text:
Showing sets of primers used to amplify the TNF-α and TNF-β to detect polymorphism
1. Introduction
Glaucoma is a complex disease that comprises a group of heterogeneous optic neuropathies characterized by a progressive degeneration of the optic nerve head and visual field defects. It starts with unnoticeable blind spots at the edges of field vision progressing to the tunnel vision and finally leading to blindness (Quigley & Broman, 2006). The disease process is insidious, and the central vision is usually not lost until the disease is advanced, a significant proportion of individuals remain either undiagnosed or undertreated. The most common forms of glaucoma are age-related, generally beginning in midlife and progressing slowly but relentlessly as the age advances (A.T.G.S.R.S.G., 2009). If detected early enough, disease progression can be slowed with drug and/or surgical treatment, emphasizing the importance of identifying the disease in its earliest stages.
Glaucoma is broadly classified into primary and secondary glaucoma based on their etiology and aqueous humor dynamics (Shields, 2005). Based on anatomy of the anterior chamber (gonioscopy), primary glaucomas are further classified as primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG). Depending on the time of onset, glaucoma is also termed as infantile, juvenile and adult type. Rarely glaucoma may be found in babies at birth this form of glaucoma called congenital glaucoma. Secondary glaucomas are characterized by the involvement of predisposing ocular or systemic diseases such as uveitis, trauma, or diabetes thereby resulting in an alteration of aqueous humor dynamics. Pseudoexfoliation glaucoma (XFG) and pigmentary glaucoma (PG) are the most frequently reported type of secondary glaucoma (Shields, 2005).
Glaucoma affects 70 million people and is the second leading cause of blindness worldwide. It is estimated that by the year 2020, this number would rise to around 79.6 million (Quigley & Broman, 2006). The prevalence of glaucoma varies widely across the different ethnic groups (He et al., 2006; Wong et al., 2006; Sakata et al., 2007; Cedrone et al., 2008; Vijaya et al., 2008a; 2008b; Pekmezci et al., 2009) and is significantly higher in blacks (4.7%) as compared to in the white (1.3%) population (Kwon et al., 2009). Primary open angle glaucoma is the predominant disease among whites and Africans (Tielsch et al., 1991; Klein et al., 1992; Rotchford & Johnson, 2002; Rotchford et al., 2003; Varma et al., 2004; Friedman et al., 2006; Sakata et al., 2007) while PACG is a major form of glaucoma in Asians (Hu et al., 1989; Jacob et al., 1998; Foster & Johnson, 2001; Casson et al., 2007). Epidemiological studies reveal that the prevalence of POAG around the world varies by about 2 orders of magnitude with the lowest estimates found among Eskimos (0.06%) residing in Alaska (Arkell et al., 1987) and the highest prevalence among African-derived people living in the Caribbean (7.1− 8.8%) (Mason et al., 1989; Leske et al., 1994). PACG is leading cause of the most of the bilateral glaucoma related blindness in Singapore, China, and India. It has been estimated that PACG blinds more people than POAG worldwide (Dandona et al., 2000; Foster et al., 2000). According to a recent study glaucoma is the major cause of blindness in Saudi Arabia. The prevalence of both POAG and PACG is higher in western region of Saudi Arabia as compared to other Asian countries (Eid et al., 2009). To date no national study has been undertaken to determine the exact prevalence of glaucoma in this country.
Elevated intraocular pressure (IOP) is a major risk factor in glaucoma which is supported by the fact that experimentally induced elevation of IOP leads to glaucoma in animals (Levkovitch-Verbin et al., 2002). The etiology of raised IOP and resulting glaucoma is not fully understood. Fluid formed by the ciliary body (aqueous humor) is removed by the trabecular outflow pathways, which includes the trabecular meshwork, the juxtacanalicular connective tissue, the endothelial lining of Schlemm's canal, and the collecting channels and the aqueous veins (A.T.G.S.R.S.G., 2009). The IOP is dependent on the rate of fluid removal, which under normal conditions matches the rate of formation. In most patients with glaucoma, the rate of fluid removal declines so that it no longer keeps pace with the rate of formation resulting in the increased IOP. The increase in IOP has been associated with genetic factors, age and oxidative stress (Tschumper & Johnson, 1990; Leske et al., 1995; Green et al., 2007). Although elevation of intraocular pressure (IOP) is often related to the optic nerve damage in glaucoma, factors other than IOP are likely to play a role in the pathogenesis of glaucomatous optic neuropathy, particularly in individuals with normal tension glaucoma (NTG).
The loss of retinal ganglion cells (RGC) is the typical pathology in glaucoma. Progressive loss of optic nerve axons and RGCs result in characteristic optic nerve atrophy and visual field defects in glaucoma patients. A number of hypotheses have been put forward that describe the sequence of events responsible for triggering ganglion cell degeneration in glaucoma. These include compromised blood flow of the optic nerve, mechanical compression due to raised IOP, loss of neurotrophic factors, autoimmune mechanisms, nitric oxide-induced injuries to the optic nerve and glutamate excitotoxicity (Mozaffarieh et al., 2008). A combination of these factors may be involved in causing glaucomatous RGC loss. Animal models also confirmed that acute IOP elevation causes blockage of brain-derived neurotrophic factor (BDNF) transport and may contribute to neuronal death (Pease et al., 2000). On the other hand the mechanisms involved in the pathophysiology and development of PACG are rather complicated and involve the anatomy of the angle, and the spatial and structural relationships between the lens and the anatomy of the angle, the iris, and the lens. Most mechanisms for PACG impute to the increasing lens thickness during aging in a relatively small eye and a shallow anterior chamber. Acute PACG has been attributed to morphometeric characteristics of the eye. Human sclera undergoes active remodeling during the development of myopia. Biochemical assays from highly myopic eyes show markedly reduced amounts of biochemical markers for collagen and glycosaminoglycans, when compared with the similar sclera of emmetropic eyes. This remodeling process occurs in concert with an increase in the production and enhanced activation of collagen degrading enzymes, particularly matrix metalloproteases (MMPs) (McBrien & Gentle, 2001). The net effect of these changes is a loss of scleral tissue at the posterior pole of the eye.
Growing evidence obtained from clinical and experimental studies strongly suggests the involvement of the immune system in glaucoma (Helpern & Grosskreutz, 2002; Wax & Tezel, 2002; Tezel & Wax, 2000; 2003). Paradoxically the role of immune system in Glaucoma has been described as either neuroprotective or neurodestructive. A balance between beneficial immunity and harmful neurodegeneration may ultimately determine the fate of RGCs in response to various stresses in glaucomatous eye (Tezel & Wax 2007). T-cell-mediated immune response may initially be beneficial to limit neurodegeneration (Schwartz & Kipnis, 2001, 2002; Kipnis et al., 2002). However, a failure to properly control aberrant, stress-induced immune response likely converts the protective immunity to an autoimmune neurodegenerative process that can facilitate the progression of neurodegeneration in some, if not all, glaucoma patients (Tezel & Wax, 2007). Expansion and secondary recruitment of circulating T cells through an antigen mediated process is supported by the evidence of abnormal T-cell subsets (Yang et al., 2001a) and increased production of serum autoantibodies to different optic nerve and retina antigens in many glaucoma patients (Tezel et al., 1998, 1999; Wax et al., 1998a, 1998b; Yang et al., 2001b) Furthermore, initial in vivo studies support the feasibility of eliciting an experimental autoimmune model of glaucomatous neurodegeneration in which RGCs progressively die in specific antigen-immunized animals by exhibiting a pattern of neuronal damage similar to that of human glaucoma (Wax &Tezel, 2009). As the role of the immune system in glaucoma is one of the surveillance, in which signal pathways of the immune system regulate cell death in response to conditions that stress retinal neurons in glaucoma. Recent studies have focused on immunological changes occurring in glaucoma pathogenesis and possible preventive therapies based along those lines have been proposed. The major targets of interest are cytokines, as these inflammatory mediators play an important role in the pathogenesis of glaucoma and may regulate RGC survival or death (Tezel & Wax, 2004).
The role of tumor necrosis factor (TNF), an important proinflammatory cytokine is a subject of interest in glaucoma studies. Both TNF-α, produced mainly by monocytes and activated macrophages and TNF-β, produced mainly by activated T-cells, play important immunoregulatory roles in various diseases. TNF-α is up regulated in several neurodegenerative disorders including optic nerve microglia and astrocytes in glaucoma (Yuan & Neufeld, 2000; 2001). It has been suggested that cell death mediated by TNF-α is a contributing factor in the progression of neurodegeneration in glaucoma (Tezel et al., 2001). TNF-α plays a critical role in optic neuropathy by initiating the inflammatory pathways through the induction of NOS-2 in astrocytes (Yuan & Neufeld, 2000). It has been observed that ischemic or pressure-loaded glial cells produce TNF-α which results in oligodendrocytes death and the apoptosis of RGC leading to glaucomatous neurodegeneration (Tezel & Wax, 2000). Furthermore, TNF-α directly induces apoptosis through TNF-α receptor-I (TNFRI) also known as death receptor which is mainly localized on the retinal ganglion cells (Tezel et al., 2001). The substantial role of TNF-α/TNFRI in the pathogenesis of glaucoma has also been supported by experimental study on mouse model of glaucoma in which induction of ocular hypertension resulted in a rapid up regulation of TNF-α followed by loss of optic nerve oligodendrocytes (Nakazawa et al., 2006). Moreover, intravitreal TNF-α injection in normal mice mimicked glaucoma like RGCs degeneration whereas anti-TNF-α neutralizing antibody or deleting the TNF-α gene blocked the deleterious effects of ocular hypertension (Nakazawa et al., 2006). Tezel et al. (2004) also reported that unilateral optic nerve crush injury in mice resulted in significantly less glial activation, TNF-α production and retinal ganglion cell death in TNFRI -knockout mice. In fact, histopathologic studies have also shown increased immunostaining for TNF-α and TNFRI in the glaucomatous optic nerve head as compared to age-matched control samples (Yan et al., 2000; Yuan & Neufeld, 2000). Interestingly, a new antiglaucoma drug (GLC756) significantly suppressed LPS-induced TNF-α production (Laengle et al., 2006a, 2006b). These observations further confirm the hypothesis of an involvement of the TNF-α /TNFRI signaling pathway in the pathogenesis of glaucoma.
The genes for TNF-α (OMIM 191160) and TNF-β also known as lymphotoxin-α (LT-α, MIM 153440), located within the MHC III region of chromosome 6, shows close linkage to the HLA class I (HLA-B) and class II (HLA-DR) genes. Studies on monozygotic twins and their first-degree relatives, using ex vivo endotoxin stimulated whole blood samples, have provided evidence that 60% of variation in the production capacity of TNF-α appears to be genetically determined (Westendorp et al., 1997). Several polymorphisms within the promoter region of TNF-α and the intron 1 polymorphism of TNF−β, in particular, have been associated with altered levels of circulating TNF-α (Sharma et al., 2006; 2008).
With regard to PACG a number of lines of evidence have suggested that both genetic and environmental factors contribute to the development of PACG (Lowe, 1972). There are various published studies that suggested a genetic basis of the PACG (Aung et al., 2008a; 2008b). PACG, as a common complex disease, has become an important target for association studies in recent years. Genetic heterogeneity is illustrated by the several loci in many glaucoma causing genes identified to date. Single nucleotide polymorphisms in several genes such as matrix metalloproteases-9 (MMP-9), Cytochrome P450 1B1 (CYP1B1), membrane-type frizzled-related protein (MFRP), methylenetetrahydrofolate reductase gene (MTHFR), myocilin (MYOC), calcitonin receptor-like gene (CALRL), endothelial nitric oxide synthase (eNOS), heat shock protein 70 (HSP70), retinal homeobox gene (CHX10), have been associated with PACG (Wang et al., 2006; Chakrabarti et al., 2007; Michael et al., 2008; 2009, Aung et al., 2008; Dai et al., 2008; Cong et al., 2009; Cao et al., 2009, Ayub et al., 2010). However, ethnic variations exist in the association between these polymorphisms and PACG, and many cases such association has not been replicated in subsequent research (Wang et al., 2006; Aung et al., 2008a). Moreover, the biological significance of the some associated single nucleotide polymorphisms (SNPs) in the development of PACG is still unknown (Michael et al., 2008). PACG has been reported to be associated with genes related to regulation of axial length and structural remodeling of connective tissues, such as MFRP, MMP-9, and MTHFR genes (Michael et al., 2008; Wang et al., 2006; Aung et al., 2008a; 2008 b; Wang et al., 2008). It has also been reported that TNF-α is capable of inducing both increased synthesis and activity of matrix metalloproteinases (MMPs) (Rajavashisth et al., 1999; Siwik et al., 2000; Uchida et al., 2000) therefore the polymorphism in TNF genes might be associated with regulation of axial length and structural remodeling of connective tissues and ultimately to the development of PACG.
Several polymorphisms have been reported in the promoter region of TNF-α gene. One of the best described single nucleotide polymorphisms (SNPs) is located at nucleotide position -308 within the TNF-α promoter region (rs1800629) and affects a consensus sequence for a binding site of transcription factor AP-2 (Abraham & Kroeger, 1999). TNF-α promoter polymorphism leads to a less common allele-A (allele 2) which has been associated with increased TNF-α production in vitro (Braun et al., 1996; Wilson et al., 1994) and higher rate of TNF-α transcription than wild type GG genotype (Wilson et al., 1997; Jeong et al., 2004). This polymorphism has been linked to increased susceptibility to several chronic metabolic degenerative, inflammatory and autoimmune diseases (Cuenca et al., 2001). It has also been reported to be involved in increased susceptibility to different eye diseases including diabetic retinopathy and glaucoma (Limb et al., 1999; Yoshioka et al., 2006; Huang et al., 2009). TNF-α ιs involved in T-cell dependent B-cell responses, T-cell proliferation, natural killer (NK)-cell activity and dendritic cell maturation. The previously published data indicate that the host's ability to produce TNF-α may play an important role in vulnerability to glaucoma.
Of interest, G/A polymorphism at nucleotide position –308 within the human TNF-α promoter region is associated with elevated TNF levels, disease susceptibility, and poor prognosis in several diseases (Messer et al., 1991; Wilson et al., 1992; Galbraith & Pendey, 1995; Patino-Garcia et al., 2000). Adenine at position –308 makes the TNF-α promoter a much more powerful transcription activator than guanine (Messer et al., 1991). The TNF-α is up regulated in several neurodegenerative disorders including multiple sclerosis, Parkinson's disease, Alzheimer's disease (Shohami et al., 1999; Yan et al., 2000; Liu et al., 2006) and in optic nerve microglia and astrocytes in glaucoma patients (Yuan & Neufeld, 2000; 2001]. Increased TNF-α level have been associated with a poor prognosis after trauma in the brain (Munoz-Fernandez & Fresno, 1998; Ertel et al., 1995), whereas a decrease in TNF-α is known to reduce nerve damage. In a rat cerebral ischemic model, exogenous TNF-α was found to exacerbate focal ischemic injuries, whereas blocking endogenous TNF-α was neuroprotective (Shohami et al., 1996).
