text:
Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus
Virology and prion diseases
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library.
Major changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold.
The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.
Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS in swine, is a member of the
In 2006 a unique syndrome with high morbidity and mortality was recognized in growing pigs in China that was originally known as porcine high fever disease (PHFD) due to its uncertain etiology
Results and discussion
Mapping short RNA-seq reads and estimating transcript expression levels
Genomic Short-read Nucleotide Alignment Program (GSNAP) was used for alignment and genome construction, and Cufflinks to determine if differential expression and changes in transcript abundance were statistically significant
Additional file 1
Cuffdiff output of all significant hits for comparison, using the HP-PRRSV rJXwn06 vs. control and the US PRRSV VR-2332 vs. control datasets, to test for differential expression and regulation amongst the two disease states. Gene annotation (symbol) was carried out using a MySQL database matching to the Ensembl Sscrofa9.56 reference genome currently supported by the Integrative Genomics Viewer (Broad Institute). Location (row.names); gene annotation (symbol); entrez gene name; treatment (sample_1, sample_2); status; abundance in FPKM (value_1, value_2); differential expression (log2_fold_change); test statistic (test_stat); p-value (p_value); false discovery rate (FDR)-adjusted p-value of the uncorrected test statistic (q_value); significance status after Benjamini-Hochberg correction for multiple-testing (significant).
Click here for file
Examination of the RNASeq data indicated that there were major changes in transcript abundance occurring in the PRRSV-infected TBLN-based unique transcripts [Cuffdiff output (Additional file
This derived catalog of expressed genes represents the first comparative analysis of the HP-PRRSV rJXwn06 and VR-2332-infected TBLN transcript abundance profiles and provides a database that informs us of genes involved in normal TBLN physiology, as well as genes whose abundance is altered by PRRSV infection.
Annotation of significantly differentially expressed genes
Gene annotation of all significant hits (Additional files
Additional file 2
Transcript sequences of all significant hits inthe HP-PRRSV rJXwn06 vs. control and the US PRRSV VR-2332 vs. control datasets.
Click here for file
Analysis of the genomic data in the context of gene ontology, by Ingenuity Pathway Analysis (IPA), allowed us to ascribe biological functional networks to the differentiated transcript abundance dataset. The top functions identified with the Ingenuity Canonical Pathway list, filtered to apoptosis, cellular immune response, cytokine signalling, humoral immune responses and pathogen-influenced signalling, based on differentially expressed genes were: granzyme A signalling, crosstalk between dendritic cells and natural killer cells, IL-10 signalling, role of pattern recognition receptors in recognition of bacteria and viruses, IL-12 signalling and production in macrophages, complement system, interferon signalling, communication between innate and adaptive immune cells, IL-17A signalling in fibroblasts, granzyme B signalling, production of nitric oxide and reactive oxygen species in macrophages, differential regulation of cytokine production in macrophages and T helper cells by IL-17A and IL-17F that were above the threshold of p value < 0.05, as calculated by Fischer's test representing the ratio of number of genes from the dataset that map to the pathway and the number of all known genes ascribed to the pathway. The genes up-regulated in the HP-PRRSV rJXwn06 infected pigs' TBLN were associated in 18 networks: from biological networks with functions associated with cell death, antimicrobial responses and cancer, with the highest network score of 37, i.e. the likelihood of genes in this network would have approximately a 10-37 chance of occurring randomly, and 21 focus molecules, i.e. the starting points for generating biological networks; to networks with functions associated with nervous system development and function, organ morphology and reproductive system disease with a score of 2 and 1 focus molecule. Many of the up-regulated networks related to cell death and inflammatory response functions fit with the results previously reported
Figure 1
Ingenuity pathways analysis summary
Ingenuity pathways analysis summary. To investigate possible interactions of differently regulated genes, datasets representing 568 genes with altered expression profile obtained from the RNAseq data for HP-PRRSV rJXwn06 were imported into the Ingenuity Pathway Analysis Tool and the following data is illustrated: The network representation of the most highly rated network (Gene Expression, Cell Death, Lipid Metabolism). The genes that are shaded were determined to be significant from the statistical analysis. The genes shaded red are up-regulated and those that are green are down-regulated. The intensity of the shading shows to what degree each gene was up or down-regulated. A solid line represents a direct interaction between the two gene products and a dotted line means there is an indirect interaction. Genes associated with Cell Death are circled with orange color.
