it.
As redes apresentam‐se como um elemento universal no desenvolvimento e entendimento da cidade, em grande parte invisíveis mas determinantes para a complexidade dos sistemas urbanos.
Por um lado, têm uma influencia formativa enquanto infra‐estruturas condicionantes e/ou
geradoras. Por outro lado, manifestam‐se em estruturas relacionais físicas, económicas, sociais e culturais. Ligações invisíveis entre as diferentes entidades e diferentes atores formando a essência coletiva do ser urbano. Enquanto teoria oferecem um instrumento intelectual, mas também operacional, que permite representar e analisar de forma sistemática a complexidade da cidade e da região.
Será apresentado um modelo demonstrativo de como estes elementos se conjugam para
oferecer um entendimento mais abrangente do fenómeno urbano. Um instrumento que opera a varias escalas do território urbano ‐ do local ao regional – interligando diferentes níveis de mobilidade e atividade humanas, oferecendo leituras múltiplas e detalhadas da realidade.

subset: commoncrawl


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One Hundred Islands – Amazing Place Of Natural Splendour
One Hundred Islands is located in Alaminos City, Pangasinan province, which is located north west of Manila with Lingayen Gulf on the East, and the South China Sea to the west. This slice of paradise is one of the most popular tourist attractions in the Philippines and is a protected National Park. The islands and islets are in Lingayen Gulf and total 124 at low tide, and 123 at high tide. However, only three of the islands have been developed to cater to tourism.
The islands are said to date back two million years ago. It has been suggested they were once corals submerged in the sea. Over time, the sea level has lowered. With natural erosion from the sea, it has left mushroom looking mounds protruding from the water.
Island hopping heaven
These islands are a popular place for tourists to go island hopping and enjoy these fascinating islands. Whilst most islands cant be visited, there are several islands that do have small sandy beaches. This is big enough to dock a small boat so you can chill out with a picnic and a few beers and go swimming in the tranquil waters. One Hundred Islands doesn't quite have as much long open beach like it's neighbour, Bolinao. However, it is island hopping heaven which Boliano doesn't have.
When we stayed there, we were fortunate enough to stay at a friends holiday house. Therefore, I'm not so familiar with hotels in the area. I do know there are a few low-cost hotels and local style resorts around. A quick internet search will uncover some option. You also have the option to camp overnight for a cost of about P200 for those that enjoy the great outdoors, and you can also hire tents for about P500.


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This picture was taken moments after the Big Reveal. I think I can count it a success! They love their new look and the easy access it affords to their books.
The rain gutters cost $5 a piece and this project took two. You have to watch the cost of brackets, joints, end caps, etc. because, as my Grandpa used to say, "That's how they get 'cha." We used as few of these items as possible and opted to just drill through the plastic in several places and miter the corners ourselves.
Just in case you're curious as to what the other side of the room looks like, here are a few more shots.
I know. Don't hate me. We have exposed brick in nearly every room in our house. I love it too!
Thanks for looking. If you like what you see, consider officially following me! Also, please leave a comment. I love them!


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Research ArticleImmunology Free access | 10.1172/JCI38332
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: firstname.lastname@example.com.
Find articles by Hou, Y. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: email@example.com.
Find articles by DeVoss, J. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: firstname.lastname@example.com.
Find articles by Dao, V. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: email@example.com.
Find articles by Kwek, S. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: firstname.lastname@example.com.
Find articles by Simko, J. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: firstname.lastname@example.com.
Find articles by McNeel, D. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: firstname.lastname@example.com.
Find articles by Anderson, M. in: JCI | PubMed | Google Scholar
1Division of Hematology/Oncology, Department of Medicine, 2Diabetes Center, and 3Department of Pathology, UCSF, San Francisco, California, USA. 4University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wisconsin, USA.
Address correspondence to: Lawrence Fong, Division of Hematology/Oncology, University of California, San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, California 94143, USA. Phone: (415) 514-3424; Fax: (415) 476-0459; E-mail: email@example.com.
Find articles by Fong, L. in: JCI | PubMed | Google Scholar
Published June 1, 2009 - More info
Chronic prostatitis is a common disease of unclear etiology and has no specific treatment. Mice deficient in the expression of the autoimmune regulator (Aire) gene, which are defective in thymic expression of self antigens and central tolerance, develop spontaneous prostatitis. In this study, we found that Aire-deficient mice developed spontaneous B and T cell immune responses to a prostate autoantigen, seminal vesicle secretory protein 2 (SVS2), which we believe to be novel. We show that thymic expression of this self antigen was Aire dependent. Moreover, prostatitis was induced in WT mice through immunization with SVS2, demonstrating that immunity to SVS2 was sufficient to induce prostatitis. The clinical relevance of this antigen was highlighted by our observation that patients with chronic prostatitis possessed specific autoantibodies against the human SVS2-like seminal vesicle protein semenogelin. These results provide direct evidence that spontaneous chronic prostatitis is an autoimmune disease and is regulated by both central and peripheral tolerance. Moreover, SVS2 and semenogelin are among the relevant autoantigens in mice and humans, respectively.
Prostatitis is a highly prevalent disease in men (1, 2). While acute prostatitis is commonly caused by bacterial infection, most patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) have no evidence of urinary tract infection. Moreover, a substantial proportion of men can have noninfectious CP in the form of asymptomatic inflammatory prostatitis that is diagnosed on prostate biopsy performed to evaluate for prostate cancer. Prostatitis is in fact the most common nonmalignant diagnosis in patients being evaluated for elevated serum levels of prostate-specific antigen (PSA), a biomarker used to screen for prostate cancer (3). The diagnosis of this syndrome relies solely on reported pain in the perineum, rectum, and/or prostate by affected men. CPPS often relapses and remits without clear triggers. Therapy for CPPS is nonspecific and usually involves empiric treatment with antibiotics whose efficacy is not clear. The etiology of CPPS is unknown. Proposed mechanisms of disease include urine reflux–inducing chemical and physical trauma, dietary factors, or alterations in the hormonal milieu, such as high levels of estrogens (4). However, accumulating evidence suggests that CPPS could be an autoimmune disease of the prostate (5). In some patients with noninfectious CPPS, elevated levels of inflammatory cytokines in seminal plasma or expressed prostate secretion have been detected (4, 6). Antigen-specific antibody and cellular responses to prostate antigens such as PSA, prostatic acid phosphatase, MAD-PRO-34, and seminal plasma have also been reported (7–9). To further elucidate the pathogenesis of human CPPS, a few rodent models of experimental autoimmune prostatitis have been developed and characterized (10). Immunization of rats or mice with prostate extracts or proteins expressed in the prostate, such as prostatic acid phosphatase, induces specific immune responses to prostate antigens and, in some models, induces prostatitis characterized by lymphocyte infiltration (11–13). Prostatitis can also be induced in mice with thymectomy on day 3 of life (14). In this model, regulatory T cells induced in vivo by the presence of specific prostate-derived self antigens are thought to inhibit prostatitis in the thymectomized mice, which supports the notion that prostatitis can be autoimmune in nature (15). While these models share some histologic features with CPPS in humans, induced prostatitis may not accurately reflect spontaneous disease. Spontaneous prostatitis has been described in several aged rat strains and even in aged NOD mice, but the mechanism by which these strains render the host susceptible to prostatitis is unclear (16, 17). To our knowledge, none of the prostate antigens identified in these mouse models have been shown to date to be relevant for the human disease.
Autoimmunity results from the loss of immunologic tolerance, which is usually maintained through central and peripheral mechanisms. The autoimmune regulator (Aire) gene is expressed primarily in lymphoid organs and plays a critical role in the development of central tolerance (18, 19). Within the thymus, Aire is expressed primarily by medullary epithelial cells. There, Aire regulates the ectopic transcription of peripheral tissue antigens and thereby controls central tolerance to self antigens by inducing the negative selection of autoreactive T cells. Aire-deficient KO mice have a functional immune system with diverse T and B cell repertoires, but spontaneously develop multiorgan autoimmunity, as seen by inflammatory infiltrates within different tissues and serum autoantibodies (20). Interestingly, Aire-KO male mice develop spontaneous CP. While autoantigens in this model have been identified (15, 20), Aire-dependent autoantigens that mediate prostatitis have yet to be identified.
