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This study aimed to investigate the ability of CpG7909 adjuvant to enhance immunogenicity and protective efficacy of a subunit vaccine composed of ESAT6-Ag85A fusion protein (Pe685a) of Mycobacterium tuberculosis.
This study aimed to investigate the ability of CpG7909 adjuvant to enhance immunogenicity and protective efficacy of a subunit vaccine composed of ESAT6-Ag85A fusion protein (Pe685a) of Mycobacterium tuberculosis.
ELISA was used to detect specific antibody and IFN-γ expression in sera; ELISPOT, to detect IFN-γ expression in splenocytes; MTT assay and FACS, to detect T-lymphocytes proliferation in spleens; and RT-PCR, to detect cytokines expression in lungs of mice after immunization. Bacterial load and histopathological lesions in lungs or spleens of mice challenged with Myco. tuberculosis H37Rv strain were analysed. Compared with incomplete Freund's adjuvant, CpG7909 induced more potent production of Pe685a-specific IgG2a/IgG1 antibody and higher expression of IFN-γ in sera, stimulated more generation of antigen-specific IFN-γ-secreting splenocytes, enhanced frequencies of CD3+CD4+ and CD3+CD8+ T-lymphocytes in spleen and increased transcription of TNF-α, IFN-γ, IL-6 and TLR9 in lung. However, lower bacterial load in lung and less severe lung pathology were not observed in CpG7909 group mice.
CpG7909 is able to enhance immunological effects of Pe685a subunit vaccine, but does not confer significant protective efficacy against Myco. tuberculosis infection.
CpG7909 as an adjuvant of subunit vaccine against Myco. tuberculosis is worthy of further investigation.
Tuberculosis (TB) caused by Mycobacterium tuberculosis is the most common infectious disease worldwide. The World Health Organization estimates that roughly one-third of the global population has been infected with TB, which is responsible for about 1 800 000 deaths annually (WHO 2009). Currently, Mycobacterium bovis Bacillus Calmette–Guerin (BCG) is regarded as the only available vaccine against TB administered annually with over 120 million doses (WHO 2004). However, the efficacy of BCG is controversial, particularly in preventing adults from pulmonary TB (Daniel 2006). To control this global threat, development of new effective vaccine and immunization strategies is urgently needed.
Subunit vaccine from potential immunogenic antigens of Myco. tuberculosis might be a promising direction (Sharma and Yoder 2011), combining the stable, nontoxic and harmless adjuvant as a carrier to exert their immune effects. Traditional adjuvants, such as incomplete Freund's adjuvant (IFA), which need to be emulsified with antigens, are toxic to humans. In recent years, CpG oligodeoxynucleotide (CpG ODN), the agonist of Toll-like receptor 9 (TLR9), has been found to be a potent adjuvant, which is able to enhance immune responses of many mammalian species including humans to a variety of antigens including peptides, soluble proteins and virus-like particles (Klinman 2006; Krieg 2006). CpG ODN directly or indirectly activates B lymphocytes, plasmacytoid dendritic cells (pDCs), macrophages and other monocytes to secrete cytokines as IL-2 and IFN-γ, resulting in strong Th1-type immune responses (Krieg et al. 1995; Ahmad-Nejad et al. 2002; Iwasaki and Medzhitov 2004; Suzuki et al. 2004). It is generally accepted that Th1-type cellular immune response plays a significant role in antituberculosis infection (Dietrich and Doherty 2009). Therefore, we suspect that CpG ODN might be a promising adjuvant of subunit vaccine to prevent from Myco. tuberculosis infection.
In this study, CpG7909, which belongs to B-type CpG and was shown to be a potent inducer of human innate immune responses (Krieg et al. 2004; Vollmer 2005; Stewart et al. 2008), was used to act as adjuvant for a recombinant ESAT-6 and Ag85A fusion protein (Pe685a), an efficient booster subunit vaccine against TB (Lu et al. 2011), to explore the possibility that CpG7909 develops into a novel adjuvant for subunit vaccine against Myco. tuberculosis.