TNF-β resembles to TNF-α in terms of several biological activities including apoptosis and gives rise to a similar pro-inflammatory response and has been shown to play a critical role in pathogenesis of many diseases. TNF-β gene polymorphism at nucleotide position 252 within first intron (A252G) (rs909253) affects a phorbol ester-responsive element. The presence of G at this position defines the mutant allele known as TNF-β * 1 (allele-1) which is less frequent allele in white subjects and is associated with higher TNF-α and TNF-β production (Messer et al., 1991; Abraham et al., 1993). Association of TNF-β +252 A/G polymorphism has been reported with various autoimmune disorders including Gravis' disease (Kula et al., 2001) idiopathic membranous glomerulonephritis, IgA nephropathy, insulin dependent diabetes mellitus (Medcraft et al., 1993), myasthenia gravis (Zelano et al., 1998), asthma diathesis (Albuquerque et al., 1998), SLE with nephritis(Lu et al., 2005), systemic sclerosis (Pandey &Takeuchi, 1999), plaque psoriasis (Vasku et al., 2000), rheumatoid arthritis (Takeuchi et al., 2005), myocardial infarction in patients with rheumatoid arthritis (Panoulas et al., 2008), and type 1 diabetes (Boraska et al., 2009). Recently TNF-β +252 A/G polymorphism is reported to be associated with both susceptibility to and mortality from sepsis (Watanabe et al., 2010; Tiancha et al., 2011).
A few studies have been undertaken to determine the association of TNF-α polymorphisms and glaucoma in different part of the world (Limb et al., 1999; Yoshioka et al., 2006; Huang et al., 2009). The results of these studies on association of TNF-α polymorphism with POAG are inconsistent. These differences in findings have been attributed to variation in sample size, ethnicity and method of diagnosis (Lin et al., 2003; Mossbock et al., 2006; Tekeli et al., 2008; Razeghinejad et al., 2009). To date there is no report available on the association between TNF-β polymorphism and glaucoma. The joint analysis of polymorphism at TNF-α and TNF-β given that both are involved in the expression of TNF-α and in a suggested mechanism for autoimmune diseases, will provide further insight into the pathogenesis of glaucoma and help in developing effective therapeutic agents. Therefore the present study was undertaken to determine the possible association of TNF-α-308 and TNF-β +252 gene polymorphisms with POAG and PACG in Saudi population.
2. Methods
2.1. Subjects
The present study was undertaken to evaluate the association of TNF-α and TNF-β alleles and genotypes in Saudi primary glaucoma patients. A total of 200 unrelated Saudi patients with Primary Glaucoma [primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG)] were recruited from ophthalmology clinic of the Riyadh Military Hospital (RMH), Saudi Arabia. The patient group consisted of 94 males and 106 females, with age at diagnosis ranging from 25 to 78 years (mean ± SD: 58 ± 14.4). The control group consisted of 200 unrelated subjects, 100 males and 100 females, ages ranging from 25 to 68 years (mean ± SD: 55 ± 11.6). The diagnosis of primary glaucoma was based on comprehensive clinical examination as mentioned below.
2.2. Preliminary examination
The preliminary examination was performed in ophthalmology clinic of RMH. Detailed demographic data and medical history were recorded for each case and control subject. The preliminary examination included oblique flashlight test, anterior segment evaluation by slit lamp, Goldmann applanation tonometry, and direct fundoscopy (all examinations performed by resident/consultant ophthalmologists). The optic disc was examined after each pupil was dilated with 2 drops of tropicamide, unless contraindicated by the presence of a shallow anterior chamber depth at the slit lamp examination. The vertical cup-disc ratio (VCDR) was estimated for each eye. The rim border was determined based on the course of the blood vessels and the gradation of color, shadows, and texture (Spaeth, 1993). A suspect appearance of the optic disc (glaucomatous-appearing optic disc; GAOD) was defined in eyes with VCDR ≥ 0.6, asymmetry of the VCDR between the two eyes ≥ 0.2, focal thinning of the neuroretinal rim, localized or diffuse retinal nerve fiber layer defect, and/or optic disc hemorrhage. A glaucoma specialist confirmed the presence of a GAOD in the participants. Fundus photography, automated perimetry, and gonioscopy were not part of the preliminary examination protocol.
2.3. Detailed examination
Patients with GAOD status and/or intraocular pressure (IOP) measurements > 21 mm Hg at the screening underwent a definitive examination. At first each subject underwent a comprehensive ophthalmic examination, including review of medical history, best corrected visual acuity(BCVA) (using Snellen distance vision chart), retinoscopy and subjective refraction (when visual acuity 20/30), slit lamp biomicroscopy, IOP measurement (using Goldmann applanation tonometry), standard automated perimetry tests, gonioscopy (using Sussman four-mirror lens), and fundoscopy examination. Gonioscopy was performed by a consultant (glaucoma specialist). Indentation gonioscopy with four-mirror Posner lens was performed in eyes with an anatomically narrow angle. The angle was graded according to the Scheie scheme (Scheie, 1957) and the peripheral iris contour, degree of pigmentation, presence of peripheral anterior synechiae, and other angle abnormalities were recorded. When the gonioscopy showed no contraindications, dilated direct fundoscopy and slit lamp biomicroscopy of the optic disc (using a 78-D lens) were performed. The VCDR was determined independently in a masked fashion by two glaucoma specialists and cases of disagreement were resolved by consensus between the two graders during the definite examination visit. There was a good agreement between the two graders in determining VCDR (0.7 or more). The presence of any notching, optic disc hemorrhages, or nerve fiber layer defects was documented. Initially, most of the standard automated perimetry tests were performed with the Octopus 101 perimeter (Haag-Strreit Octopus, Switzerland). In an attempt to avoid the influence of learning effect in visual field (VF) results, these VF examinations were not considered for assessing visual function status.
The patients were further subjected to a second VF examination using a Humphrey visual field perimeter (Humphrey Visual Field Analyzer; Carl Zeiss Meditec, Inc., Dublin, CA), with the 24-2 full-threshold or SITA (Swedish interactive threshold algorithm) standard strategy. The visual function status was determined based on this single VF test, and only reliable VF examinations were considered for analysis. An abnormal VF examination was determined by the presence of one of the following criteria: (1) glaucoma hemifield test (GHT) result outside normal limits and (2) the presence of a cluster of ≥ 3 contiguous points in the pattern deviation (PD) probability plot with P 5% or worse (within the same hemifield) (Foster et al., 2002). A reliable visual field test was defined as an examination with less than 33% of fixation losses, false positive and false negative. The glaucoma specialist verified whether the VF defects were consistent with glaucoma.
2.4. Criteria for glaucoma diagnosis
Glaucoma was diagnosed according to the International Society of Geographical and Epidemiologic Ophthalmology (ISGEO) classification, which uses three levels of evidence (Foster et al 2002). Briefly, in category 1, diagnosis was based on structural and functional evidence. It required CDR or CDR asymmetry ≥ 97.5th percentile for the normal population or a neuroretinal rim width reduced to ≥ 0.1 CDR (between 11- and 1-o'clock or 5- and 7-o'clock) with a definite VF defect consistent with glaucoma using the Swedish interactive threshold algorithm 30-2. Category 2 was based on advanced structural damage with unproved field loss. This included those subjects in whom VFs could not be determined or were unreliable, with CDR or CDR asymmetry ≥ 99.5th percentile for the normal population. Lastly, category 3 consisted of persons with an IOP ≥ 99.5th percentile for the normal population, whose optic discs could not be examined because of media opacities.
2.5. Glaucoma classification
Participants who fulfilled any of the three categories of evidence mentioned earlier were classified as having POAG or PACG
POAG: Anterior chamber angles open and appearing normal by gonioscopy, typical features of glaucomatous optic disc as defined earlier, and visual field defects corresponding to the optic disc changes.
PACG: At least two of the criteria mentioned: glaucomatous optic disc damage or glaucomatous visual field defects in combination with anterior chamber angle partly or totally closed, appositional angle closure or synechiae in angle, absence of signs of secondary angle closure (e.g., uveitis, lens related glaucoma, microspherophakia, evidence of neovascularization in the angle and associated retinal ischemia or congenital angle anomalies). Patients with signs of intracranial disease that would cause optic nerve atrophy in x-ray, computerized tomography or magnetic resonance imaging were excluded. An anatomically narrow-angle eye should have pigmented trabecular meshwork not visible in ≥ 270° of the iridocorneal angle (as assessed by nonindentation gonioscopy) and/or the presence of peripheral anterior synechiae not explained by other causes but a primary angle-closure process.
2.6. Sample collection
Venous blood was collected from the patients (POAG, PACG) and controls and stored at - 20C before extraction of DNA. The study protocol was approved by the Ethics Committee of the Hospital.
2.7. Genotyping
2.7.1. PCR amplification
Genomic DNA was extracted from the peripheral blood of patients and controls using QIAampR DNA mini kit (Qiagen CA, USA). TNF-α and TNF-β genes were amplified using amplification refractory mutation systems (ARMS)-PCR methodology (Perrey et al., 1999) to detect any polymorphism involved at position -308 of TNF-α and +252 in Interon1 of TNF-β gene respectively. The set of primers used to amplify target DNA in the promoter region of TNF-α and TNF-β genes are summarized in Table 1.
|Generic (antisense) primer
|Sense primers
PCR amplification was carried out using Ready to Go PCR Beads (Amersham Biosciences, USA). Reaction consisted of 10 temperature cycles of denaturation for 15 s at 94 °C, annealing for 50 s at 65 °C and extension for 40 s at 72 °C. Then 25 cycles of denaturation for 20s at 94 °C, annealing for 50 s at 59 °C, extension for 50s at 72 °C. Final extension was performed at 72 °C for 7 m. A positive control was included in the PCR assay by amplification of the human growth hormone (HGH) gene. The amplified product for various samples were separated on the 1.5 % agarose gel, stained with ethidium bromide and photographed.
2.7.2. Statistical analysis
The differences in allele/ genotype frequencies between patients and controls were analyzed by the Fisher's exact test. P values less than 0.05 were considered significant. The strength of the association of disease with respect to a particular allele/genotype is expressed by odd ratio interpreted as relative risk (RR) following the Woolf's method as out lined by Schallreuter et al. (1993). It is calculated only for those Alleles/ genotypes which are increased or decreased in patients as compared to control group. The RR in this study has been calculated for all the subjects.
(a) = number of patients with expression of allele or genotype
(b) = number of patients without expression of allele or genotype
(c) = number of controls with expression of allele or genotype
(d) = number of controls without expression of allele or genotype
Etiologic Fraction (EF): The EF indicates the hypothetical genetic component of the disease. The values between 0 and 1 are of significance. EF is calculated for positive association only where RR> 1 (Savejgaard et al., 1983).
Preventive Fraction (PF): The PF indicates the hypothetical protective effect of one specific allele/ genotype for the disease. PF is calculated for negative association only where RR 1 (Savejgaard et al., 1983). Values 1.0 indicate the protective effect of the allele/genotype against the manifestation of disease.
3. Results
In this case control study TNF-α and TNF-β genes were amplified to determine the allele and genotype frequencies in equal number of patients (200) and unrelated matched controls (200). The patient group consisted of POAG (135) and PACG (65) cases. The molecular analysis of the blood specimen of the patients and controls was performed in the same laboratory and at the same time. The investigator was blind to the phenotype of the subjects at the time of molecular analysis. Later on the results were separated for patient and control groups and analyzed for the determination of the frequencies of genotypes and alleles. Allelic frequencies and genotype distributions of both TNF-α and TNF-β gene polymorphisms differ between primary glaucoma (including both POAG and PACG cases) and control subjects (Table 2 - Table 9).
The results of the genotypes and alleles distribution of TNF-α (-308) polymorphism in primary glaucoma (including total POAG and PACG cases) are summarized in Table 2. The frequency of GA (-308) genotype was significantly higher (P=0.02) while the frequency of GG (-308) genotype was lower in total primary glaucoma patients as compared to controls (P=0.01).
|Genotype/ Allele
|Glaucoma (N=200)
|Control (N=200)
|P- value
|G-allele (TNF-α 1-allele)
|A-allele (TNF-α 2-allele)
No significant difference was noticed in distribution of AA genotype among the patients and controls (P=0.85). Frequency of allele A was found to be significantly increased in glaucoma patient while that of allele G was higher in control group (P=0.04).The higher frequencies of genotype GA and allele A in primary glaucoma patients as compared to controls indicated that the genotype GA and allele A are susceptible to the primary glaucoma (RR=1.63, EF=0.22 and RR=1.37, EF=0.15 respectively). The genotype GG and allele G being higher in controls indicating their protective nature for primary glaucoma (RR=0.60, PF=0.23 and RR=0.73, PF=0.15 respectively).
The results of TNF-α -308 polymorphism for total primary glaucoma cases were then stratified into POAG and PACG groups. The distribution of alleles and genotypes of TNF-α (-308) polymorphism in POAG are shown in Table 3.
|Genotype/ Allele
|Open Angle Glaucoma (N=135)
|Control (N =200)
|G-allele (TNFα 1- allele)
|A-allele (TNFα 2 -allele)
The frequency of genotype GA was significantly higher in POAG patients (P=0.03) while the frequency of GG was lower in POAG as compared to controls (P=0.04). The frequency of allele A was higher whereas the frequency of allele G was lower in PAOG patients as compared to control. However, the differences were not statistically significant for both alleles (P=0.16). The higher frequency of GA genotype in POAG indicated that the genotype GA of TNF-α -308 polymorphism might be susceptible to POAG (RR=1.66, EF=0.19) while the increased frequency of genotype GG in controls as compared to POAG patients indicated that genotype GG is resistant to POAG (RR=0.64,PF=0.16).
The frequencies of alleles and genotypes of TNF-α (-308) polymorphism in PACG is given in Table 4. The frequency of genotype GG was significantly lower in PACG as compared to controls (P=0.04). Although the frequency of genotype GA and AA was higher in PACG, the difference was not statistically significant (P=0.11 and P=0.42 respectively). The frequency of allele A was significantly increased in PACG as compared to controls (0.04) indicating that the allele A might be susceptible to the PACG (RR=1.56, EP= 0.11) while allele G might be protective (RR=0.64, PF=0.11) in Saudi patients with PACG. Similarly the lower frequency of genotype GG in PACG as compared to controls indicated that GG is protective for PACG (RR=0.55, PF=0.14).
Comparison of the distribution frequency of genotypes and alleles of TNF-α (-308) polymorphism between the POAG and PACG type of glaucoma is summarized in Table 5. The results of the genotype and allele distribution in the two types of glaucoma (POAG and PACG) show similar pattern without insignificant differences.
|Genotype/ Allele
|Angle closure glaucoma (N=65)
|Control (N=200 )
|P- value
|G-allele (TNFα 1-allele)
|A-allele (TNFα 2-allele)
|Open angle glaucoma (135) N ( % )
|Angle closure glaucoma (65)
|59 (43.7)
|26 (40)
|68 (50.4)
|32 (49.2)
|8 (5.9)
|4 (10.8)
|G-allele (TNFα 1-allele
|186 (68.9)
|84 (64.6)
|A-allele (TNFα 2-allele)
|84 (31.1)
|46 (35.4)