In the present study, we determined the role of central tolerance in the development of spontaneous prostatitis. We identified a prostate-specific protein, seminal vesicle secretory protein 2 (SVS2), against which reactive CD4+ T cells and antibodies developed spontaneously in Aire-KO mice. We demonstrate, for the first time to our knowledge, that thymic expression of this autoantigen was Aire dependent. We also show that immunization with SVS2 broke tolerance to this antigen and induced autoimmune prostatitis in Aire-sufficient WT mice, demonstrating that central tolerance to this antigen was incomplete in WT mice. Furthermore, we show that patients with CPPS possessed immune responses to semenogelin (Sg), the human counterpart to SVS2. These results may provide new approaches to the diagnosis and treatment of CPPS.
Aire-KO mice develop spontaneous lymphocytic infiltration in the prostate. Aire-KO mice spontaneously develop multiorgan autoimmunity, including that of the eye, salivary glands, ovaries, and stomach (20). Inflammation in the prostate was also observed in most Aire-KO mice (21), but to our knowledge, the nature of immune recognition for autoimmune prostatitis in Aire-KO mice has not previously been reported. We first examined the histological changes in the prostates of Aire-KO mice. In Aire-KO mice, the inflammation observed included cellular extravasation into the interstitial tissue between the glands as well as infiltration into glandular epithelium of both the dorsal and ventral prostate glands, leading to effacement of the usual glandular architecture in severe cases (Figure 1A). Moderate to severe prostate inflammation (scored as shown in Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI38332DS1), including intraepithelial infiltration of prostate glands by lymphocytes, was observed in 5 of 7 male Aire-KO C57BL/6 (B6) mice at 10–20 weeks of age, but was completely absent in all WT littermate controls (Figure 1B). Immunohistochemistry performed on Aire-KO prostate glands demonstrated predominance of CD4+ T cells among the infiltrating cells in the prostates (Figure 1C). CD8+ T cells were also observed, but were fewer in number than CD4+ T cells. Nevertheless, both CD4+ and CD8+ T cells infiltrated the glandular epithelia of these prostates. CD19+ B cells were seen at low frequency; when present, they resided within the stroma of the prostate tissue. In contrast, WT mice lacked lymphocytes within either the glandular cell layer or the stroma (Figure 1C).
Spontaneous development of autoimmune prostatitis in the Aire-KO mouse. (A) H&E staining of representative prostate sections from Aire-KO and WT mice. The urethra (u) and ductus deferens (dd), 2 ducts that course through the prostate glands, are denoted. Scale bars: 300 μm. (B) Mononuclear infiltration in H&E-stained prostate sections from 7 Aire-KO and 5 WT littermate mice on B6 background (18–20 weeks old), graded blindly by a pathologist using a scoring system adapted from grading used in evaluating human prostate tissues (see Supplemental Table 1). Data points represent individual mice, and significance of differences was assessed by 2-tailed Student's t test. (C) Immunohistochemical staining for CD4, CD8a, CD19, and anti-rat IgG in cryostatic sections of prostates from 20-week-old Aire-KO and WT mice. Staining is from 1 representative each of 6 Aire-KO and 5 WT mice. Scale bars: 150 μm.
Aire-KO mice develop spontaneous immune responses to SVS2. In order to define the relevant prostate autoantigens that mediate this prostatitis, we screened sera of Aire-KO mice for autoantibodies to proteins present in the protein lysates derived from mouse prostate tissue. Sera of Aire-KO and WT B6/NOD F2 mice (generated as described in Methods) were used in Western blots to determine whether autoantibodies were present. Aire-KO B6/NOD F2 mouse sera were used to facilitate the identification of relevant autoantigens because these mice develop a more aggressive autoimmune phenotype (21). Autoantibodies against prostate-derived proteins were detected in most Aire-KO sera (Figure 2A). Some sera showed broad reactivity, whereas others showed more limited and/or weaker reactivity, consistent with heterogeneity in the immune response. Nevertheless, common bands were also evident across multiple mice, which indicates that there were immune responses to proteins shared by the majority of the Aire-KO mice. These common bands within the prostate lysate were not evident in immunoblots to lysates from other tissues — such as the eye, salivary glands, or lacrimal glands — in which Aire-KO mice also develop spontaneous inflammation (Supplemental Figure 1A). In an effort to purify and identify the targeted antigens, sera from Aire-KO mice with strong autoreactivity to the lysates and from WT mice were used to make immunoprecipitation columns. These columns were used to enrich for antibody-binding proteins from pooled prostate tissue extracts. Enriched proteins from both Aire-KO sera– and WT sera–coupled columns were concentrated and subjected to gel electrophoresis. Multiple distinct bands visualized by silver staining that were unique to the Aire-KO sera column were excised and analyzed by mass spectroscopy (Supplemental Figure 1B). We successfully identified SVS2 (GenBank accession no. 78070545), with 29% sequence coverage of the protein, through peptide mass fingerprinting (PMF) from both the 26- and 38-kDa bands (Figure 2B). SVS2 is an androgen-regulated protein expressed by the prostate and secreted into seminal fluid, where it serves as a major clotting protein involved in the formation of the copulatory plug (22).
Aire-KO mice possess spontaneous autoantibodies to SVS2. (A) Immunoblotting of whole prostate extracts was performed with the sera from male Aire-KO and WT mice on the B6/NOD F2 background. Each lane of the multiscreen immunoblot represents the reactivity of sera from an individual mouse, denoted by number. Gel bands stained by sera from multiple Aire-KO mice were excised and analyzed by mass spectroscopy. (B) Multiple microsequenced peptides were derived from SVS2. The peptide sequences identified through PMF are denoted by underline and were predominantly within the N terminus of the protein. (C) Immunoblots of recombinant MBP-SVS2 and purified SVS2 from semen plasma with sera from Aire-KO and WT B6/NOD F2 mice. MBP and OVA were negative controls. (D) Anti-SVS2–positive sera from Aire-KO mice were preincubated with either MBP-SVS2 or MBP before being used in the immunoblots against the prostate extract. WT and heterozygous Aire+/– B6/NOD F2 sera were used as controls.
To confirm that SVS2 was the protein recognized by autoantibodies in the Aire-KO mice, we generated purified SVS2 through 2 approaches. Mouse-derived SVS2 protein was purified from the seminal vesicle fluid according to a protocol previously used to purify human Sg (23), a protein that has structural similarities to mouse SVS2 protein. This purification procedure yielded the expected 38-kDa protein. Alternatively, recombinant SVS2 was produced as an 80-kDa fusion protein with maltose-binding protein (MBP) expressed in E. coli. The recombinant MBP-SVS2 fusion protein was purified from bacteria lysate with an amylose column. Recombinant MBP was also expressed and purified from E. coli and used as a control in subsequent experiments.
Sera from Aire-KO mice contained autoantibodies that recognized both the purified, semen-derived protein and the recombinant protein by Western blot (Figure 2C). Aire-KO sera did not bind to the control MBP or to another irrelevant protein, OVA. The absence of SVS2 reactivity in the WT sera further supports the importance of Aire regulation in autoreactivity to this antigen. To confirm that the prostate extract–reactive autoantibodies were specific to SVS2, we preincubated the Aire-KO sera with varying concentrations of MBP-SVS2 before immunoblotting the prostate extracts. Increasing concentrations of MBP-SVS2 abrogated immunoreactivity to the bands, while preincubation with MBP did not alter this immunoreactivity (Figure 2D). We therefore conclude that SVS2 is a prostate autoantigen targeted by autoantibodies in Aire-KO mice. Moreover, these results also indicate that some of the recognized lower-MW proteins represent fragments of SVS2.
SVS2 expression in the thymus is Aire dependent. To determine the distribution of SVS2 expression, RNA was isolated from different tissues and assessed by real-time PCR to quantify the expression of SVS2 mRNA. Consistent with prior reports (24), in Aire-KO mice, SVS2 was specifically expressed in the prostate gland, but was essentially undetectable in other organs tested, including tissues in which spontaneous inflammation can develop (Figure 3A). Next, we sought to determine whether SVS2 is expressed in the thymus and whether its thymic expression is Aire dependent. We purified CD45– thymic stromal cells from WT and Aire-KO mice and analyzed the expression of SVS2 by quantitative real-time PCR. SVS2 was expressed at substantially higher levels in the WT thymus than in the Aire-KO thymus (Figure 3B). This result demonstrated that thymic expression of the SVS2 gene was indeed Aire dependent.