Specific pathogen-free, six- to eight-week-old, female BALB/c mice (provided by Lab Animal of Tongji Medical College) were randomly divided into four groups, PBS group, CpG7909 group, CpG7909/Pe685a group and IFA/Pe685a group used as positive control (n = 10). 200 μl PBS, 50 μg CpG7909 (TCG TCG TTT TGT CGT TTT GTC GTT, synthesized by Sangon Biotech, Shanghai, China), mixture of 50 μg CpG7909 and 50 μg Pe685a protein (kindly provided by Prof. Xionglin Fan, Tongji Medical College, China) and emulsion of 100 μl IFA and 50 μg Pe685a were respectively injected intraperitoneally in the same area of mice. Immunization was repeated three times with 2 week intervals, and antigen dosage reduced to half at the last immunization. Five mice per group were sacrificed for immunoassay at 2 weeks after the last immunization. The remaining mice were used for challenge with Myco. tuberculosis H37Rv virulent strain.
Sera were collected from each mouse at 2 weeks after the last immunization, and levels of the anti-Pe685a antibodies were determined using enzyme-linked immunosorbent assay (ELISA). Plates were coated with Pe685a (2 μg) in a coating solution (15 mmol l−1 Na2CO3, 34·5 mmol l−1 NaHCO3, 0·02% NaN3, pH 9·6), incubated at 4°C overnight and then blocked with 2% BSA in PBS for 60 min at 37°C. Serum samples were applied in serial 10-fold dilutions from 1: 10–1: 106. After incubating the plates for 2 h at 37°C, biotin-conjugated anti-mouse IgG1 (RMG1-1, Biolegend, San Diego, CA, USA) and IgG2a (RMG2a-62, Biolegend) antibodies were added for the detection of specific antibodies and were incubated at room temperature for 45 min. To detect bound antibodies, the OPD substrate was added, and the reaction was stopped by the addition of 50 μl of 2 mol l−1 H2SO4. The optical density was measured at 492 nm.
Sera were collected from each mouse 2 weeks after immunization and diluted to appropriate concentrations. Mouse IFN-γ ELISA kit (DKW12-2000-048, Dakewe Biotech Company, Shenzhen, China) was used to determine the IFN-γ expression in sera following the manufacturers' instructions, using the provided standard curve reagents. Controls and samples were run in duplicate to assure consistency. Intrasample variability was less than 10%. The optical density was measured at dual wave length (405 and 630 nm).
Splenocytes were isolated as described below. Briefly, spleens were removed, and splenocyte suspension was filtered through a 200-mesh stainless steel sieve prior to being centrifuged at 1500 g for 5 min. Pellets were resuspended in 8 ml of red blood cell lysis buffer (Beyotime Institute Biotech, Haimen, China) and incubated at 37°C for 8 min. Splenocytes were collected with centrifuge at 1500 g for 5 min and resuspended at 1 × 106 cells ml−1 in complete culture medium (RPMI1640 supplemented with 10% FBS, 100 U ml−1 penicillin, 100 U ml−1 streptomycin). MTT assay was performed as per the manufacturers' instructions (Beyotime Institute Biotech). Briefly, splenocytes (2 × 104 well−1) were seeded into a 96-well microplate and were cultured at 37°C, 5% CO2 for 16 h with stimulation by Pe685a (2 μg well−1). MTT staining liquid (10 μl well−1, 5 mg ml−1) was added and incubated for 4 h. Formanzan dissolved liquid (100 μl well−1) was then added and incubated until the purple crystals dissolved. Controls and samples were set in duplicate to assure consistency. The optical density was measured at 570 nm. Stimulation index (SI) was introduced as evaluation index to estimate the proliferation of lymphocytes. The calculation method is as follows: SI = (samples OD570-blank controlled OD570)/(negative control OD570-blank controlled OD570). SI > 2 was considered as specific lymphocytes proliferation.
Of 2 × 104 well−1 splenocytes were seeded into a 96-well microplate and cultured at 37°C, 5% CO2 for 4 h with stimulation by Pe685a (2 μg well−1). Cells were removed into epoxy epoxide tubes, washed and stained with PE-CyTm7-labelled CD3e (anti-mouse), APC-labelled CD8 (anti-mouse) and PE-labelled CD4 (anti-mouse) (BD PharMingen, San Diego, CA, USA) for 30 min at 4°C in dark. Isotype controls were set up for every group. Cells were analysed by FACScan LRSII instrument (BD FACSCalibur flow cytometer, BD, San Jose, CA, USA).