Our studies on TNF-β gene polymorphism at position +252 of intron1 showed that the frequency of GG was significantly higher in primary glaucoma (including total POAG and PACG cases) as compared to controls (P=0.001) while the frequency of GA genotype was significantly lower in glaucoma patients (P=0.001). Allele-G was predominantly distributed in glaucoma patients (P=0.001) whereas frequency of allele A was significantly higher in control group (P= 0.011). Though there was difference in the distribution of AA genotype among the primary glaucoma and control, the difference was not statistically significant (P=0.25) (Table 6).
|Glaucoma (N=200)
|Control (N=200)
|G-allele (TNFβ 1-allele)
|A-allele (TNFβ 2-allele)
The increased frequencies of genotype GG and allele G of TNF-β (LTα) - interon1 +252 polymorphism in patients group indicated that genotype GG/allele G is susceptible to the primary glaucoma (RR=3.235, EF=0.491and RR=1.396, EF=0.152 respectively) whereas the decreased frequencies of the genotype GA and allele A in glaucoma patients showed their resistant/ refractory nature (RR=0.344, PF=0.432 and RR=0.715, PF=0.52 respectively).
On stratification of results for two types of glaucoma (POAG and PACG), the distribution of genotypes and allele of TNF-β (LTα) - interon1 +252 polymorphism retained almost the same pattern in POAG and PACG as was in the results for the total primary glaucoma group (Tables 7, 8). The frequency of genotype GG and allele G were higher in POAG as compared to controls (P=0.001 and P=0.05 respectively) while the frequencies of genotype GA and allele A were higher in controls (P=0.001 and P=0.05 respectively).
The increased frequencies of genotype GG (RR=3.28, EF=0.435) and allele G (RR=1.376, EF=0.119) in POAG as compared to controls (Table 7) indicated that genotype GG and allele G are susceptible to the POAG in Saudis. The reduced frequencies of genotype GA (RR=0.326, PF= 0.386) and allele A (RR= 0.726, PF=0.119) in the POAG patients as compared to controls showed that genotype GA and allele A of TNF-β intron1+252 polymorphism are resistant for POAG.
|Genotype/ Allele
|Open angle glaucoma (N=135)
|Control (N=200)
|G-allele (TNFβ 1-allele)
|A-allele (TNFβ 2-allele)
The distribution of genotypes and alleles of TNF-β intron1+252 polymorphism is shown in Table 8. The frequency of genotype GG was higher in PACG as compared to controls (P=0.001) while the frequencies of genotype GA was higher in controls (P=0.001). The higher frequencies of genotype GG in PACG as compared to controls indicated that genotype GG is susceptible to the disease (RR=3.142, EF=0.299) while genotype GA might be refractory for PACG as the frequency of GA was higher in controls RR=0.385, PF=0.229). The frequency of allele G was also higher in PACG and that of allele A was lower in PACG as compared to controls however, the difference in the frequency distribution of allele A and G was not significant in PACG (P=0.08).
Comparison of the distribution frequency of genotypes and alleles of TNF- β (LT-α) interon 1+252 polymorphism between the POAG and PACG is summarized in Table 9. The results clearly indicated that the genotype and allele distribution in the two types of glaucoma (POAG and PACG) have similar pattern with minor insignificant differences.
|Angle closure glaucoma (N=65)
|Control (N=200)
|G-allele (TNFβ 1-allele)
|A-allele (TNFβ 2-allele )
|Open angle glaucoma (135)
|Angle closure glaucoma (65)
|47 (34.81)
|22 (33.85)
|65 (48.15)
|34 (52.30)
|23 (17.04)
|9 (13.85)
|G-allele (TNFβ 1-allele)
|159 (58.89)
|78 (60.00)
|A-allele (TNFβ 2-allele)
|111 (41.11)
|52 (40.00)
The comparison between the associations of TNF-α 308 polymorphism in glaucoma patients in various ethnic populations is given in Table 10. The distribution trend shows ethnic variations. TNF -α (-308) polymorphism is strongly associated with POAG in Saudis, Chinese and Iranian but not in Caucasians and Japanese.
4. Discussion
As early as 1873, Dooremal for the first time observed that the eye is one of the immune privileged region of the body which was later described by Streilein (1999). Multiple factors have been suggested to contribute to the immune privilege of the eye (Niederkorn, 2006) such as the blood- aqueous- barrier and the fact that eyes do not have a lymph drainage system. Aqueous humor is for example, capable of suppressing cytokine production by activated T-lymphcytes (Mochizuki et al., 2000).
The anterior chamber of the eye has an active immunomodulation (Streilein, 2003). It has also been suggested that autoimmune damage to the optic nerve may occur directly by autoantibodies or indirectly by a "mimicked" autoimmune response to a sensitizing antigen, which in turn injures retinal ganglion cells (Wax, 2000). In a study by Joachim et al. (2007) IgG antibody levels were found to be significantly higher in the aqueous humor of patients with POAG and PEXG as compared to controls, suggesting the role of multiple immune factors in glaucoma.
|No. of
|TNF –α (308) polymorphism
|Present study
|Primary glaucoma
|Associated with TNF –α-308 GA
|Lin et al., 2003
|Associated with TNF –α-308 AA
|Fan et al., 2010
|Associated with TNF –α-308G
|Razeghinejad et al., 2009
|Associated with TNF –α-308A
|Funayama et al., 2004
|No association
|Mossbock et al., 2006
|No association
Growing evidence obtained from clinical and experimental studies over the past decade strongly suggests the role of immune system in the pathogenesis of various types of glaucoma. It has been observed that immune mediators are not usually the primary causative agents but play a critical role in the progression of the disease (Funayama et al., 2004; Joachim et al., 2007). In addition, it has been reported that components of the immune system involved in the pathogenesis of glaucoma are also involved in neurodegeneration following brain injury. In such cases inflammation occurs in response to glutamate, reactive oxygen species (ROS), nitric oxide (NO), and cytokines including tumor necrosis factor-α (TNF-α), which are released from activated microglia or macrophages (Schwartz, 2007). As glaucoma is a disease of old age and involves optic nerve neuropathy, it has been proposed that both genetic as well as epigenetic factors are involved in the progression of the disease. Such factors cause a decrease in the cellular viability and self renewal capacity, which results in the generation of dysfunctional microglia. Such age-related attrition may contribute to the development of neurodegenerative diseases by diminishing glial neurosupportive functions. Secondary degeneration by the immune components leads to the neurodegenerative injury in glaucoma (Levkovitch-Verbin et al., 2003).
TNF-α is considered to be a neuroprotective component of the immune system, because it activates the ubiquitous transcription factor NF-κB through binding to the high affinity TNF receptors (TNF-R2), which in turn mediates the expression of a wide range of genes essential for neuronal survival. Contrary to its neuroprotective role, TNF-α can also serve as a neurodegenerative factor when it binds to the low affinity death receptors TNF-R1 and activates the mitochondria mediated apoptotic pathway (Lilienbaum & Israel, 2003; Marchetti et al., 2004, Tezel & Yang, 2005). Thus a delicate balance between the two pathways determines the survival of the cell, and any shift in equilibrium might have deleterious effects. An increased expression of TNF-α can shift the balance toward TNF-R1 signaling, as seen in glaucoma, and thus promote retinal ganglion cell death.
In the present study we found a strong association of GA genotype of TNF-α (-308) polymorphism with primary glaucoma, suggesting its role in the pathogenesis of the glaucoma. The results of our study showing the association between TNF-α-308 polymorphism and glaucoma are in agreement with some of the published data on Asian populations. A recent study from Iran showed that inheritance of high producer TNF -α-308 A allele is susceptibility factor for development of POAG (Razeghinejad et al., 2009). A highly significant association of POAG and TNF -α-308 AA genotype has also been observed in the Chinese population (Lin et al., 2003). These investigators suggested that the close association between TNF-α 308 A/AA and POAG could be used as genetic marker for the disease mapping and identifying subjects susceptible to glaucoma. On the other hand in another study from China the frequency of allele G of TNF-α 308 was higher in patient group than controls and it was suggested that variants in TNF were risk factors for POAG in the Chinese population (Fan et al., 2010). The variations in association of TNF-α(-308) polymorphism with POAG (Table 10) could be due to racial differences, sample size, poorly characterized controls and clinical heterogeneity between different samples. Khan et al. (2009) found a close association between TNF-α 308 polymorphism and pseudoexfoliation glaucoma (PEXG, a type of secondary glaucoma) in Pakistani population. These findings clearly suggest that transcriptional regulation of TNF-α is essential to circumvent the deleterious effects of over expression by transcriptional up regulation. The results of this study clearly suggest that excessive production of TNF-α associated with the G-308A polymorphism may have an important role in the development of glaucoma and may act as a genetic susceptibility factor driven by a high TNF-α expression, which would subsequently lead to immune responses causing the onset and /or progression of the disease. This hypothesis is further supported by the findings of several earlier investigators showing upregulation of the expression of TNF-α and TNF-α receptor-1 in the retina and optic nerve head in glaucomatous eyes (Yan et al., 2000; Tezel et al., 2001; Yuan & Neufeld, 2000). Agarwal et al. (2000) reported that the G to A transition at position −308 results in a six- to sevenfold increase in transcription of TNF-α as compared to normal basal level. Similarly Abraham & Kroeger (1999) demonstrated the functionality of TNF-α-308 polymorphism in the reporter gene assays with a significant up regulation of up to five fold in the constructs of TNF1 (G-allele) and TNF2 (A-allele). A recent study by Sawada et al. (2010) demonstrated that TNF-α levels were significantly elevated in the aqueous humor of glaucoma patients as compared to controls.
Contrary to our findings on Saudi population, studies on Japanese and Austrian Caucasian populations showed no association between TNF-α polymorphism G-308A and primary glaucoma (Funayama et al., 2004; Mossbock et al., 2009). These differences may be attributed to the variation in ethnicity, sample size, poorly characterized controls and clinical heterogeneity of the patients. However, Funayama et al. (2004) noticed a definite interaction between polymorphism in Optineurin (OPTN) and TNF-α genes that would increase the risk for glaucoma in Japanese which indirectly suggest the participation of immune mechanism in glaucoma. The variation in Japanese population to some extent may be attributed to the fact that G-308 A polymorphism is very rare in this population (Allen, 1999).
The role of immune system in glaucoma is further substantiated by the fact that TNF-α mediated activation of matrix metalloproteinases (MMPs) in aqueous humor of the eye results into optic neuropathies. Several in vitro studies have shown that TNF-α is capable of inducing both increased synthesis and activity of MMPs (Rajavashisth et al., 1999; Siwik et al., 2000; Uchida et al., 2000). The MMPs are a family of zinc-dependent endopeptidases that have been reported to cause the degradation of the extracellular matrix (ECM) (Stamenkovic, 2003) and involved in wide range of normal and pathological conditions. Uncontrolled activation of MMPs is counterbalanced by specific tissue inhibitors of metalloproteinases (TIMPs), and a delicate balance of MMPs and TIMPs is required for physiologic ECM turnover. An impaired balance between MMPs and TIMPs may thus contribute to the development of glaucoma (Schlötzer-Schrehardt et al., 2003; Määttä et al., 2005). The higher frequency of Allele-A in our patients clearly suggest increased secretion of TNF-α which lead to increased synthesis and activity of MMPs. Increased expression of MMPs has been associated with structural remodeling of connective tissues of eye which might lead to the development of PACG (Papp et al., 2006). Further studies are warranted to confirm the role of MMPs in TNF-α mediated genesis of glaucoma and other eye diseases.
In our study the GG genotypes of TNF-β (+252) polymorphism were significantly over-represented in glaucoma patients as compared to the controls (P= 0.001) while GA genotype was significantly higher in controls as compared to glaucoma patients (P=0.001) indicating that GG, genotypes at +252 were susceptible to glaucoma (RR=3.235, EF=0.491), whereas GA was found to be refractory (RR=0.344, PF=0.432). Similarly the higher frequency of allele-G of TNF-β (+252) polymorphisms in patients (P=0.011) indicated that Allele G is associated with glaucoma. TNF-β (+252) polymorphism has earlier been reported to be associated with a number of autoimmune diseases (Albuquerque et al., 1998; Zelano et al., 1998; Pandey et al., 1999; Vasku et al., 2000; Kula et al., 2001; Lu et al., 2005; Takeuchi et al., 2005; Panoulas et al., 2008; Boraska et al., 2009; Watanabe et al., 2010; Tiancha et al., 2011) however, this is the first report on the association between TNF-β polymorphism and glaucoma. The presence of allele G at +252 position defines the mutant allele also known as TNF-β* 1 (allele-1) is reported to be associated with higher TNF-α and TNF-β production (Messer et al., 1991; Abraham et al., 1993). Therefore the higher frequencies of allele G and genotype GG of TNF-β (+252) along with increased genotype GA of TNF-α (308) polymorphism might act in tandem to increase TNF- α secretion in ocular tissue of the patients that might lead to the development of glaucoma.
Several studies have shown the relationships between increased TNF-α levels and several ocular diseases including retinopathy (Doganay et al., 2002; Zorena et al., 2007). Sugita et al. (2007) detected significantly elevated levels of TNF-α and TNF-α receptors in the ocular fluid of patients with active uveitis. In an animal model of high intraocular pressure, elevation of TNF-α precedes the loss of retinal ganglion cells and oligodendrocytes. In addition, retinal cell degeneration has been observed by administering of TNF-α even without the elevated intraocular pressure (Nakazawa et al., 2006). TNF-α contributes to this process by adversely affecting oligodendrocytes (Butt & Jenkins, 1994) which increases the susceptibility of axons to excitotoxicity in the optic nerve head and retinal ganglion cell death (Coleman, 2005). It has been observed that both mRNA and protein levels of TNF-α or TNF-α receptor-1 (TNF-R1) are raised in the retina of glaucomatous eyes as compared to normal eyes, and therefore it was suggested that cell death mediated by TNF-α is a contributing factor in the neurodegeneration in glaucoma (Tezel et al., 2001). Using animal models, Nakazawa et al. (2006) showed that ocular hypertension or increased intraocular pressure induces TNF-α upregulation in the retina, which in turn leads to RGC degeneration. It has also been observed that anti-TNF- α antibodies can prevent death of RGCs suggesting that reducing the expression of TNF- α would be beneficial in treating glaucoma.
5. Conclusion
This study clearly showed that the TNF-α (-308) and TNF-β (+252) polymorphisms are significantly associated with the susceptibility to primary glaucoma (POAG and PACG) in Saudis and could be used as a genetic marker for disease mapping. To the best of our knowledge this is the first study showing an association of TNF-β (+252) polymorphism with POAG and PACG. Immune hypotheses of glaucoma go very well with the findings of this study performed on the polymorphisms of TNF-α and of TNF-β genes. These cytokines might play a crucial role in the signaling glaucomatous neurodegeneration. The TNF-α (G-308A) and TNF-β (A+252G) polymorphisms alter and modulate the production of cytokines such as TNF–α which is consistently proposed to be involved in the RGC degeneration. Moreover, TNF has been found to be associated with the regulation of glial cells and stimulation of the synthesis and secretion of nerve growth factors (NGF).Therefore understanding the role of gene polymorphism like TNF-α (-308) and TNF-β (+252) in various ethnic populations could be helpful in predicting the predisposition to glaucoma and might help in developing novel therapeutic strategies for the management of this disease.