SVS2 is highly expressed in the prostate and is an Aire-dependent antigen expressed in the thymus. (A) Expression levels of SVS2 were quantitated by RT-PCR on cDNA prepared from diverse tissues. Values are normalized to cyclophilin expression for each tissue and expressed in arbitrary units; exact values are shown above bars. Error bars denote SD. ND, not detectable. (B) Quantitative real-time PCR assay for the SVS2 expression in the CD45– thymic stroma cells of Aire-KO and WT mice. Values (mean ± SD) are normalized to cyclophilin and are representative of 2 experiments performed.
Autoimmune prostatitis is mediated by CD4+ T cells. Adoptive transfer experiments were performed to determine the immune effectors that could mediate prostatitis. Splenocytes from Aire-KO B6 mice were pooled; depleted of CD4+ T cells, CD8+ T cells, or CD19+ B cells; and adoptively transferred into immunodeficient RAG-KO B6 mice. Mice that received CD8+ T cell–depleted or CD19+ B cell–depleted splenocytes developed prostate inflammation with pathological characteristics similar to those of Aire-KO mice (Figure 4A). Adoptive transfer of CD4+ T cell–depleted splenocytes, however, failed to induce this level of prostatitis (Figure 4A). These results indicate that autoimmune T cells mediate the spontaneous prostatitis seen in Aire-KO mice, in which CD4+ T cells play a dominant role.
Spontaneous T cell responses to SVS2 in Aire-KO mice. (A) Splenocytes derived from Aire-KO B6 mice were depleted of CD4+ or CD8+ T cells or of CD19+ B cells by FACS. Sorted cells (5 × 106 cells) were adoptively transferred into male RAG-KO B6 mice. H&E staining of representative frozen prostate sections demonstrate that cells depleted of CD8+ T cells and CD19+ B cells, but not CD4+ T cells, were capable of transferring disease into RAG-KO mice. Results are representative of 2 separate adoptive transfer experiments, each with 2 mice per group. Scale bars: 150 μm. (B) Splenocytes (200,000 per well) from Aire-KO and WT mice were assessed for T cell responses against MBP-SVS2 (10 μg/ml) or MBP (10 μg/ml) by IFN-γ ELISPOT. Values are mean ± SD. SFU, spot-forming unit. (C) Results from individual Aire-KO and WT mice (n = 7 per group) of the experiment described in B. (D) IFN-γ ELISPOT assays were also performed with purified semen-derived SVS2 (10 μg/ml) and OVA (10 μg/ml) (n = 5 per group). In C and D, data points represent individual mice, horizontal bars denote means, and significance of differences was assessed by 2-tailed Student's t test.
Based on these results, we next sought to determine whether Aire-KO mice possess endogenous SVS2-reactive T cells. We assessed splenocytes from Aire-KO and WT mice for antigen-specific responses by IFN-γ ELISPOT. This approach allowed us to detect specific responses to MBP-SVS2. While there was heterogeneity in the immune responses to MBP-SVS2 in Aire-KO mice, these responses were significantly different from the lack of response observed in WT mice (Figure 4, B and C). Because Aire-KO mice also had higher background reactivity to MBP than did WT mice, we assessed additional mice for immune responses to nonrecombinant, purified proteins. Again, we detected spontaneous T cell responses to semen-derived SVS2 only in the Aire-KO mice (Figure 4D).
To confirm that SVS2-reactive T cells in fact mediate prostatitis, we generated an SVS2-reactive CD4+ T cell line by stimulating splenocytes from male Aire-KO mice in vitro with purified SVS2. Upon restimulation with SVS2-MBP, the SVS2-reactive T cell line produced IFN-γ, but not IL-17 or IL-10 (Figure 5A). An OVA-reactive T cell line was generated in parallel as a control (Figure 5B). Adoptive transfer of the SVS2-reactive T cell line into RAG-KO B6 mice induced prostatitis (pathology score of 4; see Supplemental Table 1) in 3 of 3 treated mice, whereas adoptive transfer of the OVA-specific T cell line resulted in no inflammation (Figure 5C).
Functional capacity of SVS2 T cell lines. (A) SVS2-specific T cell lines were generated from Aire-KO male mice with semen-derived SVS2. After 2 rounds of in vitro restimulation with SVS2, this T cell line was restimulated in vitro with MBP-SVS2 or MBP, stained for CD4, and stained intracellularly for IFN-γ. Gated CD4+ T cells were also assessed for IL-10 and IL-17. (B) An OVA-specific T cell line was induced from male Aire-KO mice in parallel using the same approach as in A. Following 2 rounds of in vitro restimulation with OVA, this line was stained for CD4 and stained intracellularly for IFN-γ after restimulation with OVA or MBP. In A and B, numbers represent the percentage of cells within the respective gates. (C) Cells of the SVS2-specific T cell line or OVA-specific T cell line (1 × 106 cells) were adoptively transferred into male RAG-KO B6 mice. Prostates were assessed by immunohistochemistry for anti-CD3 staining. Results are representative of 3 mice per group. Scale bars: 150 μm.
Tolerance to SVS2 is broken in WT B6 mice. The results from the Aire-KO mice indicated that central tolerance plays a major role in preventing autoimmune prostatitis. In order to determine whether central tolerance to SVS2 is complete, we examined whether SVS2 is immunogenic in WT B6 mice by immunizing them with MBP-SVS2. In doing so, we could also determine whether inducing an immune response to SVS2 is sufficient for triggering prostatitis. WT mice were challenged with either MBP-SVS2 or MBP, emulsified in CFA. After 2 booster immunizations with the corresponding protein in incomplete Freund adjuvant (IFA), sera, splenocytes, and tissues were harvested for analysis. To minimize cross-reactivity to potential contaminants in the recombinant antigens, semen-derived SVS2 was used to perform the in vitro assays. Autoantibodies against the semen-derived SVS2 were detected in the sera of 7 of 14 mice (Figure 6A). None of the 14 MBP-immunized mice displayed autoantibody activity against SVS2 (Figure 6B). The pooled splenocytes from immunized mice were also assessed for T cell responses by IFN-γ ELISPOT. The splenocytes from MBP-SVS2–immunized mice demonstrated specific reactivity to the semen-derived SVS2, while the splenocytes of MBP-immunized mice had no specific immune response against SVS2 (Figure 6C). Depletion of CD4+ T cells from the MBP-SVS2–immunized splenocytes by fluorescence-activated cell sorting (FACS) eliminated the SVS2-specific response, while depletion of CD8+ T cells had essentially no effect. These results demonstrate that tolerance to this self antigen was broken and that both antibody and CD4+ T cell responses were induced in Aire-sufficient mice.
Induction of SVS2-specific antibody and T cell response by immunizing WT B6 mice with MBP-SVS2. (A and B) WT B6 mice were immunized with either (A) MBP-SVS2 or (B) MBP emulsified in CFA. Sera from individual immunized B6 mice were used to immunoblot semen-purified SVS2 or OVA. Each blot represents an individual animal. For each experiment, shown is 1 group of 4 mice, representative of 14 total mice immunized. (C) The pooled splenocytes from 4 mice that were immunized with MBP-SVS2 or MBP were assessed by IFN-γ ELISPOT for reactivity to purified SVS2 (10 μg/ml), OVA (10 μg/ml), or PBS. Splenocytes depleted of CD8+ or CD4+ T cells were also assessed. Values are mean ± SD. Results are representative of 2 experiments.
SVS2 immunization induces prostatitis in WT B6 mice. Next, we examined the prostates of immunized mice for the induction of prostatitis. Although the severity of inflammation was less than that seen in Aire-KO mice, 10 of 14 MBP-SVS2–immunized mice nevertheless had intraprostatic CD3+ T cell infiltration (Figure 7, A and B). These results demonstrate that tolerance to SVS2 was broken in WT mice, with pathologic consequences.