Mouse IFN-γ ELISPOT kit (U-CyTech biosciences, Utrecht, the Netherlands) was used to determine the number of IFN-γ-expressing cells in splenocytes following the manufacturer's instructions as described previously (Shi et al. 2010). Briefly, the splenocytes were diluted to a concentration of 1 × 106 ml−1 with Lympho-Spot serum-free medium for rodent (Dakewe Biotech Company) containing an appropriate antigen (2 μg well−1 Pe685a). About 2 × 105 cells were added to the wells of the ELISPOT plate. IFN-γ spot-forming cells were enumerated using an ELISPOT reader (Biosys Bioreader 4000 PRO, Karben, Germany). For each animal, the number of spots in wells with medium alone was subtracted from the number of spots in test wells. The mean number of antigen-specific IFN-γ spot-forming cells (per million cells) for each group was determined.
Total RNA was extracted with TRIzol reagent (Invitrogen,Carlsbad, CA, USA) from about 100 mg of lung tissue. For RT-PCR, 2 μg RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Invitrogen) to obtain cDNA samples. The resultant cDNA was stored at −70°C until PCR amplification. Cytokine-specific primers (Sangon Biotech) were listed as followed (Table 1). For a comparative internal standard, primers of β-actin were added to each PCR mixture according to the procedure described previously (Patten et al. 2003). The final PCR mixtures contained 1 μl cDNA, 10 μl 2 × PCR Mix (Dongsheng Biotech, Guangzhou, China), 2 μl sense and antisense primers (100 mmol l−1) and sterile water to 20 μl. PCR was performed by the following procedures: 95°C for 5 min, 94°C for 30 s, 55/60°C for 30 s, 72°C for 45 s, total 30 cycles, at last 72°C for 10 min. PCR products were visualized by UV light after electrophoresis. Analysis was performed using the Gene Genius Bio Image system and Gene Tools from syngene. β-actin amplification was performed on each individual sample as an internal positive control standard.
|Sequences of primers
|Length (bp)
|Temperature (°C)
Four weeks after the last immunization, BALB/c mice were challenged with 1 × 106 CFU of virulent Myco. tuberculosis H37Rv through a lateral tail vein. Four weeks postchallenge, five mice per group were sacrificed for efficacy comparison. The spleens and lungs were removed aseptically, homogenized and cultured for CFU of Myco. tuberculosis on Lowenstein–Jensen medium. Left lung lobes from different vaccinated mice were fixed in PBS-buffered formalin and embedded in paraffin. Tissue sections were prepared and stained with haematoxylin and eosin (H&E) stain. A pathologist with no prior knowledge of the experimental groups recorded the result under a light microscope and scored as described previously (Shi et al. 2010). Briefly, sections were evaluated according to the four histopathological parameters, including peribronchiolitis, perivasculitis, alveolitis and granuloma formation and noted as absent, minimal, slight, moderate, marked or strong, which was scored as 0, 1, 2, 3, 4 and 5 respectively; thus, the maximal sum was score 20.
One-way analysis of variance was used to compare the difference between groups of mice, and P-value <0·05 was considered statistically significant.