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Angry. Hurt. Resentful because of one's bad experiences or a sense of unjust treatment.
I think that everyone has felt this way at some point in their life. I think it is one of the worst human emotions that we could expierence. It can drive people to extreme measures at the drop of a hat. Maintaining a bitterness or a grudge inside you is equivalent to drinking a toxin and expecting it to affect another person.
I am guilty of holding grudges and harboring bitterness and resentment. I don't need a jury to inform me of that. I've been through many events that should have destroyed me. The part that eats away at me from time to time is the thought that each experience that went sour is usually worse than the previous one. I'm slowly improving my negative outlook on the military and certain people in my past that have been malicious. Forgiving others and letting bygones be bygones is a hard pill to swallow. I wish that I could be the type of woman that maintains her lady-like demeanor in the face of monstrosity.
I just can't be.
I just apologize for losing my temper and cut all ties with the offender. They cease to exist to me. Eventually, the rage inside me subsides and their past offenses don't cause me to become spitting mad anymore. Moving on with life feels good. It makes me happy. I am so close to moving onto the next chapter of my life.
Unfortunate, there are some events that have occurred to me that will probably cause me to remain bitter. Thank you, Uncle Sam for that. It's a pleasure.
Yea, writing this has made me feel a little salty. What's y'alls advice for removing the bitterness from your life?
via Daily Prompt: Bitter