This study produced transcriptional profiles of TBLNs from non-infected, HP-PRRSV rJXwn06 and US PRRSV VR-2332-infected pigs that provides insight into immune dysregulation elicited by the virus on host transcript abundance levels necessary for a effective immune response.
This RNA-Seq compendium extends the analyses of previous gene expression atlases performed using Affymetrix GeneChip technology and provides an example of new methods to accommodate the increase in transcriptome data obtained from next generation sequencing
It is well established that many pathogens cause changes in expression of specific genes that act to protect the host and clear the infection. PRRSV strains differ in their dysregulation of the immune response to infection and delay in development of a protective immune response in vaccinated pigs
Cells and virus
MARC-145 cells were cultured in minimum essential medium (EMEM, SAFC 56416C) with 10% fetal bovine serum at 37°C, 5% CO2. Wild-type (
Swine study
The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Animal Disease Center-USDA-Agricultural Research Service. Thirty-two 10-week-old cross-bred pigs were obtained from a U.S. high-health herd and were found to be free of PRRSV and influenza virus antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) kits (HerdChek PRRS 2XR; IDEXX Laboratories, Westbrook, Maine) and NP ELISA (MultiS ELISA, IDEXX, Westbrook, Maine), respectively. Pigs were also confirmed negative for porcine circovirus type 2 by quantitative real-time PCR
RNA isolation
Following humane euthanasia, tracheobronchial lymph nodes (TBLN) from in vivo HP-PRRSV rJXwn06 (N = 8), US PRRSV VR-2332 (N = 7), or sham-infected pigs (N = 8) were harvested at 13 days post-infection and total cellular RNA was prepared as follows. One gram of TBLN from each pig was collected immediately upon necropsy, minced and stored in RNAlater (Life Technologies, Grand Island, NY) at −80°C until homogenized for extraction of total RNA with MagMAX™-96 for Microarrays Total RNA Isolation Kit (Applied Biosystems, Carlsbad, CA) using the manufacturer's protocol. The integrity of the RNA was confirmed with a 2100 Bioanalyzer and RNA 6000 Nano-chip (Agilent, Santa Clara, CA). The samples used had an average RNA Integrity Number (RIN) value of 7.8 and 28S:18S rRNA ratio of 1.9.
cDNA library construction
cDNA libraries were constructed from pooled total cellular RNA from the TBLN in each treatment group using TruSeq Sample Prep Kits (Illumina Inc., San Diego, CA) and sequenced by 2 × 100 paired-end sequencing on an Illumina HiSeq 2000 instrument.
RNA-Seq (Whole Transcriptome Shotgun Sequencing) pipeline
In order to analyze the Illumina reads, a series of bioinformatics methods were used to investigate gene expression profiles in TBLN during PRRSV infection with HP-PRRSV rJXwn06 and US PRRSV VR-2332 at a snapshot of 13 dpi. This was carried out with the construction of a RNAseq analysis pipeline (Figure
Figure 2
Computational pipeline
Computational pipeline. RNAseq analysis pipeline comprised of GSNAP for alignment and Cufflinks to determine if differential expression, and changes in transcript abundance were statistically significant (adapted from
Ingenuity pathway analysis (IPA)
Datasets representing genes with altered expression profile derived from RNAseq analyses were imported into the Ingenuity Pathway Analysis Tool (IPA Tool; Ingenuity® Systems, Redwood City, CA, USA;
The basis of the IPA program consists of the Ingenuity Pathway Knowledge Base (IPKB) that is derived from known functions and interactions of genes published in the literature. Thus, the IPA Tool allows the identification of biological networks, global functions within the host and functional pathways of a particular dataset. The program also gives the significance value of the differentially expressed genes, the other genes with which it interacts, and how the products of the genes directly or indirectly act on each other, including those not involved in the microarray analysis. The networks created are ranked depending on the number of significantly expressed genes they contain and also list diseases that were most significant (Figure
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LCM: study conception, data collection and analysis, research design, manuscript writing. DF: research design, data analysis. AA: data analysis. DOB: RNAseq interpretation discussions and data analysis. BG: infectious clone and production of virus stocks. KML: study conception, animal study execution, virus stocks, data collection, and manuscript preparation and writing. JNH: data collection. SNS: data collection. H-CY: infectious clone of HP-PRRSV strain JXwn06. KSF: study conception, rescue of HP-PRRSV infectious clone, and production of virus stocks. MEK: study conception and manuscript preparation and writing. All authors read and approved the final manuscript.