To determine what type of the immune response against Pe685a was induced by CpG7909 adjuvant, we first evaluated specific IgG2a and IgG1 antibody productions in sera of immunized mice. It was shown that both CpG7909 and IFA stimulated significant secretion of IgG2a and IgG1 against Pe685a (Fig. 1a). Nonspecific antibodies were found in PBS and CpG7909 control mice. However, the IgG2a level resulting from CpG7909-/Pe685a-immunized mice was significantly higher than that resulting from IFA-/Pe685a-immunized mice (Fig. 1a). IgG2a/IgG1 ratio was also established. Mice immunized with CpG7909/Pe685a presented higher IgG2a/IgG1 ratio than that did in IFA-/Pe685a-immunized mice (Fig. 1b). Antigen-specific IFN-γ-secreting splenocytes from immunized mice were further evaluated (Fig. 1c). Splenocytes from mice immunized with CpG7909, CpG7909/Pe685a and IFA/Pe685a produced a significant secretion of IFN-γ when compared with cells from nonimmunized mice. Splenocytes from CpG7909-immunized mice and IFA-/Pe685a-immunized mice produced similar quantities of IFN-γ. However, the splenocytes of mice immunized with CpG7909/Pe685a presented significantly more secretion of IFN-γ than those of other mice group did. The level of IFN-γ expression in sera from immunized mice was also evaluated, and highest concentration of IFN-γ production in sera was also observed in CpG7909-/Pe685a-immunized mice (Fig. 1d). The above results showed that apparent Th1-type immune response was induced in CpG7909-/Pe685a-immunized mice.
Proliferation of sensitized T-lymphocytes in splenocytes challenged by specific antigen was detected by MTT, and data were presented as SI (Stimulation index) value. With the stimulation by Pe685a (2 μg well−1), CpG7909 stimulated more potent production of specific T-lymphocytes induced by Pe685a than IFA (Fig. 2a). Furthermore, the proportion of CD3+CD4+ and CD3+CD8+ T-lymphocytes in spleen was measured with FACS. CpG7909 stimulated more proportion of both CD3+CD4+ and CD3+CD8+ T-lymphocytes in spleen induced by Pe685a, whereas IFA only enhanced proportion of CD3+CD4+ T-lymphocytes in spleen induced by Pe685a, which indicated that CpG7909 is able to stimulate higher frequencies of CD3+CD4+ and CD3+CD8+ T-lymphocytes than IFA (Fig. 2b,c).
Cytokine transcription in lungs was detected with semiquantitative reverse transcription PCR. Expression of target genes was normalized with β-actin transcription. The expression levels of TNF-α, IFN-γ, IL-6 and TLR9 in lungs of vaccinated mice were determined by grey value ratio. As shown in Fig. 3, compared with IFA-/Pe685a-immunized mice, mice vaccinated with CpG7909/Pe685a induced higher expression of TNF-α, IFN-γ, IL-6 and TLR9 (Fig. 3). The expression of IL-10 and IL-4 was also measured, and there was no statistical difference between experimental groups (data not shown). These results showed that CpG7909 induced stronger expression of TNF-α, IFN-γ, IL-6 and TLR9 than incomplete Freund's adjuvant.
Four weeks after virulent H37Rv challenge, mice were killed and bacterial load and histopathological lesions in lungs or spleens of mice were analysed. Lung tissues from different groups of infected mice were fixed, sectioned and stained with H&E stain for assessment of pathological changes (Fig. 4a). There were extensive pathological changes in the lung from mice of each group. PBS control mice had the highest score (score 12) of lung pathology, with marked peribronchiolitis, perivasculitis and alveolitis. Slight damage in alveolar tissues with aggregated, relatively large number of lymphocytes, peribronchiolitis and perivasculitis was also observed in lungs of mice vaccinated with CpG7909/Pe685a, which was assessed as score 7. Mice from IFA group had the lowest score (score 3), and pathological changes in lung tissue were much less, compared with PBS and CpG7909 control group and CpG7909/Pe685a group (Fig. 4b). The clear hierarchy of protective efficacy in different groups of vaccinated mice, evaluated with bacterial load in lungs and spleens, was consistent with the extent of histopathological lesions (Fig. 4c), which indicated that CpG7909 adjuvant was not able to confer more significant protective efficacy against Myco. tuberculosis infection than incomplete Freund's adjuvant.