Induction of prostatitis in WT B6 mice with SVS2 immunization. (A) Male B6 mice were immunized s.c. with MBP-SVS2 or MBP emulsified in CFA. After 2 booster immunizations with the respective proteins in IFA, the prostates were analyzed by immunohistochemical staining for CD3+ cells in frozen prostate sections. Scale bars: 150 μm. (B) Lymphocyte infiltration in the prostate of each immunized mouse in A was scored blindly by a pathologist. (C) Pooled splenocytes from the mice immunized with MBP-SVS2 or MBP were adoptively transferred (AT) i.v. into RAG-KO mice. After 4 weeks, the prostates were analyzed by immunohistochemical staining for CD3+ cells. Scale bars: 150 μm. (D) Lymphocyte infiltration in the prostate of each mouse in C was scored by a pathologist. In B and D, data points represent individual mice, and significance of differences was determined by 2-tailed Student's t test.
To confirm that the induced prostatitis is T cell mediated, we performed adoptive transfer experiments. Pooled splenocytes from MBP-SVS2–immunized mice were also adoptively transferred into RAG-KO B6 mice. MBP-immunized splenocytes were adoptively transferred into RAG-KO B6 mice as controls. At 4 weeks after transfer, the prostates of recipient mice were assessed for prostatitis. The intraprostatic CD3+ T cell infiltrates into the glandular epithelium were observed in all mice that received SVS2-MBP–immunized splenocytes (Figure 7C). None of the mice that received MBP-immunized splenocytes displayed any prostatitis (Figure 7, C and D). These results further support the role of the T cell response induced by SVS2 in mediating disease.
CPPS patients develop an autoimmune response to human Sg. While no clear human homolog for SVS2 exists, SVS2 is a member of a family of prostate-specific proteins collectively known as rapidly evolving substrates for transglutaminase (24). Similar prostate-specific proteins also exist in humans. By the same method used to purify SVS2 from mouse semen, we purified one such protein, Sg, from human semen plasma and examined whether CPPS patients possess antibodies to this autoantigen. As expected, the Sg preparations contained both isoforms: the 52-kDa Sg1 and the 71-kDa Sg2. Sera from 26 of 39 patients with the clinical diagnosis of CPPS had autoantibodies to these proteins, in contrast to 8 of 39 sera from age-matched normal males (Fisher exact test, 2-sided P < 0.0001; Figure 8A). As part of their clinical management, 16 of the 39 assessed CPPS patients had undergone prostate biopsies; of those, 9 were found to have prostatitis. Interestingly, all patients with biopsy-proven prostatitis had autoantibodies to Sg (Table 1). The specificity of these autoantibodies was confirmed against recombinant human MBP-Sg1 and MBP-Sg2 fusion proteins (Figure 8B).
CPPS patients possess autoimmune response to human Sg. (A) Immunoblotting of purified Sg was performed with the sera from CPPS patients and age-matched male blood donors. Each lane of the multiscreen immunoblot represents sera from an individual sample. Shown are 15 representative sera from each group. The 52- and 71-kDa bands denote Sg1 and Sg2, respectively. Rabbit anti-human Sg (anti-hSg) and human nonspecific IgG (hIgG) were used as positive and negative controls, respectively. (B) Immunoblots of recombinant MBP-Sg1, MBP-Sg1, and Sg purified from semen plasma were used to detect IgG antibodies within sera from a patient with CPPS and an age-matched normal male donor. Polyclonal rabbit anti-human Sg Ab was used as positive control. MBP and BSA denote irrelevant protein controls.
Presence of prostatitis on biopsy
Through the identification of an Aire-dependent autoantigen, we demonstrated that loss of thymic expression of SVS2 led to the development of spontaneous peripheral SVS2-specific T cells. These T cells produced IFN-γ, not IL-10 or IL-17, in response to antigen, consistent with a Th1-dominant immune response. Moreover, adoptive transfer of SVS2-reactive T cells was sufficient to induce autoimmune prostatitis. These results demonstrated that central tolerance may play an important role in preventing spontaneous autoimmune prostatitis and were consistent with other studies showing that Aire maintains self tolerance by regulating peripheral-tissue antigen expression in the thymus (25, 26). In our model, we found that CD4+ T cells were the primary mediators of this prostatitis, which was also consistent with findings in other inflamed tissues in Aire-KO mice (25).
We identified SVS2 as a target of spontaneous autoantibodies Aire-KO. Other autoantigens have been implicated in rodent prostatitis, such as prostate steroid binding protein (PSBP) in immunized rats and NOD mice (12, 17, 27) and EAPA-1 and EAPA-2 identified in day-3 thymectomized mice (15). Nevertheless, these models do not clearly demonstrate that prostatitis is an aberrant autoimmune response. Our results represent the first identification to our knowledge of an Aire-dependent prostate autoantigen.
Despite the high prevalence of spontaneous prostatitis and SVS2-specific antibody responses in Aire-KO males, only a subset of mice had detectable SVS2-specific T cell responses. This heterogeneity could reflect limitations in the sensitivity of detecting SVS2-specific T cells, particularly since we assessed immune responses in the spleen, as we were unable to dissect lymph nodes draining the prostate. Peripheral tolerance could also play a role in suppressing immune responses to this antigen, potentially through an Aire-dependent (28) or -independent mechanism, such as by autoantigen-specific Tregs (29). Nevertheless, the fact that Aire-KO mice developed spontaneous prostatitis with such high prevalence indicated that Aire-independent mechanisms of peripheral tolerance are insufficient to protect against autoimmunity. Again, other antigens could play a role in inducing prostatitis, while immune responses to SVS2 could involve a bystander antigen recognized through epitope spreading. Nevertheless, by demonstrating induction of prostatitis in Aire-sufficient mice through immunization with SVS2, we showed that SVS2 can in fact be an autoantigen involved in the initiation of autoimmune prostatitis. As a result, our findings also indicate that central tolerance to SVS2 is incomplete in these WT mice and support a role of peripheral tolerance in suppressing spontaneous autoimmune prostatitis, not unlike what is seen with other previously described autoantigens such as myelin basic protein and proteolipid protein (17). While high-avidity autoreactive T cells would presumably be deleted from the repertoire in these WT mice, the T cells that do escape the thymus would presumably possess low-avidity TCR for this antigen. The lower histologic scores for the inflammation in immunized WT mouse prostates compared with the spontaneous prostatitis in Aire-KO mice are therefore not surprising. Nevertheless, the inflammation we observed in immunized WT mice was localized to the glandular epithelium, where SVS2 is expressed; in fact, this inflammation more closely resembled the pathologic prostatitis seen in humans.
Prostatitis has not been described as a typical clinical manifestation in men with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is caused by mutations in Aire (30, 31). Nevertheless, CP is frequently asymptomatic, so this clinical manifestation may not yet be appreciated. Moreover, there can also be considerable heterogeneity in the clinical presentation and immune responses in these individuals, as has been seen with autoantibody reactivity against an identified autoantigen (32).
The diagnosis of human CPPS relies upon patient symptoms, and not on any objective measure of disease. When these patients undergo prostate biopsies, marked prostate inflammation is found in anywhere from 33% to 88% of patients (33, 34). While this variability may result from the different biopsy techniques (35), a substantial proportion of patients with symptoms nevertheless have negative biopsies. The identification and characterization of the autoantigen involved in the disease could resolve some of the controversy concerning the autoimmune mechanism underlying CPPS. While antibody responses to several other prostate antigens have been previously described (7–9), these antibodies were detected infrequently. To our knowledge, human homologs for the other identified autoantigens in rodent models of prostatitis (e.g., prostatic steroid-binding protein and spermine-binding protein) have not been identified. Although the direct human homolog of rodent SVS2 has not been identified, we focused on SVS2-like seminal vesicle proteins, such as Sg, that have previously been characterized in human seminal plasma and have the same pattern of tissue expression (36–38). In spite of the amino acid sequence disparity between SVS2 and Sg, these proteins share similarities in gene structure, 3-dimensional structure, and/or functional domains; they are collectively referred to as the rapidly evolving substrates for transglutaminase; and Sg was purified with the same approach used to purify SVS2 (24).