subset: commoncrawl


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who better to lead an otherworldly stoners caravan than the goddess of pop herself? this shirt takes the classic stoner metal sleep baseball shirt, & puts cher at the head of the pack.
high-quality vinyl stickers meant for indoor or outdoor use. measures appx. 3"x3.5".
*stickers are shipped via usps mail with no tracking unless purchased with a larger item. please let me know if you would like tracking for an additional fee.*


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Herbed Parmesan Popcorn with Brazil Nuts
Make your own seasoned popcorn for an anytime snack. Ours uses rosemary and garlic powder and, of course, Parmesan, for a salty, cheesy flair.
Make your own seasoned popcorn for an anytime snack. Ours uses rosemary and garlic powder and, of course, Parmesan, for a salty, cheesy flair.
This fiery hot sauce is fantastic served as a dip for oven-roasted chicken fingers, on the side of broiled or grilled steak, spread sparingly on sandwiches and burgers or mixed with whole-wheat couscous studded with chickpeas, chopped dried apricots, tomatoes and scallions.
Fresh dill and Dijon pack this turkey scallopine with mouthwatering flavor, while kale provides both a fiber and antioxidant boost. It's a balanced meal with less than 200 calories per serving!
Smaller spring potatoes, also known as new potatoes, tend to be sweeter, making this an ideal time of year to try a variety other than America's beloved Russet (otherwise known as the Idaho potato). Our Herbed New Potatoes is a side that's both simple and elegant.
Top Chef's Angelo Sosa credits his Dominican father for his adventurous cooking style. His beet carpaccio is drizzled with herb-laced Italian honey and a dollop of Greek yogurt.
Texan chef Mark Paul's exclusive recipe for the traditional, slow-cooked Italian favorite replaces veal shanks with lamb, offering up a warming dish that's sure to elicit more than a few smiles!
Serve this party-perfect greek feta dip with veggies, as a spread for burgers and sandwiches, or with chicken or veggie skewers.
The soufflé has a reputation for being fussy, but the truth is that anyone can whip up this impressive dish. With reduced egg yolks and just a tablespoon of extra-virgin olive oil, this soufflé also fits comfortably into your healthy lifestyle.
A fricassee is a dish made by browning meat, sprinkling it with flour and briefly stewing it, creating a sauce (often creamy) in the process. We've streamlined the method and cut out the fat, so the flavor of fresh tarragon is at the forefront.
Tangy and sweet goat's milk chèvre forms the savory, calcium-rich base of this visually appealing appetizer.


text: We offer a comprehensive woodworking machinery refurbishing service for a variety of machines. Our service is in depth and tailor made to our customers requirements. Drawing on our experienced technicians skills, our service is second to none. The refurbishing of wood working machinery is an environmental sound and cost effective way of investing in machinery or prolonging machinery longevity. We only use the finest European components, materials and parts. Call on 01706 873344 for more information.


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The following pages display my rental units - both long-term rentals in Gardena, California, and short-term (seasonal) rentals in Palm Springs, California.
Click on ABOUT GARDENA for information on the city of Gardena, California and the surrounding cities such as Torrance, Redondo Beach, Hermoda, and Manhattan Beach. For information on my rental units in Gardena, click on FOURPLEX to view photos and to read about amentities offered on the different four (4) units - from a 1 bedroom, 1 bath to 3 bedrooms, 2 1/4 bath townhouse. For more information on my smaller property, which many have said, "feels like a house," click on DUPLEX. Both units offer off-street parking (garages and/or carports), laundry hook-upps (duplex) or on-site laundry (fourplex). Some units come with storage units.
If you are looking for a relaxing escape to the desert in Palm Springs, click on MESQUITE COUNTRY CLUB which offers stunning views of the golf course and mountains from the unit, pools and spas, and golf course. For a well-maintained, mid-century 1958 building, look no further than DESERT SKIES, located in the sought after area of Canyon Corridor in South Palm Springs, CA.
There is no smoking, including but not limited to, e-cigarettes, cigars, VAPS, marijuna (marijuna edibles OK), etc., and no pets - sorry, not even a "tiny dog." It makes for a more harmonious living situation with neighbors.
Renters must obtain renters insurance. Owner is not liable for damage to renter's personal belongings caused by, but not limited to, fire, flooding, earthquake, etc. or by any theft, vandalism, or damage, caused by the renter, family members, guest or others.