CpG7909, the first CpG to enter clinical trials in 1999, has been shown to augment production of specific antibody as adjuvant for vaccine including hepatitis B, flu, anthrax and malaria vaccines (Amlie-Lefond et al. 2005; Vollmer 2005). CpG7909 is known to directly activate B cells and indirectly activate B cells through enhancing expression of cytokines such as IL-6 and IL-10 and then promote activated B cells to produce antibodies (Krieg et al. 1995; Jego et al. 2003; He et al. 2004). CpG ODN can also activate plasmacytoid dendritic cells (pDCs), macrophages and other monocytes to secrete cytokines as IL-2 and IFN-γ, resulting in strong Th1-type immune responses (Krieg et al. 1995; Ahmad-Nejad et al. 2002; Iwasaki and Medzhitov 2004; Suzuki et al. 2004; Thapa et al. 2012). In our study, higher level of IgG2a/IgG1 antibodies specific to ESAT6-Ag85A fusion protein, a subunit vaccine that has been confirmed to possess good immune protective effects on mice infected with Myco. tuberculosis (Lu et al. 2011), was detected in sera of CpG7909-/Pe685a-immunized mice, compared with IFA group mice, suggesting that CpG7909 might be able to activate more B cells than incomplete Freund's adjuvant, to increase immunological effect of mycobacterial subunit vaccine. Furthermore, higher level of IFN-γ in sera and more secretion of IFN-γ by spleen cells were detected in CpG7909-/Pe685a-immunized mice, compared with IFA-/Pe685a-immunized mice. These results showed that CpG7909 induced a strong Th1-type immune response against Pe685a in immunized mice, which is consistent with the previous reports.
The present work showed that CpG7909 enhanced the proliferation of spleen lymphocytes stimulated by ESAT6-Ag85A fusion protein, and it was found that higher proportion of CD3+CD4+ and CD3+CD8+ T-lymphocytes, especially CD3+CD4+ T cells, were induced in CpG7909-/Pe685a-immunized mice, compared with IFA-/Pe685a-immunized mice. Previous studies have shown that CpG7909 has the strong ability to induce specific CD8+ T-cell production (Rothenfusser et al. 2004; Amlie-Lefond et al. 2005). Our results of apparent proportion of CD3+CD4+ T-lymphocytes induced by CpG7909 might be related to ESAT6-Ag85A fusion protein, which has ever been confirmed to induce Th1-type response (Lu et al. 2011). Or other mechanisms might be involved in it such as activation of CD4+ T cells regulated by plasmacytoid dendritic cells stimulated by CpG7909 (Moseman et al. 2004; Jarnicki et al. 2008; Kuball et al. 2011). Therefore, CpG7909 effectively enhanced ESAT6-Ag85A fusion protein-mediated specific cellular immunity, by stimulating higher frequency of CD4+ T-lymphocytes.
We further detected transcription of TNF-α, IFN-γ, IL-6 and TLR9 in lung, the main target organ infected by Myco. tuberculosis. Previous studies have shown that CpG7909 enhanced TNF-α, IFN-γ and IL-6 expression through binding with TLR9, followed by B-cell activation and T-cell differentiation (Krieg et al. 1995; Jego et al. 2003; Gelman et al. 2006; Tross and Klinman 2008). It was found that CpG7909/Pe685a induced a significant higher expression of TNF-α, IFN-γ, IL-6 and TLR9, while IFA/Pe685a induced slight increase in TNF-α, IFN-γ and IL-6 expression in lungs. These cytokines played an important role in specific antibody production and CD4+ and CD8+ T-cell proliferation induced by CpG7909. IL-6 is involved not only in B-cell activation, but also in stimulation of T-cell proliferation and CTL activation (Jego et al. 2003). IFN-γ induces Th1-type T-cell differentiation (Liu et al. 2008). TNF-α is able to promote macrophage activation, cytokine expression and Th1-type T-cell differentiation (Zhang et al. 2002). Collectively, from above results, we suspect that CpG7909 might be able to enhance immunological responses induced by ESAT6-Ag85A fusion protein, through activation of a variety of cytokines, to prevent pneumonia infection from Myco. tuberculosis in some extent.