Interestingly, we found that two-thirds of sera from patients with CPPS possessed autoantibody against human Sg. However, when we narrowed our assessment to patients that had undergone prostate biopsy, all of the patients with biopsy-proved prostatitis possessed autoantibodies to Sg. Antibodies to this antigen were detected in patients without prostatitis, but their presence could stem from prior inflammation in the prostate that was no longer reflected in the biopsies. In addition, 20% of our normal male blood donors possessed anti-Sg autoantibodies. These individuals were assessed for prostatitis symptoms as part of routine blood donor screening and not based on a prostate symptom–directed history. Inflammation within the prostate can be present in asymptomatic men. Nevertheless, our data demonstrated that autoimmune recognition of Sg could be a feature of CPPS. Detecting immune responses to this autoantigen may be useful in the diagnosis of CPPS, but this will need to be formally tested prospectively. More importantly, our demonstration of this mechanism underlying CPPS in humans, which we believe to be novel, may lead to therapeutic approaches for this enigmatic disease, specifically, targeting Sg as an autoantigen and treating CPPS as an autoimmune disease. Moreover, identification of the relevant autoantigens in CPPS could provide future opportunities for antigen-specific treatments. Because prostatitis can also lead to spuriously elevated PSA levels in affected individuals, diagnosing CPPS could potentially improve the clinical utility of this blood test, which is used to diagnose prostate cancer. Finally, because CPPS may also promote the development of prostate cancer (39), the diagnosis and treatment of CPPS could possibly affect the incidence of this common malignancy.
Mice and reagents. Aire-KO mice were generated as previously described (25). The Aire-KO male mice used in these experiments were backcrossed into the B6 and NOD Lt/J backgrounds for more than 10 generations and generated using heterozygous breeders. WT B6 mice used in the experiments were littermates of the Aire-KO mice. Aire-KO B6/NOD F2 mice were derived from intercrosses of Aire-KO B6 and Aire-KO NOD Lt/J mice (40). All mice were housed in a pathogen-free barrier facility at UCSF. Experiments complied with the Animal Welfare Act and NIH guidelines for the ethical care and use of animals in biomedical research and were approved by the UCSF Animal Care and Use Committee. Tissue culture experiments were performed in RPMI 1640 media (Cambrex) supplemented with 10% fetal calf serum (BioWhittaker), penicillin/streptomycin (Sigma), and nonessential amino acids (Cambrex).
Antigens. The natural mouse SVS2 and human Sg were purified from mouse and human semen plasma, respectively, as described previously (23). In brief, fresh semen plasma were collected in 40 mmol/l Tris/HCL buffer (pH 9.7) with 4 M urea, 25 mmol/l EDTA, 30 mmol/l DTT, 3 mmol/l benzamidine, and 0.5 mmol/l Pefabloc (Boehringer) at 4°C and was applied on a heparin-sepharose column (0.7 × 2.5 cm; GE Healthcare) equilibrated with 0.1 mol/l Tris (pH 8.5), 2 M urea, 5 mmol/l EDTA, 3 mmol/l DTT, 3 mmol/l benzamidine, and 0.2 mmol/l Pefabloc buffer. After application of the sample, the column was washed 5 times with 2 ml equilibration buffer, then 4 ml of 0.1 M NaCl. The column was eluted with 4 ml of 0.25 M NaCl buffer. The eluant was desalted and concentrated with 30-kDa–cutoff Centricons (Millipore). The recombinant MBP-SVS2 and MBP-Sg1 proteins were made according to the protocol of the pMal Protein Fusion and Purification System (New England Biolabs Inc.) with the following modifications. SVS2 gene that was cloned from mouse prostate and Sg1 gene that was cloned from human prostate biopsy were subcloned in frame with MBP and transformed into TOP10 chemically competent cells (Invitrogen). Plasmid DNA isolated was transformed into BL21 (DE3)pLysS bacteria to express the fusion protein. Eluted proteins were concentrated with Amicon Centricon columns with a 10-kDa cutoff and washed 2 times with PBS. For ELISPOT assays, purified MBP, MBP-SVS2, and MBP-Sg1 proteins were passed through a ProteoSpin endotoxin removal kit (Norgen Biotek Corp.). Protein concentrations were measured with BioRad Dc assay (BioRad) using BSA as the standard.
Immunoprecipitation. Immunoprecipitation of autoantigens was performed using protein G agarose coupled to WT or Aire-KO sera as described previously (41). In brief, whole prostate tissues were homogenized in 0.15 M NaCl, 0.05 M Tris (pH 8), and 0.1% CHAPS (Sigma-Aldrich). Protein agarose G–coupled columns were washed in 30 ml PBS, and prostate extracts from RAG-KO mice prepared in CHAPS buffer were passed through the matrix. Columns were washed with 30 ml PBS and again with 30 ml of 10 mM phosphate (pH 6.8). Eluates were collected by passing 0.5 ml of 100 mM glycine (pH 2.5) over the column and collecting the flow through. Eluates from multiple runs were pooled and concentrated in a centrifugal protein concentrator (Vivaspin).
In-gel digestion and PMF. SVS2 was identified by provisional PMF as previously described (25). In brief, gel bands were excised, destained (stain-stripped) 3 times in 50% acetonitrile and 25 mM ammonium bicarbonate (pH 8), dehydrated with 100% acetonitrile, and dried in a Speed-Vac (Savant). Excised gel fragments were digested for 16 h at 37°C. Peptides were extracted, and PMF was used for preliminary protein identification. Mass spectra were produced by MALDI-TOF (Voyager DE STR; Applied Biosystems), representing protonated molecular ions (MH+) of tryptic peptides from the proteins present in each gel spot. The mass spectra were internally mass calibrated using 2 trypsin autolysis products present in the digest mixture. Preliminary protein identities were established by matching the experimentally determined peptide masses to those produced by an in silico tryptic digestion of the Swiss-Prot protein database (http://us.expasy.org/sprot/) within the window of experimental mass measurement accuracy. The PMF data-searching algorithm (available through MS-Fit; http://prospector.ucsf.edu/cgi-bin/msform.cgi?form=msfitstandard) was used to perform the database searches.
Immunoblotting. Mouse or human sera were screened for the presence of autoantibodies by Western blotting. Rabbit anti-human Sg antibody (Santa Cruz Biotechnology Inc.) was used as a positive control. Secondary goat anti-mouse, goat anti-rabbit HRP antibody (Upstate Biotechnologies) and goat anti-human IgG HRP antibody (Invitrogen) were used in the respective assays. The resulting films developed with either Upstate Visualizer or ECL chemiluminescent substrate. For competition studies, Aire-KO B6/NOD F2 sera were preincubated with serial dilutions of MBP-SVS2 or MBP in TBS-T with 1% nonfat dry milk for 2 hours at room temperature before use as the primary reagent to blot the membranes. The concentrations of protein used in these experiments were 30, 7.5, 1.88, and 0.47 μg.
Real-time PCR. Real-time PCR was performed on cDNA prepared from DNAse-treated RNA derived from dissected tissues or FACS-purified CD45– thymic stroma cells, as previously described (42). Aire- and cyclophilin-specific primers and probes were used as previously described (25). SVS2 Taqman primers and Fam-labeled MGB probes were purchased from Applied Biosystems (catalog no. Mn01251795_g1). Reactions were run on the HT7900 sequence detection system (Applied Biosystems).
ELISPOT analysis. Splenocytes were harvested from Aire-KO mice, WT B6 mice, or immunized WT B6 mice, and the release of IFN-γ was measured by ELISPOT assay. Briefly, MultiScreenHTS-IP plates (Millipore) were coated overnight with 2 μg/ml anti-mouse IFN-γ mAb or anti-human IFN-γ mAb (BD Biosciences) at 4°C. The plates were washed with PBS and blocked with RPMI medium containing 10% FCS for 2 hours at 37°C. Mouse splenocytes were added to each well with the different antigens and incubated for 18 hours in RPMI complete medium. The plates were washed with PBS before adding 2 μg/ml biotin-labeled anti-mouse or anti-human IFN-γ mAb (2 μg/ml; BD Biosciences) and incubated overnight at 4°C. After washing and further incubation with horseradish peroxide–conjugated avidin (diluted 1:100; BD Biosciences) for 1 hour at room temperature, the plates were washed and developed using 3-amino-9-ethylcarbazole substrate solution (BD Biosciences). Positive spots displayed in the plate membranes were examined using an automated ELISPOT reader (AID; Autoimmun Diagnostika GmbH). The number of spot-forming cells was the average number of spots in duplicate or triplicate wells.