We thank the following members of the Virus and Prion Research Unit at the National Animal Disease Center: J. Huegel, J. Crabtree, A. Burow, D. Adolphson, S. Anderson, M. Kappes and A. Vorwald for technical assistance. We also gratefully acknowledge D. Alt of the Genomics Unit at the National Animal Disease Center, and A. Severin of Iowa State University NGS Bioinformatics, for assistance in data analysis. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States
General overview of PRRSV: a perspective from the United States
Porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression
In utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by Streptococcus suis type II
Pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs
Synergism between porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis in swine [In Process Citation]
Laboratory investigation of PRRS virus infection in three swine herds
A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome [In Process Citation]
Genetic, geographical and temporal variation of porcine reproductive and respiratory syndrome virus in Illinois
Associations between genetics, farm characteristics and clinical disease in field outbreaks of porcine reproductive and respiratory syndrome virus [In Process Citation]
Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus
Comparative pathogenicity of nine US porcine reproductive and respiratory syndrome virus (PRRSV) isolates in a five-week-old cesarean- derived, colostrum-deprived pig model
Reproductive failure of unknown etiology
Genetic perspectives on host responses to porcine reproductive and respiratory syndrome (PRRS)
Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development
Lelystad virus and the porcine epidemic abortion and respiratory syndrome
Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark
An infectious cDNA clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome
The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence
Experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus
De novo assembly and analysis of RNA-seq data
Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation
Genenames.org: the HGNC resources in 2011
Cloning and characterization of porcine resistin gene
Porcine S100A8 and S100A9: molecular characterizations and crucial functions in response to Haemophilus parasuis infection
Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells
Citrullination by peptidylarginine deiminase in rheumatoid arthritis
Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis
Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling
HP-PRRSV challenge of 4 and 10-week-old pigs
In-depth global analysis of transcript abundance levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus
Molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo
Lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs
Infection with Porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs
Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus
In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants
Coinfection of pigs with porcine respiratory coronavirus and Bordetella bronchiseptica
Uncovering the complexity of transcriptomes with RNA-Seq


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text: My small forum has only been around for a few years but in that time it has been mentioned on BBC TV and radio during Formula One sessions. We've also had some high profile visitors such as Formula One teams, etc. Most recently, the ex-Renault F1 technical director Pat Symonds, wrote an article for Motor Sport Magazine and used all of our overtaking data as the basis for it. As the data is available to members only, I can only presume he is a member. I haven't worked out which one though Anyone else got any forum related claims to fame?


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- To demonstrate his olfactory prowess, Brian predicts various events, saying to Lois "Your toast is ready". The toaster immediately pops up two slices of toast. In the very next scene, the toaster is shown with no toast in it.
Previous Episode's Goofs /// The Thin White Line's Goofs \\\ Next Episode's Goofs


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The Survival Template
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Customer Reviews
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- Print Length: 41 Pages
- Language: English


text: SHIMMER Volume 55 kicks off with this contest between Jessie McKay and Yuu Yamagata. The veteran Yamagata is still in search of her first SHIMMER win, having suffered losses at Volumes 53 and 54. Jessie too had hard luck at the past two volumes, but she has plenty of experience bouncing back in SHIMMER. Who will pick up their first win of the weekend?


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Smartphone add-on tests for HIV and syphilis in 15 minutes
February 5, 2015
A low-cost smartphone accessory that can detect three infectious disease markers from a finger prick of blood in just 15 minutes, performing all mechanical, optical, and electronic functions of a lab-based blood test.
That's what team of researchers led by Samuel K. Sia, associate professor of biomedical engineering at Columbia Engineering, has developed.
It performs an enzyme-linked immunosorbent assay (ELISA) triplexed immunoassay… read more


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The Madison Skate Shoe features
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Business Tip — A number of web sites out there will tell you how to come up with a workable business model for your e-commerce web site. Here's an untypical way to sell your content.
Much about everywhere, you'll read you're going to make money by selling:
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Sometimes, you'll simply read you're going to save money — or sell more offline — because your web site explains each and every aspect of your product or service.
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text:
Life Technologies to Collaborate with Merck Serono
News Jul 17, 2013
Life Technologies Corporation announced that the company has signed an agreement to collaborate with Merck Serono, a division of Merck KGaA, Darmstadt, Germany, for current and future companion diagnostics projects. The current agreement constitutes the first collaboration between the two companies and represents another step in Life Technologies' strategy to develop its diagnostic business through internal development, collaborations and select acquisitions.