Finally, we evaluated CpG7909-protective effect on mice infected with Myco. tuberculosis. However, it was found that CpG7909 did not reduce bacterial load in lung and spleen and attenuate histopathological lesions in lungs. In previous studies, it has been reported that CpG1826 and CpG2041 could not confer significant protection against Myco. tuberculosis infection compared with IFA in mice, although both of them can enhance production of specific antibodies and IFN-γ as adjuvant to CFP antigen of Myco. tuberculosis (Fonseca et al. 2007). A mixture of Ag85B, HspX and fusion protein CFP-10/ESAT-6 (C/E) with a combination of CpG has also been found to elicit both humoral and cellular immune responses in mice, whereas it played only a minor role in control of disease progression in guinea pigs challenged with Myco. tuberculosis (Chen et al. 2010). However, another group reported that a low dose (1 μg) of CpG ODN-mixed MPT-1 antigen of Myco. tuberculosis has been found to play better protective role in mice against Myco. tuberculosis infection when compared with IFA (Silva et al. 2009). Contradictory mechanism of immunological and protective effect of CpG adjuvant remains to be further investigated.
Mice immunized with IFA/Pe685a displayed better protection from Myco. tuberculosis infection, despite poorer immune response indicated that comprehensive factors such as CpG decoration, immunization dose, antigen-binding proportion, immunization cycle and other factors might be considered. Incomplete Freund's adjuvant was confirmed to induce a mixed Th1-/Th2-type response that plays an important regulatory role in disease development (Hossain et al. 2001). In our research, lower level of IgG2a/IgG1 and IFN-γ in serum and less secretion of IFN-γ by spleen cells detected in IFA-/Pe685a-immunized mice might indicate the balance of Th1- and Th2-type immunological response induced by IFA, which might contribute to the protective efficacy of IFA/Pe685a immunization. How to develop CpG as an effective and safe adjuvant of subunit vaccine against Myco. tuberculosis requires further investigation. Furthermore, in the use of CpG as adjuvant, the immune pathological injury, which might be induced by CpG, needs further detailed research.
The authors would like to thank Prof. Xionglin Fan (Tongji Medical College, China) for providing ESAT6-Ag85A fusion protein and reviewing manuscript and Prof. Yanhong Liao (Tongji Medical College, China) for reviewing manuscript. This work was sponsored by Huazhong University of Science and Technology Research Fund Innovation to Hanju Huang (M2009041).
The authors declare that they have no conflict of interest related to the manuscript.


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Hindustani Covenant Church
|Hindustani Covenant Church
|Evangelical, Congregationalist
|Rev Dr Steven David
National Council of Churches in India
|Present in 12 states: Maharashtra, Karnataka, Uttar Pradesh, Orissa, Goa, Andaman and Nicobar Islands, Jammu and Kashmir, Andhra Pradesh, Gujarat, Uttranchal, Haryana and Bihar.
|Mission Covenant Church of Sweden
|17 October 1963
in Pune
|111 (2012)
|22976 (2012)
|119 (2012)
|Aid organization
|Social projects in many places through Covenant Social Service
|Secondary schools
Hindustani Covenant Church (HCC) is an evangelical, congregationalist denomination in India. It has 111 local congregations and is represented in twelve states. The highest authority is the Church Council, which meets once a year and consists of representatives from all congregations. The Church Council elects the Executive Committee. The Church Headquarters is in Pune. Ministers are educated at Union Biblical Seminary (UBS) in Pune, which educates ministers to a number of denominations.
At the World Mission Conference in Tambaram, India, in December 1938, the Mission Covenant Church of Sweden President Axel Andersson and the missionary Gustaf Ahlbert examined the possibilities to start mission work in India. Already in 1939 the MCCS decided to start mission work among Turkish peoples and Muslims in India and the following year the first missionaries arrived in Mumbai. The mission work was registered under the name of the Swedish Hindustani Mission. Later on work was also taken up in Pune and Sholapur. The first person baptised was a refugee from Xinjiang, Noor Muhammed, a physician who was given the name Luke. He helped with the translation of the Old Testament into Uighur. On October 19, 1963, 22 delegates from the three congregations in Pune, Mumbai and Sholapur, among them 6 missionaries, assembled and founded the Hindustani Covenant Church (HCC). Rev. B. Thoma became its first moderator and remained at this post up to his retirement in 1986 when he was succeeded by the present moderator, Rev Steven David.