Generation of antigen-specific T cell lines. Aire-KO B6 male mice were immunized with the purified SVS2 (200 μg/mouse) emulsified in an equal volume of CFA (Difco). The antigens were injected s.c. into the footpads. Mice were boosted twice with the same amount of antigen emulsified in IFA at 10-day intervals. At 10 days after the second boost, single-cell suspensions from draining lymph nodes of SVS2-immunized mice were incubated with purified SVS2 (50 μg/ml). After 24 hours, mouse IL-2 (50 U/ml), IL-7 (10 ng/ml), and IL-15 (10 ng/ml; all from Peprotech) were supplemented into the media. Subsequent restimulations occurred at weekly intervals by the addition of purified SVS2-pulsed, irradiated B6 splenocytes to the cultures followed by the same cytokine supplementation 24 hours later. The OVA-specific T cell lines were generated in parallel in separate mice through the same procedure. After 2 cycles of restimulation, the dead cells were removed by centrifugation with Ficoll (Histopaque-1083; Sigma-Aldrich), and the cultured cells were harvested for further analysis and adoptive transfer experiments.
Intracellular cytokine staining of antigen-specific T cells. Antigen-specific T cell lines were incubated with the irradiated B6 splenocytes pulsed with MBP-SVS2 (50 μg/ml) for 24 hours. GolgiStop buffer (BD Biosciences) was added at a final concentration of 1 μg/ml for the last 4 hours of incubation. The cells were then harvested for intracellular cytokine staining. Briefly, the cells were first stained with allophycocyanin-conjugated anti-mouse CD4 antibody (BD Biosciences) and fixed with 4% formaldehyde in PBS for 10 min. After subsequent permeabilization with 1× BD perm/wash buffer (BD Biosciences) for 15 minutes, the cells were stained with PE-conjugated anti-mouse IFN-γ (eBioscience), PE-conjugated anti-mouse IL-10 (Biolegend), or FITC-conjugated anti-mouse IL-17a (BD Biosciences) antibodies. The cells were assessed by flow cytometry and analyzed with FlowJo (version 8.0.3; TreeStar).
Adoptive transfer experiments. Splenocytes were harvested from Aire-KO B6 mice or immunized B6 mice. CD4+ or CD8+ T cells or CD19+ B cells were depleted using FACS. Briefly, cells were stained with PE-conjugated anti-CD4, -CD8, or -CD19 antibodies (BD Biosciences — Pharmingen) for 30 minutes on ice, and then the PE-negative CD4-, CD8-, and CD19-depleted cells were collected (FACSAria; BD Biosciences). Sorted depleted cells (5 × 106 cells) were injected i.v. into RAG-KO B6 mice. Animals were aged 40 days after the transfer, sacrificed, and analyzed. In additional experiments, 1 × 106 cells from either SVS2-reactive or OVA-reactive T cell lines were injected i.v. into RAG-KO B6 mice. Animals were aged 20 days after the transfer, sacrificed, and analyzed.
SVS2 immunization experiments. Male B6 mice at 25 weeks of age were immunized with MBP-SVS2 (200 μg/mouse) or MBP (200 μg/mouse). Both proteins were emulsified in an equal volume of CFA (Difco) supplemented with Mycobacterium tuberculosis H37Ra. The antigens were injected s.c. on the back. Mice were boosted twice with the same amount of antigen emulsified in IFA at 2-week intervals. At 2 weeks following the second boost, tissues and sera were harvested for further analysis or adoptive transfer experiments.
Histopathology. For H&E staining, prostate tissues were either fixed in 10% neutral-buffered formalin and embedded in paraffin or frozen in OCT tissue embedding compound (Tissue Tek) at –80°C. After sectioning, tissues were stained with H&E (Sigma-Aldrich). For immunohistochemical staining, prostates were snap-frozen in OCT, sectioned at 6 μm, and processed for staining using Vectastain Elite ABC Kit (Vector Laboratories) with biotinylated anti-rat antibody against rat anti-mouse CD3 (clone CT-CD3; Caltag Laboratories), CD4 (clone H129.19; BD Biosciences — Pharmingen), CD8a (clone 53-6.7; BD Biosciences — Pharmingen), or CD19 (clone 1D3; BD Biosciences — Pharmingen), followed by streptavidin peroxidase conjugate. Colorimetric detection was visualized using DAB substrate (Pierce). Histology was assessed by a pathologist blinded to mouse phenotype or treatment, who scored lymphocytic infiltrates as described in Supplemental Table 1.
Human subjects. Sera were obtained from patients with clinical histories of CPPS at the University of Washington Medical Center. The study protocol was reviewed and approved by the University of Washington Institutional Review Board, and individuals provided informed consent prior to participation. Clinical assessment of prostatitis in the prostate biopsies was performed by the center's pathology department. Control sera were obtained from male volunteer blood donors without histories of prostatitis symptoms. Sera were stored in aliquots at –80°C until used.
Statistics. For the mouse study, differences between groups were evaluated using 2-tailed Student's t test, and differences were considered statistically significant at P < 0.05. For human studies, differences between groups were evaluated by Fisher exact test, and differences were considered statistically significant at P < 0.05.
This work was supported by NIH grants AI056388 and CA102303. The authors thank Marcella Fasso and Dil Kapadia for critical review of the manuscript; the UCSF Biomolecular Resource Center Mass Spectrometry Facility and the Sandler Family Foundation for mass spectrometry; and the UCSF Prostate SPORE Tissue Core for providing guidance in preparing tissue sections and staining.
Conflict of interest: The authors have declared that no conflict of interest exists.
Nonstandard abbreviations used:Aire, autoimmune regulator; B6, C57BL/6; CP, chronic prostatitis; CPPS, chronic pelvic pain syndrome; FACS, fluorescence-activated cell sorting; IFA, incomplete Freund adjuvant; MBP, maltose-binding protein; PMF, peptide mass fingerprinting; Sg, semenogelin; SVS2, seminal vesicle secretory protein 2.
Reference information: J. Clin. Invest.119:2031–2041 (2009). doi:10.1172/JCI38332


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We are seeing a leak of updates to Android 8.0 Oreo on Samsung Galaxy S8, S8 + and Note 8 in the last hours. The forums, in particular, those of Reddit, burn with every information and the last one is that the update of the Galaxy S8 + adds support for Dolby Atmos.
Without a doubt, it is great news. The tide of updates filtered in the last hours, caused by the end of the Android betas adds a new and interesting information for those who have a Samsung Galaxy S8 +. At the moment, it is still a stable version of Android 8.0 Oreo for American teams, that is, for those who have Snapdragon 835, exactly like the one that appeared yesterday for the Galaxy S8 and Galaxy Note 8.
Apparently, Samsung phones enjoy a version that works much better than the previous Oreo betas as it also has the latest security patches this January 2018. But, without doubt, the great addition is, as we have said, the presence not only of support for Dolby Atmos but the adjustments to manage it.
Dolby Atmos on the Galaxy S8 + thanks to Android 8.0 Oreo
While it is true that the Dolby Atmos equalizer is already present in Android phones for a few months, only reached a certain number of manufacturers. It has always been possible to add to the rooting of the terminals and by installing it … but the vast majority has never seen it working on their devices.
Dolby Atmos allows, among other things, to change the equalization settings and the emulation of the surround sound when we are using headphones. Specifically, you can find automatic pre-settings for movies, music, and voice. In the beta of Oreo for the Galaxy S8 + still have to refine certain aspects because, it seems, still has failures. It does not work properly and, among other things, when activated it can no longer be deactivated.
This shows that, for now, we are not facing the final version of Android 8.0 Oreo for Samsung and that there will still be a time for a "traditional" OTA. That is why, in the case that you have a model with Snapdragon 835 processor (if you have a European unit with Exynos or try it), you can try this beta although keep in mind that it is very likely that you will find more failures.


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My BlogLog account seems to have magically disappeared. I was thinking about cancelling it anyway but I guess I don't have to. Now I can stalk all of you incognito.