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Thousands of New Zealanders took to the streets this weekend to remember those close to us with dementia and to help make this a more dementia-friendly country.
Memory Walks were held in 21 locations around New Zealand as part of World Alzheimers Month. The aim is to raise further awareness and acceptance of dementia as a widespread condition.
A 2012 global survey of people who have been diagnosed with dementia (including New Zealanders) revealed that 40% experience being stigmatised by the condition. They report not being included in some aspects of everyday life, feeling marginalised by society, and experiencing loss of friends and isolation.
Twenty percent are so afraid of the stigma that they conceal their diagnosis from others.
"In this environment, dementia is often viewed with the same fear that used to accompany HIV or cancer," said Catherine Hall, Chief Executive, Alzheimers NZ.
"The Memory Walks are about changing that perception, because with the right awareness, acceptance and support services, people with dementia can lead fulfilling lives for many years", she said.
Champion for Dementia, Kerry Prendergast and New Zealand's former Sale of the Century television presenter Steve Parr, joined the Memory Walk in Wellington. It was Steve's first walk for dementia and he has flown from Brisbane specially to take part.
"My mother Jeannie is in the advanced stages of dementia and it has been heart-breaking to watch the effects on the gentle, but strong and independent mother I know," he said.
"Having now seen the shocking statistics on dementia – about 60,000 Kiwis have the condition and two in three people are affected by this in some way – I wanted to show my support and help raise awareness and reduce the stigma."
"There's so much we can do together to be more dementia friendly – we can reach out to people we know with the condition, we can include them in our lives and activities, we can focus on their abilities instead of their disabilities," said Catherine Hall.
Some of the people shown in photographs on the Alzheimers NZ website and social media channels are stock photography models.
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Ctrl + 1 Home
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Article Marketing Can Make You A Success – Here's How
February 20, 2018
Article marketing is an increasingly popular method of hosting keyword-rich articles on article websites that will then syndicate this content. Having keywords will get your website more views. This article will help you to learn the fundamentals of article marketing.
Use the DNF (do not follow) option on your webpage if allowing unrestricted users to comment on your pages. When users leave spam in comments (links to unrelated sites),the attribute for "no follow" will let the web crawlers know and the links will not be followed. This practice prevents you linking to sites which may and can affect your site's reputation in a negative manner.
Write evergreen material. Avoid topics that are only true the moment or day you write about them. The goal is to have content that will deliver, even if it is over a year old. If they're able to go over older articles and can still leave satisfied, they will also probably keep up with your new material.
Have your readers provide feedback to your emails. People like feeling useful and giving their inputs. Encouraging feedback allows readers to contribute. Your readers will appreciate this, and you'll get tons of tips on how to improve your article marketing tactics.
Blogging is one of the best new ways to market your business; a blog can help you secure a place as a thought leader in your industry. Try to include articles that are witty or show your leadership skills. If you are on top of your industry then your customers will be able to understand that you are informed. This will get you more sales.
Give your visitors information that is useful. The better they like your writing and articles, the more likely they will be to come back again and again. Especially if they feel like you are offering them a lot of great useful information.
Resist any inclination to flood indexes with the same exact article. Instead use as many indexes as you can with a variety of articles on each one. It's very tempting to post the same article over many indexes. Search engines are also aware of this technique and will penalize your ranking for it.
Start each article with a well-written, attention-grabbing paragraph. The first paragraphs of an article are very important for both readers and search engines. Putting crucial information in that first paragraph ensures that both are satisfied. Keep things interesting and don't overdo it. It is important to hook them on your content until they get to the very end.
Make certain you submit a large number of articles. If blogging is your primary marketing method, then pay attention to how often you post articles. If you want to promote a single keyword, you must submit lots of articles that contain it. If you're posting in a popular niche, you'll need at least 15 articles in the public network: 5 to directories and 10 to blog networks. To make sure this ends in a high page rank, top it all off with at least four dozen posts to private blogs.
As explained in the beginning of the article, the concept of article marketing is really just another way to market your website and bring increased recognition and traffic your way. Traditional advertising and marketing methods can be ineffective when used on the internet and new marketing methods are being created. You can use this information to get the most out of your advertising and marketing efforts.


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John Walters of Seven Days joins Dave for a review of Statehouse happenings. Then Chris Curtis of the attorney general's office stops by to talk about the latest from the FDA on maple and honey labeling.
Then we have Jay Diaz from the ACLU on Governor Scott's policy of blocking users from his Facebook page. And former Vermont Bar Association President Daniel Richardson rounds up recent Vermont and U.S. Supreme Court decisions.
← All Posts for Show The Dave Gram Show


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Sunnyside Cleaning Service LLC has provides the most comprehensive cleaning services to the Rayland, OH area for more than a decade. With incredibly low prices, we are your one stop shop for all your cleaning services from floors and construction sites to janitorial and businesses.
Our experts at Sunnyside Cleaning Service LLC know how to clean. They are experienced and professional and provide you and your business with spot on services. We specialize in construction cleanup along with industrial cleanup, janitorial services, and floor care.
We are locally owned and operated and have 20 plus years of experience in the cleaning industry. Fully licensed and insured, you can count on our team to professionally clean your business offices, commercial construction site, or any place that needs qualified cleaning services.
The cleaning crew we employ understands floor to ceiling cleaning and take great pride in knowing we do a job well. We guarantee our cleaning services and are on call for 24/7 emergency services.
We are the top choice when it comes to a commercial cleaning company, industrial cleaning services, and commercial or industrial floor cleaning. With prices that beat our competitors, you will love all of our services.
Our floor cleaning team has experience in tile, grout, and carpet cleaning. We offer an all-encompassed floor care plan that provides VCT flooring care along with stripping, waxing, and buffing.
You can count on us to take care of all your cleaning needs from inside out. We are the foremost authorities in commercial and industry cleaning, and we do a good job; guaranteed.
We also offer services that include wall washing, window cleaning, and clean up services for exterior. From wash outs to windows, Sunnyside Cleaning Service LLC is here for you.
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Rayland, OH 43943
Telephone: (740) 859-3196


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I was developing the game using AndEngine. I had two activity Welcome screen(Welcome.java) and Game Screen(Game.java) when user clicks on Start button in Welcome Screen he should be moved to next activity.
I end up with the following code. You can also use the following code to change the activity in AndEngine.
Place this code somewhere on suitable event such as onClick() event after which you want to change the activity.
Welcome.this.startActivity(new Intent(Welcome.this, Game.class)); Welcome.this.finish();
Change the class name to yours and fix the import Error that will occur.
And finally add this code in AndroidManifest.xml before </application>
<activity android:name="com.example.myGame.Game"> </activity>
Don't forget to change your package name and Class name.


text: Rob Browning wrote: > I'm about to be working hard on the emacs21 package which needs to be > done immediately, so expect to see a lot of noise out of me shortly. Good stuff! > On the bright side, I expect that *many* of the long-standing emacs20 > bugs will be ready for retirement -- I'm wondering if I should bother > reassigning them to emacs21 and then close them, or if I should just > tag them 'wontfix. There are a _lot_ of them. How long will you maintain an emacs20 package once the much better emacs21 is out? It needs a lot of work, but I guess they should be checked again on emacs21 and: fixed on emacs21 -> tag 'wontfix. not fixed on emacs21 and an upstream bug -> forward it upstream not fixed on emacs21 and a Debian bug -> fix it. Some of the bugs seem misplaced. I think we should do stuff like #40474 now (Emacs packages should use --no-site-file --no-init-file for compilation) but I'm pretty sure that's up to the add-on elisp packages and not the emacs20 package. Would you like a group of developers help maintain the emacs20/21 packages? Peter


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When you go on vacation, sometimes the best part is when you go home. Day 26 is about ensuring that your soul feels like is has a home and that you nurture this place in order to feel grounded and secure. I LOVE my home. Everyday when I drive into the driveway I say, "I am so grateful to be living in this beautiful home with my loving husband and amazing children." I truly never get tired of being in awe of where I live. The energy I put into this gratitude statement is then returned to me as my home truly loves me back. I can feel it! I encourage you to begin to develop a relationship with the place you live. It may not be your dream home, and that is ok, but the more you start to nourish the feelings you have for where you live, the happier you will become. If you truly hate where you live, then move! I know that you may feel that you can't – but guess what, if there is a will there is a way! Keep me posted! ~Blessings, Lisa
Earth: Day 26 – A Home for the Soul
There are four things that the soul requires in a home:
1. It needs a sense of belonging, to feel connected to the land or to a place.
2. The soul needs a place to feel safe, so you can be yourself without fear or hesitation.
3. It yearns for harmony with the greater cycle of nature.
4. Your soul needs sacred space.
When your home has these things, your life will be filled with inner peace.
Affirmation for the Day: I am at home, no matter where I am.
Today: Wherever you are, affirm that you are "at home." For example, if you are in you car, affirm to yourself, "I am at home in my car." If you are in your office say, "I am at home in my office." If you are on a bus say, "I am at home on the bus." Also, periodically affirm, "I am at home in my body." If you are with other people, affirm to yourself, "I am at home with … (Sue and John, or my children, or my husband, or my wife, etc.)"
Level 1: Bring Nature into Your Home
The survival of the earliest humans depended on their ability to live in balance with the natural world and their homes reflected an awareness of this fact. Modern homes are often separated from nature and the soul has suffered as a result.
Today, bring nature into your environment so that the "beauty in your home leads the heart to the holy mountain." Here are some ideas. Put up a photo of nature. Gather some leaves and make an arrangement of leaves, moss, pine-cones, and stones on a table. Tie feathers on strings and let them dangle where a breeze can softly move them. Put a big plant by the front door. Take a walk and find some items from nature and arrange them into a mandala on a coffee table. Play CDs of nature sounds: waterfall, rain, ocean surf, wind, crickets, or birds, etc
To leave me a comment, click on the {comment} link below. ~Blessings, Lisa
p.s. always check back, as I read every comment and like to respond back to you!