The non-exclusive agreement covers an initial project for oncology and provides for a long-term collaboration across a potentially broad range of Life instrument platforms and a wide range of therapeutic areas. Financial terms of the agreement were not disclosed.
"Life Technologies' combination of platforms and capabilities are unique in the companion diagnostics space, particularly in oncology," said Ronnie Andrews, President, Genetic & Medical Sciences at Life Technologies. "With our recent acquisition of Compendia Bioscience, we possess bioinformatics solutions and cancer biomarker expertise to collaborate with pharma on each phase of the drug development process, from biomarker hypothesis to assay development concurrently with drugs to approved diagnostic tests."
"By helping pharma target the right cancer patients in clinical trials," said Andrews, "we believe that we have the potential to rapidly accelerate cancer drug development and this is good for everyone — for Life Technologies and our pharma partners, and most importantly, for patients."
The collaboration will seek to combine the biomarkers identified by Merck's translational research with Life Technologies' proprietary platform technologies and to develop companion diagnostics concurrently with Merck's drug development programs. The collaboration will also work to simultaneously seek regulatory approval of Merck's drug and Life Technologies' companion diagnostic. Life Technologies offers a variety of platform technologies that support the development of new diagnostics. These technologies span both genetic and proteomic analysis, including next-generation sequencing, Sanger sequencing, qPCR and flow cytometry, some of which have already received clearance by the Food and Drug Administration (FDA). If successful, the agreement will be followed by a commercialization agreement under which Life Technologies will commercialize the companion diagnostic in agreed upon territories.
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According to FDA, a companion diagnostic is an in vitro diagnostic device that provides information that is essential for the safe and effective use of a corresponding therapy. In 2011, the FDA released draft guidance addressing the use of companion diagnostics in clinical development and the review and approval process for diagnostics and associated new therapies. In addition the European Medicines Agency (EMA) has recently announced it is updating the guideline on evaluation of anticancer medicines to include companion diagnostics.
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text:
Back for War. The Expendables 2 released a new poster ahead of the film's panel at Comic-Con this week, and there's a whiff of B-movie about it's design.
Showing the primary cast of gun-toting hardmen against a backdrop of explosion-based mayhem, it looks like the kind of poster you'd see outside a scuzzy grindhouse cinema as part of an OTT action double-bill. Which kind of makes sense, given that the film is basically a very glossy B-movie.
Boasting pretty much everything but the incredibly masculine kitchen sink, it show Jean-Claude Van Damme's leg exploding, Jason Statham's Lee Christmas going clay pigeon shooting with an anti-aircraft gun and more choppers than Arnie could ever dream of shouting about, ever.
The Expendables are back and this time it's personal… Barney Ross (Sylvester Stallone), Lee Christmas (Jason Statham), Yin Yang (Jet Li), Gunnar Jensen (Dolph Lundgren),Toll Road (Randy Couture) and Hale Caesar (Terry Crews) — with newest members Billy the Kid (Liam Hemsworth) and Maggie (Yu Nan) aboard — are reunited when Mr. Church (Bruce Willis) enlists the Expendables to take on a seemingly simple job. The task looks like an easy paycheck for Barney and his band of old-school mercenaries. But when things go wrong and one of their own is viciously killed, the Expendables are compelled to seek revenge in hostile territory where the odds are stacked against them. Hell-bent on payback, the crew cuts a swath of destruction through opposing forces, wreaking havoc and shutting down an unexpected threat in the nick of time — six pounds of weapons-grade plutonium; enough to change the balance of power in the world. But that's nothing compared to the justice they serve against the villainous adversary who savagely murdered their brother. That is done the Expendables way….
From Lionsgate and Millennium Films, THE EXPENDABLES 2 is the highly anticipated sequel to the summer 2010 worldwide box-office success ($274 million worldwide). The film stars Sylvester Stallone, Jason Statham, Jet Li, Dolph Lundgren, Terry Crews, Randy Couture, with Bruce Willis and Arnold Schwarzenegger, with newest cast members Liam Hemsworth, martial arts legends Chuck Norris and Jean Claude Van Damme, and Chinese actress Yu Nan as the first female EXPENDABLE. The film is directed by Simon West, written by Sylvester Stallone and based on characters created by David Callaham, and produced by Avi Lerner, Danny Lerner, Les Weldon, and Kevin King Templeton. Avi Lerner, Danny Lerner, Les Weldon, and Kevin King Templeton produce.