What is the deal with doctor's offices that will not call you back??? I've left two messages with my PCP regarding my asthma that is darn near going to kill me but they have yet to return my call. One of these messages was left LAST WEEK!! It's frustrating.
My good friend, D, found out today that she is having a baby girl! Congrats miss D and let the pink fest begin! I wonder who decided on pink as the token color for girls and blue for boys? If I ever have a baby I'd like to change that to red for a girl and green for a boy as I like those colors better.
I've been tagged for a meme by the lovely Newscoma so look out for seven amazing and astounding facts about moi coming to this blog in the very near future. Pay attention and you might learn something.


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— Donald J. Trump (@realDonaldTrump) June 12, 2018
STEALTH JEFF on Twitter: "Dear Stupid people wondering why Trump didn't treat Kim as an inferior: we got this summit because Trump understands the Asian practice of FACE and you don't. Dear Stupid people wondering why we should trust Kim now: Because he'd LOSE massive face if he reneges."
BREAKING: 9th Circuit Court orders Kim Jong Un to continue his development of nuclear weapons.
— Mike Pampeo (@SecOfState70) June 12, 2018
F- – – Trump!???
As a student of history, let me tell you what a Hitler-like Nazi-like President of the United States would have done these past 500 days:
He would have had Chuck Schumer put into a concentration camp. He would have had medical experiments done on Nancy Pelosi and Maxine Waters. He would have had Robert Mueller and his entire investigatory team murdered in the middle of the night, the same night, with long knives. Lisa Payne and Peter Strzok would be in concentration camps now, working twenty-hour days for the Bayer Aspirin or Ford Motor companies, businesses who in the 1940s got rich off Nazi-provided slave labor. Jeff Sessions would have been fired. Writers at the Washington Post and New York Times would be dead or in concentration camps. Instead of wrangling with Jeff Bezos, the Government would now own Amazon. And CNN. And Time-Warner. Several or all of the four Democrat Supreme Court justices would be in concentration camps or dead. Not only would ten million illegals have been rounded up by now, but they would not necessarily have been deported because they, too, would be in cattle cars now, en route to concentration camps, gas chambers, and ovens for the Final Solution of the border issue. There would be no haggling and negotiating for votes in the House or Senate; rather, the Congress would have been disbanded, the Constitution suspended, and we now would be invading Mexico instead of negotiating over NAFTA and steel and aluminum tariffs. And — for snarky extreme-right conservatives who might be mumbling "Hmmm, not so bad" — many of those snarky conservatives, too, now would be dead or en route to ovens because their neighbors may have ratted them out in order to grab their houses after their arrests. And, yes, their kids also would be dead.
Chef Rush, the WH Chef Who Makes Sure Trump Gets His Protein So, while I don't think it's terribly important whether President Trump loves McDonalds' or not, rest assured that a lot of his meals are coming from this guy, and when he says you eat the broccoli and lean beef, you eat the broccoli and lean beef.


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The PMI® Delaware Valley Chapter is a dynamic organization of project management professionals dedicated to serving the business and educational community of the Chapter through the advancement of project management.
The PMI Delaware Valley Chapter provides a forum for project management professionals to:
- Promote the principles of the Project Management Institute® through networking with project management professionals.
- Share project experiences.
- Provide and receive training.
- Support project management professionals in their certification efforts.
Service Area
The PMI Delaware Valley Chapter serves the Greater Philadelphia Area, South Jersey, Delaware and Eastern Shore Maryland.
PMI Delaware Valley Chapter Mailing Address
P.O. Box 438
Drexel Hill, PA 19026
Advance the mission and objectives of the Project Management Institute within the Delaware Valley Chapter. Develop a growing and committed membership of local project management professionals through an aggressive recruiting plan and retention.
- Promote professional project management principles and techniques with local businesses, universities and professional associations.
- Create and deliver an educational program that strengthens local project management professionals' skills and supports the Project Management Institute Certification Program.
- Provide world-class PMI certification and training by hiring the best instructors and by developing the best training materials.
PMI Hardship Provision
A "Hardship Provision" is available to any PMI member whose financial condition has changed drastically and sufficiently so that a member would have difficulty renewing their membership. A member must be in good standing for at least three previous years to qualify for the hardship provision. Under this provision, a member is granted a waiver for one year of PMI membership dues and one year of dues for membership in any two PMI Components of which the applicant was a member the previous year. The Hardship Provision may be granted for a maximum of two years for each member. This information can also be located on the back of the renewal invoice that members receive. To apply for consideration send a letter explaining the nature of the hardship, along with the invoice, to the attention of the Manager, Membership Services prior to the membership expiration date. Please remember that any incorrect or untrue statements made by a member are in violation of the PMI Member Ethical Standards-Member Standards of Conduct and may lead to the initiation of an ethics complaint in accordance with the PMI Member Ethics Case Procedures.
PMI is a registered mark of the Project Management Institute, Inc.


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I enjoyed hearing Nina Platt's tech-savvy librarian's perspective on limitations of search, and the skills that a good librarian has. (I also enjoyed chatting with her and her new employee at Legal Tech08).
I certainly agree with her that, if you're trying to find anything at all unusual or difficult to find, a librarian or skilled researcher is going to be miles ahead of a typical search engine. The libriarian / researcher can grasp and then supply the real context to the query, through iterative questions that the user hadn't thought or didn't know to ask.
I serve as something like a librarian with respect to my firm's internal work product resources, and I have found myself using a similar "patter" when people call looking for a sample brief or statistics on patent litigation. I can help much faster and more effectively if I know why they are asking, including (usually) the specific procedural stage of the case, the jurisdiction, and which side we're representing. Usually the first email doesn't have everything I need to search effectively.
I can't help but wondering if the combination of social search and faceted / guided navigation will bring search a little closer to what a librarian does, by showing some of the possible contexts and refinements. See my previous posts here and here on Vivisimo's entry into the field.
With such a system, not implemented at any law firm yet so far as I know, if the clueless associate searches for "motion to dismiss", they'd get a half-million results. If they can then see and refine / filter by tags other less clueless associates applied such as "jurisdiction" "forum non conveniens" "particularity", not to mention metadata attributes such as forum, they may be led down the right path almost as accurately as a libriarian or practice support / KM attorney might take them.


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5 Ways to Save on Summer Cooling Bills
If you are like many, every year you resolve to cut back on your energy costs. You decide that, once and for all, you are going to "toughen up" and run the air conditioning less frequently. You will finally notch up your thermostat that extra degree or two and sweat it out.
Yet when August rolls around again, where does all that resolve go? It is HOT, and all you want is to be COOL.
Happily, this year we have some tips that will help you stay cool AND save on your summer cooling bills!
P.S. Remember, you still have the rest of this month to enter for a chance to win a FREE air conditioner: read more about the contest HERE!
Tip 1: Use those fans
Big fans, small fans, ceiling fans, floor fans—all fans are welcome in the hottest month of the year.
Not only can using fans help you feel cooler than the actual reading on your thermostat, but fans can keep your indoor air fresher and cleaner through continuous circulating.
The one fan you DON'T want to use continuously is the one on your air conditioning unit. This fan will keep the air circulating, for sure, but it will also run up your energy bill to the tune of $50+ per month and increase the humidity in your indoor air.
Continuously running your A/C unit's internal fan will also increase wear and tear on the unit, which can mean earlier and more expensive maintenance and repairs. Keep your fan setting on the thermostat at Auto, particularly if the AC is off during cooler hours of the day. Opt for a low-cost floor or ceiling-mount fan instead to keep air circulating and keep it spinning.
NOTE: If you use ceiling-mount fans, be sure you have the fan blades spinning in the right direction for summer! Most ceiling fans have a small switch near the light fixture. The summer setting pushes the air down towards the floor to cool the central areas of each room. You will be able to feel if the fan is on the right setting by standing beneath it. If there is air blowing on you, it is on the right setting.
Tip 2: Take your kitchen outdoors
"Heat stress" is a medical term to describe what happens when indoor air gets too hot. Symptoms of heat stress can include rashes, fatigue, fainting, exhaustion, and even the potentially deadly heat stroke.
One of the biggest causes for elevated indoor air temperature is cooking inside...
When your oven or stove heats up, your house heats up correspondingly.