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It is the opinion of financial regulators that Forex brokers have been running wild in their zeal to acquire customers. Some have even gone as far as offering free day trading through the use of no deposit bonuses just to get new clients signed up. Now, some regulators are looking to implement new regulations to curb these practices and soon trading conditions won't be what they used to be. If you want to become a Forex trader, now is the time to do so because once these new laws come into effect, the industry will be completely different.
Changes in Forex regulation
Perhaps the first Financial regulator to impose rules on the Forex market was the CFTC in 2010 through the Dodd-Frank Act. This regulatory body noticed that there were several financial markets that were decentralized thus widely unregulated and sought to put an end to that. While they could not regulate these markets through an exchange, stricter regulations would work just as well, and they did.
Today, there are only a handful of Forex brokers based in the US, yet cases of fraud have gone down as a result of the regulations. At the moment, the highest number of Forex brokers are based in Cyprus, and there are 2 main reasons:
- A license from CySEC allows brokers to operate in the entire Eurozone and solicit clients from all around the world except the US
- Regulations on Forex trading by CySEC had been quite lenient allowing small Forex brokerage firms to establish themselves
On the 30th of November 2016, CySEC sent a circular to all Forex and binary options brokers informing them that bonuses will not be allowed completely. As for leverage, the default level will be 50:1, beyond which the client would have to undergo an appropriateness test. The CySEC circular stated that bonuses and high leverage are putting traders at an unnecessary risk.
These new regulations were supposed to come into effect on the 30th of January 2017, but the deadline was pushed forward. CySEC is still insistent on implementing these new rules, and there is only some little time left before the bonuses are gone for good.
Brokers offering free day trading
There aren't many Forex brokers who allow free day trading at the moment, but some notable and trustworthy brokers include XM and Plus500. XM brokers offer a free $30 no deposit bonus which you get once your account has been approved. Z.COM offer $25, again, once your account is approved.
Just remember that the no deposit bonus is not a gift or free money, but it is meant just to give you a taste of real trading. Any broker can offer a demo account, but the downside is that there is no actual reward. Every dollar you make is virtual and there is no incentive to make an effort. With the bonuses offered by XM and Z.COM, though, whatever you make with the bonus is actual money, and that should be all the motivation you need.
It has all the benefits of actual rewards and none of the risk found in stacking actual money. What's more, both of these brokers have excellent services that you could explore with the bonus even before you decide to invest actual money.
Soon, all this is going to end as soon as CySEC implements these new regulations and now is the time to try free day trading. All signs point toward regulating the Forex market and perhaps even centralizing it the way it was in the past. But this is still a long way down the road; right now, let's just enjoy free day trading as long as we still have it.


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This compilation video of dogs protecting their owners babies is really cute and heart warming…in a way. Yes, we love that these dogs want to protect the baby. But here it seems to have gone too far, don't you think?
If your huge dog is growling at you and showing teeth when you try to get near your baby, maybe it's time to set some new rules.
Youtube commenters were basically on the same page, except a few who think it's great:
"This is a problem. It means your dog has assumed the lead role and will go to the extremes of protecting the child from it's actual parents."
"Just relax and enjoy the video. You don't have to be a bunch of right fighters. I grew up with a dog who wouldnt let my dad and I play fight. She would attack him every time we tried because she thought he was trying to hurt me. Guess what we did? Encouraged that behavior. It meant she would always keep me safe, no matter what. "
"When it comes to the big Rottweiler and the other dog telling the father "No you can't touch the baby," that's when it is a problem. I did not think that was cute at all. When your pets come between you and your baby then you got a problem in front of you."
"It's normal for dogs to protect children from strangers or be more protective of their owner when they are pregnant (hovering, not growling) when a dog protects a child from their parent either it thinks they are going to harm the child or it thinks they are too incompetent to care for the child. If neither of these things are true then you have a problem. If they are true, then you are the problem."


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Dynamics AX
Take your business to the next level
In a competitive global marketplace, managing change, keeping an eye on every aspect of your business and seamlessly operating across multiple locations and countries requires an enterprise level financial management system.
With Microsoft Dynamics AX from MG Technology Group, you get all the benefits of a tier one ERP solution to standardize critical business processes, provide greater visibility across your entire organization and simplify regulatory compliance without spending millions of dollars.
Microsoft Dynamics AX from MG Technology Group gives your business the power to:
- Invest in a scalable, customized ERP solution – with the flexibility to respond to market changes, mergers, acquisitions and product launches
- Manage global manufacturing and supply chain – keep everyone on the same page for more efficient work supporting global operations
- Stay compliant – reduce your risk and liability with an ERP system that can respond to changing requirements at home and abroad
- Compete on a global scale – get better company insight and standardize operations for simplified management of a complex, international organization
Microsoft Dynamics AX gives you the insight to play in the global marketplace, and the power to compete against corporate giants with agility they can't match.
Because your Microsoft Dynamics AX solution is customized to your needs and able to grow and change as your business does for a fraction of the cost of a tier one, you get both competitive and capital advantages.
Find out what Microsoft Dynamics AX from MG Technology Group
can do for your growing business.


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"A very strong way to get in front of the right people"
Sponsoring or exhibiting at the AI for Business Summit gives you the opportunity to showcase your products, services and solutions.
You will get the chance to make face to face contact with business leaders and key decision makers who want to be at the forefront of AI.
- Make valuable contacts - Meet with 200+ qualified, senior people - share your knowledge and grow your network of contacts
- Hand-picked speaker faculty - Hear from and meet industry leaders that are developing and implementing major AI strategies and projects
- Spark innovation - Where will the next AI innovation and great idea come from? Join the representatives from the whole ecosystem and be part of the conversation
- Interactive formats - Big Debates, End-User Case Studies, Hosted Round Tables, Innovation Spotlights and more
- Networking - Enjoy the best of formal and informal networking, and continue the conversation at the after party
For more information on ways to engage with delegates at the AI for Business Summit, please contact Damien Yong on firstname.lastname@example.com or call +61 (0)2 8188 7513.


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I love watching HGTV. Having been a General Contractor and dabbling in interior decorating, I have to say that I'm addicted to real estate and rehab shows. House Hunters, Fixer Upper, Addicted To Rehab, Property Brothers are all of my favorites.
I guess that is why Jeff and I felt ok about purchasing an RV project. There is a certain thrill and satisfaction that comes from making something out of nothing. But I have to say, this project has been one of those labors of love that is often seen in rehabbing an old Victorian home along some of our countries most historical main streets.
The old dilapidated, leaning home that people thought was a total tare down is resurrected into a magnificent showpiece, lovingly restored back to its former glory. Usually at a cost that exceeds what new construction would have run all in the name of love.
I think that is where we are at with our Domani.
We should have probably taken a bulldozer to her the day the roof was pulled off. But she had bones, class and character that couldn't be overlooked nor found in new construction. And now that we are nearing the end and we have had a chance to spend some quality time with our girl, she has made an impression on our hearts. She has become our labor of love!
After 3 1/2 months of pulling our hair out restoration, it's all finally coming together. And we couldn't be more pleased with our home on wheels.
Things I love about the Domani:
Counter space: Every RV'er knows that countertops are a premium in campers. Though still small by housing standards, I have twice as much counter space than before. And, they are solid surface rather than plastic.
Lobster Pot Sink: These sinks are popular in Airstreams. Frankly, I have not heard of too many people liking them. But me? I'm a fan! It's deep, it's wide and there is plenty of room to wash a pan without mopping the floor at the same time.
Bedroom Door: It's the simple things that contribute to happiness. Having a door that closes off the bedroom from your spouse watching howling piano playing puppies on facebook at 4:30AM is one of them.
Open shower & sink, enclosed toilet: We have an open shower/sink area to the bedroom with an enclosed toilet closet. Jeff and I are both vertically endowed, so having a sink area that allows brushing our teeth without hitting our elbows feels palatial.
Closet & Cabinet Space: The Domani is only 32' long so there was some bedroom closet space sacrificed along the front of the coach that typical 5th wheels have. But I do have a tilt out laundry bin and 24 upper cabinets in the living room/kitchen that more than make up for it.
Full Length Mirror Closet Door: I didn't know that I wanted a full-length mirror until I had one. It's very helpful when you are putting on a t-shirt, shorts and flip flops to make sure that the fit is just right…. she says sarcastically!
Storage Bay: Holy mother of storage! Jeff and I both could fit in there with enough room to roll around in. I'm thinking of turning it into my office.
Larger Frig: Who doesn't like more refrigerator space. Our frig now has an extra shelf over our previous frig, which is like going from an apartment sized refrigerator to a side-by-side.
Big Windows: In the Domani we have lots of big open windows and skylights that let the light flood in. We removed the old smelly accordion blinds and are replacing them with MCD roller shades (more on that in the next post). The views are going to be great once we get this baby out on the road!
Sofa and Two Chairs: Before, we had a jack-knife sofa which was adjacent to the kitchen. So when we wanted to put our feet up they ended up on the kitchen counters. (The multipurpose kitchen sink/ottoman). Now we actually have a sofa long enough to stretch out on and two very comfortable chairs. The space between the sofa and chairs is cavernous so no more footsies with Jeff while making dinner.
What now?
We are disappointed that we did not make it to Iowa for RAGBRAI this year. The repairs just kept us stationary a while longer.
We have now moved on to the MORryde parking lot for a new hitch, basement slide out tray and new axles and will hopefully finish up here end of next week.
We have our sites set on getting our girl back to Arkansas for a wedding in September before heading out west for the fall and winter.
It's been a long time coming and we are itching to get on the road. I've booked our campsites for the Xscapers Albuquerque Balloon Fiesta and the Grand Canyon, both in October, which gives us some sense of hope that we will be rolling out of Elkhart very soon. We've kissed the frog and she has turning into princess charming. Now lets ride this thing off into the sunset!
Can't wait!