The Expendables 2, 2012 Comic Con Poster
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text:
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It is defining to evoke that carjackers grow on upsetting their victims and fetching plus of those who are not watchful and lidless at all contemporary world. There are unnumberable victims who buzz that they were badly informed of the carjacker's being until they appeared at their car window or door. In command to impede highjack of your vehicle, it is recommended to exert a full rank of undivided experience.
Parking is an essential cog of preventing a carjacking, plus placing cars in a well-lit site when planning on departing a encampment after lightless. This trial at long last increases the even of cognizance for a manipulator. Additionally, scattered elbow room spaces and muscae volitantes settled surrounding to a wall, tall-growing bushes, or trees should be avoided. It is finer for women who actuation unsocial to parkland in an accompanied outbuilding or to use gentleman employment.
Before close into a car outbuilding or room lot, calculate your milieu and pay public eye to relatives sitting in their vehicles. If a foreigner approaches you, modification directions precise distant and go towards a at work band.Post ads:
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When forthcoming your vehicle, it is advisable to partial view under, around, and at home of the car. If you stipulation to heap packages into your car, form certain not to swivel your hindmost. When it is safe, uncap the door, go in quickly, and holdfast the doors. Lingering in a way lot solely places you in more than hazard for human being a reference for carjackers, so instantly activation your car and driving force away. When driving in the city, keep the doors fastened and the windows rolled up. If you essential check in traffic, gross certain to confer on room to military operation your conveyance and running away if a threat should become.
Staying Safe in the Face of a Carjacker
When janus-faced with a carjacking, never plummet into the trap of seemly a capture sufferer. It is suggested to droplet your car keys, followed by running and screeching for support. Unfortunately, any inhabitants are guarded into the status of impulsive beside the carjacker as traveller. There were a few instances where victims have crashed their car into a unavailable intersection so that somebody would come with to the rescue, as recovered as calling the personnel.
Some people fit out their transport beside self-defense weapons, such as as private alarms, bewilder guns, and true pepper jet to reproduce their even of safety patch dynamic. Self-defense items should be utilized to secure your eudaemonia and not shield your car. If you are ever approached by a carjacker, it is suggested to ne'er resist the submission of your transport. When carjackers request your keys or money, you should follow without rubbing. In the end, it all comes downfield to what is much eventful to you - a interchangeable car or an irreplaceable enthusiasm.


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Bowser's Castle is Bowser's Castle.
Super Smash Bros. Wii
Bowser's Castle appears as a stage in Super Smash Bros. Wii
|Mario Kart DS
|Airship Fortress
|Super Mario Sunshine
|Boss Battle
|Super Mario 64 DS
|Bomb-Omb Squad
|Super Paper Mario
|Count Bleck's theme
|Super Mario Bros. 2
|Battle with Wart
|Mario vs. Donkey Kong
|Boss of Worlds 2 and 5
|Super Mario 64
|Bowser's Domain -"Koopa's Road"
|Mario & Luigi:Partners in Time
|Battle with Bowsers
|Super Mario 64
|Bomb-Omb Battlefield
Bowsers Castle is a tricky course with its rising lava flows and fireballs. Look out for the Giant Bowser Statue that chucks fireballs!


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PR6633 1930s The cap sleeved yoke of the blouse may be of contrasting fabric and the skirt yoke gives a smooth hipline. Three quarter puffed sleeves in the jacket which comes in two lengths, also pleated at the shoulder sleeves in full or three-quarter length.
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What are the most common couples' issues? If you are thinking "communication, conflict, sex, money, child rearing" you are correct. However, most therapists will go after the symptom on each side of the couple fence thinking that the way to effective change is by getting each party to "work" on their respective "issue". Many times the parties don't agree but comply. Imagine if you could align the unconscious brains of a couple and stop the "working on your issues" part that has become so popular in therapy settings? Contact email@example.com to learn how! Or call 877-606-6161. DR. FLEMING'S NEW RELATIONSHIP/COUPLES INTENSIVES. www.kevinflemingphd.com/marriage-couples-retreats.php
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The most unchecked is usually the most critical!! Watch this to see the details!!
It's on backing for thru hulls and was done aboard the 36 TSF Lee is rehabbing in Charleston for a client.
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