This makes summer the perfect time to take your kitchen outdoors. By using an outdoor grill (or even building your own solar oven—a great kid-friendly project), you can save up to double on energy bills, cut down wear and tear on your air conditioner and enjoy eating healthy grilled food.
Tip 3: Program your thermostat
If you really like your home and office to stay cool, you may struggle with this tip. But you don't have to change a thing about the temperature settings for when you are at home or at work—you can still keep those as cool as you like.
The goal here is to program your thermostat to cycle off when you are not at work or at home. This accomplishes two money-saving goals.
First, your A/C unit will work more efficiently to cool and dehumidify your indoor air when it cycles back on, because it will have to push more energy right away and work harder, which is how the unit was designed to work.
Second, your A/C unit won't have to run continuously, which will save on your annual A/C-related wear and tear and maintenance costs.
Here are a few other ways to program your thermostat to save:
Save an estimated $8 per month for each degree you program your thermostat above 78°F (25.5°C).
Program your thermostat to turn off one hour before you leave the house and to turn back on one hour before you return home again. While you are gone, program the thermostat for 82°F (28°C). This will save you an estimated 10 percent on monthly energy bills.
Ask your energy company about peak and off-peak rate packages. If you can cut back on energy use during peak hours by switching to an off-peak plan, you can save more on energy costs each month.
Keep the hot stuff (lamps, electronics, etc.) away from the thermostat. Any extra heat source can make the thermostat think it is hotter inside than it is, which will cause your air conditioner to run (and spend your cash) unnecessarily.
Tip 4: Go green on the sunny side
Everyone appreciates shade during the summer, and your air conditioner is no exception. Here are two things to give your A/C unit some extra shade relief:
Cover the unit with an awning to keep the direct sun off of it.
Plant some fast-growing bushes or trees near the unit to provide some natural shade (however, be sure to keep the immediate area around the unit clear with at least 4 feet for ventilation).
Tip 5: Give your A/C unit a retirement party
If your HVAC unit is more than a decade old, you may not be able to control energy expenses. The newer Energy Star-certified HVAC units are simply better at cooling than their predecessors.
A well-timed replacement can save you 20 percent or more per month.
Enter to Win a FREE A/C!
Now through the end of August, Shipton's is giving away a FREE brand-new Energy Star air conditioner to one lucky winner! One winner has already been chosen and the next will be announced the first week of September.


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Instant oatmeal and rum in an alder thicket provide shelter from the storm.
Aaron Teasdale
One evening in mid-June in the remote fishing port of Yakutat, Alaska, a young Tlingit girl and her mother walk past two men hunching over strange bicycles and a spread of colorful equipment outside the town's single-gate airport. "Where are they going on those bikes?" she asks her mother, which is an excellent question considering that Yakutat has only a few short Jeep tracks and no roads or trails leading to another town (because there is no other town).
"I have no idea, honey," her mother replies.
From the patio of a nearby shack, aspirationally named the Yakutat Lodge, a woman in a Bud Light sweatshirt takes out a cigarette and says, "What're you guys doin'?" Ryan Krueger and I point to the fat, four-inch-wide tires on our bicycles and explain that we can ride on sand, that our tiny rafts will allow us to cross the many rivers we will encounter, and that once we figure out how to actually affix our tents and sleeping bags to our bikes and stuff five days of provisions into our backpacks, we intend to ride to the ocean and down its uninhabited coast to see what there is to see.
She considers our dry bags, paddle shafts, bulging bike wheels, and various sacks of food."
"You ever done this before?" she asks dubiously."
"Actually," I say, pausing in my third attempt to strap a deflated raft to my handlebars, "this is our first time."
She says she'll pray for us.
[Keep reading at Sierra Club]
[Keep reading at Sierra Club]


text: ANKARA, February 17: While terming the bilateral relations between Pakistan and Turkey as exemplary, leadership of both the countries agreed to further strengthen existing cooperation in the fight against terrorism and to jointly target anti-terror activities such as curbing the sources of funding for terrorists. This was agreed in a meeting between Deputy Prime Minister of Turkey Mr. Jamil Chechek and Deputy Speaker National Assembly of Pakistan Mr. Faisal Karim Kundi in a meeting here today. Mr. Kundi is heading a seven-member parliamentary delegation that includes Members National Assembly Mr. Usman Muhammad and Ms Shireen Arshad, Senators Mr. Muhamad Kazim Khan, Ms Simeen Yusuf Siddiqui, Mr Mohammad Saleh Shah and Mir Lashkar Raisani. Mr. Kundi said terrorists always have the backing of external forces and a joint fight involving friendly countries supported by the United Nations could help in eradicating this menace from our societies. The Turkish Deputy Premier informed that Turkey is victim of terrorism since the last 40 years and is sharing the expertise and information with the brotherly country of Pakistan. Mr. Kundi conveyed the appreciation of Pakistani nation for Turkish support for flood affectees of Pakistan. Earlier, in a meeting with the President of Turkish Cooperation and Development Agency (TIKA) Mr. Musa Kulaklikya, the visiting Pakistani delegation was briefed about the ongoing developments projects that TIKA is undertaking in Pakistan. He informed that a group of experts would be visiting Pakistan to further explore the possibilities of Turkish technical support for building dams in Pakistan. Mr. Kundi invited TIKA to consider projects in the fields of potable water for rural areas, handicrafts including training of master trainers, education exchange, and mining.


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Dr. Bea Marie Abordo graduated at Centro Escolar University - Manila. During her college years, she was an active participant in Dental Missions to improve her skills and be able to help a lot of people. To improve on her dental skill and practice, she has attended a number of seminars both local and international. She attended the 3rd Asian Sympsium on Advanced General Dentistry in Singapore, "New Trends and Innovations in Esthetic Dentistry" by the Philippines Academy of Esthetic Dentistry (PAED), and she is currently studying Cosmetic Dentistry at PAED Batch 27.
Dr. Bea has always been proud to be part of the GAOC Dental team. She believes that GAOC is the best because learning never stops. There's something to learn everyday with every cases they encountered that molds them to be the best and enables them to deliver the best possible treatment every patient deserves.


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We are always looking for signs of recovery during an economic downturn, and almost any chink of light is enough to make us think that things aren't quite as bad as people say they are. Unfortunately for every little bit of good news there always seems to be far more gloomy indicators hot on their heels.
For example we recently heard that house prices were set to increase by 16% over the next few years, great headline but for many people this prediction will only take them back to where they were when they bought their hoses 3 years ago. Inflation is rampant and the threat of raised interest rates is ever present, all this leads to a lack of confidence by consumers who keep their money in their bank accounts and believe they can't afford to do the things they originally planned to do. In the car business this has a dramatic effect on sales and will often mean that certain cars become virtually unsellable.
Buyers are tending to fall into 2 categories; those looking for a much more economical, cheaper proposition and the other looking for the absolute bargain of the century. This leads to a fall in volumes and erosion in profitability and dealers finding themselves massively overstocked tend to start distress selling in order to keep a tight control of their stock management.
Of course, for the car buyer, this can be positive, they can be confident they can get a great deal and that there is plenty of choice but for sales execs, with major pressure being put on them, it can lead to desperation measures and cutting corners by telling customers exactly what they want to hear just to get the sale closed.
Showroom and online footfall is very definitely down and car dealers will need to look at alternative ways in which to entice buyers into their showrooms and by looking at different marketing methods perhaps this can be achieved. It is vital for them to stand out from the crowd and make a compelling case for a buyer to choose them over a rival, although I am sure even now there will be car buyers who will have an instance of trying to get served in a showroom and being virtually ignored.
At the same time finance refusals are again on the rise and it appears that lenders are still very nervous about loosening the purse strings which has an adverse effect on sales rates but also staff morale.
For a sales exec to spend a long time with a buyer delivering a great experience and agreeing a deal only to be told 2 days later that the customer doesn't qualify for funding must be very disheartening for both buyer and sales exec alike.
During times like now experienced dealers with dynamic managers and staff will navigate through these choppy waters, because they understand that by doing what they have always done will always get them the same result.
Doing things differently (but well) and standing out from the crowd should ensure that customers will keep coming to buy their products and services. Unfortunately we can't tell you what that difference is but you'll know it when you see it!