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Choose the Hotel Cristina for your wedding day and you'll enjoy a fabulous setting with panoramic views over St Aubin's bay.
There are many places you can choose for weddings in Jersey, but few wedding venues offer such breathtaking views as a backdrop to the special day. From the Terrace and restaurant you and your guests can look out over the entire St Aubin's bay.
Our guests also enjoy the gardens and in summer everything looks spectacular!
Over the years we've had the pleasure to host many weddings. Our guests have enjoyed our venue for their Jersey wedding. Don't take our word for it; you can read the testimonials in our
We understand that every wedding is different and would be delighted to show you around the Hotel Cristina, to discuss your requirements in detail.
Our dedicated team are on hand to guide you through all your preparations and to help you with anything you may need to make your Jersey wedding memorable. Our dedicated wedding coordinator to assist you with all hotel arrangements through to your wedding day.
We supply the romantic views, you just say 'I do' - we have years of experience of hosting wedding parties. Jersey weddings for 2020 are filling fast, so please do contact us to find out our availability and to view our menu packages.
For more information, you can download our wedding brochure or contact Robbie Grant.
Wedding Brochure Email us Call 01534 758024
With our Loyalty card every time you book direct, eat or drink in one of our hotels, bars or restaurants in Jersey you will receive five points for every £1 you spend.
Each point is equal to 1p so for every £1 you spend you get 5p back!
2018 winners:
Best service from leisure tourism & accommodation
Best service from a team
People's choice award
Outstanding contribution to customer service
2019 winners:
Best service from leisure tourism & accommodation
Dolan Hotels - Outstanding contribution to customer service
Dolan Hotels is one of Jersey's longest established hotel groups with 4 star luxury Jersey hotels in the beautiful St. Brelade's Bay and St. Aubin's Bay. Whether you're visiting Jersey UK for a luxury weekend break, a family holiday, wedding or honeymoon, our stunning hotels in Jersey will cater for your every holiday need.
One of Jersey UK's best country house hotels with 59 elegantly furnished rooms and amazing views.
A modern family owned hotel located above the bay of St. Aubin with panoramic views of the surrounding area.
4 Star hotel located right in the heart of St Brelade's Bay, celebrated for its clean bathing and watersports.
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It happens every spring, but this year, the questioning has been particularly intense. Every month of April, on occasion of the London Book Fair, newspapers publish articles speculating if it still makes sense to publish books.
Only 3% of the population read books, so what? At the turn of the 21st century, one thousand
book titles were published for every feature-length film made. Today,
the ratio is one to six hundred. The number of films produced every
year has increased and, at the same time, the number of published books
has diminished.
Are you missing the point completely?
"We live in a visual world," sociologists argue.
"In many areas, the written word is becoming a relic of previous
centuries." Media analysts blame the trend on video-games and portable
DVD players. Others simply say that reading requires too much effort
after our long work schedules.
In my view, those commentators are
missing the point completely. Despite the abundance of cheap visual
entertainment, readers' motivation remains strong. The reason why people
read books has nothing to do with the demands of society and everything
to do with individual psychology.
The key to consistent thinking
media, due to its structure and economics, is unable to express minority
views in a consistent, intellectual manner. In this respect, all has
been tried and all has failed. Complex ideas cannot be transmitted
without the written word. No photograph and no film can replace a chain
of reasoning built in clear sentences.
television, and radio, despite the growing number of channels, can only
thrive when they aim at large audiences. They can offer multiplicity in
the multitude, but no original ideas. Digital video has reduced the
budget necessary to make a film, but not the distribution costs.
Actors, good lighting, and a decent soundtrack are still expensive.
Books, on the other hand, can still be published and distributed
Something new and beneficial
In a film, special effects cannot cure
the problems of a weak scenario. Even great acting is unable to sustain
a filmed story that doesn't make any sense. How long ago is it since
you saw a really thought-provoking film? How often do you gain deep
insights from watching television? The written word remains the ideal
means to transmit innovative, creative ideas.
The good news about reading
is that 3% of the population still remain avid readers. That's not a bad proportion at all. A strong audience
for writers is still there, and it is not going to become smaller in the
foreseeable future.
Do people read internet blogs for the same
reason that they love books? Is it because they want to read original
ideas? Do they do it in order to enjoy some fresh writing? I suspect
that, for most, the main drivers are the joy of discovering something
new and beneficial, as well as a steadfast refusal to join the other 97%.
For more information about rational living and personal development, I refer you to my books.
[Text: http://johnvespasian.blogspot.com]
[Image by jucanils under Creative Commons Attribution License. See the license terms under http://creativecommons.org/licenses/by/3.0/us]


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Auum Pets
This Auum Pets supplement, an Omega-3 formulation, has been specially designed for dogs and cats. Not only does it provide the necessary balance of Omega-6:Omega-3 ratio, it has vitally important fat soluble Vitamins A, D and E. It is completely "fish oil free".
Auum Pets is fashioned after the original diet of the Canadian Eskimo Dog. These special sled dogs led Admiral Perry to discover the North Pole in 28 days. They are capable of pulling 13 hours a day in -40C temperatures.
Because their natural raw meat diet is enriched with seal blubber, they consume massive amounts of Omega-3 and associated fat soluble vitamins A, D and E. Their extreme endurance, staggering strength and the three beautiful layers of rich fur, are testaments to a diet rich in mammalian Omega 3.
The ideal omega 3 fat soluble supplement for both dogs and cats!
- Mammalian omega 3 contains EPA, DHA and DPA
- Added D3, vitamin A (beta carotene and retinal palmitate) and vitamin E
- DPA has a neuronal affinity enhancing nerve function
- Contains the correct ratio of omega 6:omega 3 for mammals – a 1:1 ratio
- This powerful omega 3 helps to correct dietary imbalances and reduce inflammation
- Mammalian omega 3s have the configuration that allows for phenomenal uptake
- In addition to anti-cancer properties, D3 helps regulate calcium intake and promotes normal bone development
For more product information on Auum Pets click here.
"Our 15-year-old Jack Russell Terrier, 'Iggy', recently incurred an unfortunate head injury. This was of great concern to my wife and I, as you can imagine. Soon after we started adding a teaspoon of the Auum Omega 3 mammalian oil in his morning food, we noticed a remarkable acceleration in Iggy's healing." — Brian Coburn
For more testimonials, click here.
For purchasing, click here.
Dr. Jan Dadson DVM, VP Veterinary Medicine
Dr. Dadson operated Wingrove Veterinary Services (Guelph) for thirty years. He provided mixed animal services to the surrounding area becoming experienced in soft tissue as well as orthopedic surgery, diagnostic ultrasound, provincial emergency meat inspection, and all aspects of a full service veterinary clinic. He organized health care and procedures for one of the first Elk farms dedicated to the collection of Elk velvet and was the vet of record for the largest importer / exporter of reptiles in Canada providing regulatory backup and supplies. He became known as "the Boxer Vet" due to providing services to one of the premier Boxer breeders in North America.
After selling his practice Dr. Dadson operated a mobile practice providing care to dogs and cats in their owner's homes. He has investigated treatment protocol for degenerative myelopathy (DM). DM is an ascending paralysis of dogs, which appeared as a new entity in his final year at his Wingrove practice. His continuing clinical investigations with Auum Pets formulations, beginning in 2012, resulted in a written paper outlining his parallel observations and assessments reflected in both animal and human patients using the Auum omega-3 supplements. Although not yet published it was referenced at the University of Toronto conference on Alternative Methods for Treating Autism and ADHD in Children.


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Kquartz is a brand of Quan Stone Established in 2009,Website:http://www.aquartzcountertops.com, we supply both Man-Made and Natural Stone products. Our main product is Engineered Quartz Stone in slab of size of 300x140cm and 320x160cm, also tile in size 600mmx600mm or other sizes as per client request. The thickness can be 12mm, 15mm, 20mm and 30mm. Our monthly production is 50,000 square meters. We also supply Composite Marble in Slab of size 320x160cm or 300x120cm and tile in size 800mmx800mm or other sizes as per client request. Monthly production can reach to 100,000 square meters. Our Natural Stone products are kitchen Countertop and Bathroom vanity top, Strip, Stair, Window Sill, Column and other special works as per client designs. We have SGS Certificate and Reaction to Fire Text Reports issued by SGS for both Quartz Stone and Composite Marble. Also we have strict Quality Control System to ensure each slab that the clients receive is the correct quality without defects. Thanks for the stylish designs, consistent quality, most competitive price and best service, our products enjoy a good reputation among our customers around the world, such as USA, UK, Canada, Australia, New Zealand, Germany, France, Poland, Ireland, Spain, U.A.E, South Africa... Whether you are a wholesaler or you have projects for new building or you want to remodel your existing home, our specialists will give you the attention you deserve.
|Gems and Jewellery
|Business Type
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|Firm Type
|Pvt Ltd.
|Expand Level
Countertop ,Quartz countertops , quartz stone , kitchen countertop, quartz kitchen countertop , artificial stone , artificial stone countertops , quartz color ,engineering stone ,bathroom countertop. Quartz vanity top . table tops ,granite countertops . quartz stone countertop
Yasumine road No. 987, 1, 508
Xiamen () - (China)
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Game Tags:
Game Plays: 5207
Once upon a time there was a little troll who lived in a dark forest. He was a clever little chap and an extremely kind fellow. He loved his garden and his hobby was constructing machines. Life was slow and Peaceful, until one day he received a letter… The game features a cute little troll as a main hero, lots of really big guns as your weapon of choice and an abundance of different abilities that will make your little big adventure fun and unforgettable.
Game Not Working?? Please Let Us Know!!!


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I have hesitated for years whether I needed a personal site as public access to me didn't involve using a map or a magnifying glass.
I am on all the relevant social sites and I am the most approachable person I know.
However, as it has been proven to me quite recently, linking all my crazy career interest together was no so easy, so this is the resulting factor. A tree from which all subsequent branches can then sprout.
Here is where you will not only find out what makes my engine run but the current design as well as future models.
Enough with the analogies! Point is on these pages, you will find out; Who I am. What I do. Where I want to go from here. There is not much, I know BUT hey...I haven't finished yet!
If you still need to find out more (God bless you if you do) detour to Contacts!