Social work[edit]
HCC has a quite large social work. This is coordinated by a body called Covenant Social Service (CSS). Social work has comprised humanitarian aid in case of catastrophic events like floodings and earth quakes, community development, health care and education. In 1981, HCC founded St. Luke's Medical Society (SLMS), in order to provide health care. SLMS has two hospitals, a smaller one in Nannaj near Sholapur and another one in Aurad, near Gulbarga in Karnataka. HCC also works with education. The Church runs three High schools, in Gulbarga, Nannaj and Sholapur and one Junior College of Science in Solapur. There are also a number of informal education projects for the poorest children. Besides, CSS offers courses leading to certain professions in order to help people to find a sustainable livelihood.
See also[edit]
- Fält, H. (2004).Mission i religionernas land, Svenska Missionskyrkan, Stockholm.
- Svärd, Lydia (1979).Förbundskyrkan i Indien, Gummessons, Stockholm.


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TwentyFifteen Interview 02/20: Lim Weixiang speaks to Leonard Goh
How did you get interested in photography?
When I was a student, my friend had free tickets for a documentary about Magnum co-founder Robert Capa. The film really left a significant impression on me, but film and development costs then were too expensive for me to pursue photography actively.
When I graduated from the National University of Singapore in 2007, I bought my first digital camera – a Nikon D40 – and that was when I became more serious about photography. Making photos became an excuse for me to indulge my innate curiosity. I think we all want an outlet to express ourselves. Photography happens to be my medium of choice.
How and when did you get interested in documenting the coastline of Singapore?
It happened by accident. I was at Punggol Beach with my then-girlfriend to see if we could find some sort of resolution and while we were there, I made a picture of her and her dog, Coco, looking out wistfully to sea.
When the roll of film was developed, I thought I might have something there, so I continued going out to the coastline to shoot. I found the process interesting and, to a certain extent, cathartic.
From the way you describe the your ex-girlfriend, right down to the details of the dog, it sounds like if you weren't there with her then, you wouldn't have started on OUR COASTLINE.
Would you say that love is the primary driver for this project?
Yes, it began in some ways as a 'break-up' project. We could have gotten married, but I froze and I couldn't understand why.
So around December 2011, I started going out to the coastline, hoping for answers, and the photos initially, I would say, reflected that: people or landscapes which looked lost.
While it started off as an emotional exercise, it slowly evolved over the last year and a half, to include my feelings about living in Singapore, like the state of our education system or our ageing population – other kinds of 'searching'.
Tell us a little something about some of the people in the photos.
I hope it's not me projecting. But the people in the photos, they all seem to be either looking for something or waiting for something, which I guess resonated with me as I was going through a hard time emotionally when I began.
When I made the photos, I was looking for photos that had resonance with how I felt. It was a bag of feelings, of longing for the past, for simpler times. I was questioning my own decisions and wondering what the future would hold. Photography was an outlet for those emotions.
What are some challenges you faced in creating OUR COASTLINE?
In a way, the photos are random encounters along Singapore's coastline. To bring cogency to the images, I tried to get a similar look to the photos and one important aspect was how the images were lit. I had to wait for the right kind of weather, which would give the right kind of light, before I could head out to the coastline to make photos.
Also, because the photos are based on a certain feeling (as I mentioned earlier), there were many occasions when I would spend hours at a certain part of the coastline searching and waiting for the shot that would encapsulate how I felt, but ended up with no photos to show because the scenes just didn't look or feel right.
How did you decide on which subjects to capture or focus on for this project?
I chose my scenes based on how they made me feel. It was an intuitive process. If the scene didn't feel right, I wouldn't capture it. I found that I knew instinctively, even before I made the shot, if it was going to be a keeper.
When I switched to the digital medium later, I could afford to take more risks and made more frames – which meant the project evolved to become more than an emotional process. It became more of a documentary and I began to capture the quirky side of life along the coastline.
You started OUR COASTLINE shooting in medium format but abandoned it in the end. Can you tell us more about this choice or process?
Initially I used a medium-format camera, the Mamiya C330, because I wanted to try a different format, and also have the option of being able to make big prints.
I found that shooting with a TLR slowed down the picture-making process. There are only 12 frames in each roll of film and that forced me to be more selective. It was good training.
Using the TLR was also stealthier. Most people nowadays don't recognise the TLR as a camera anymore, so I could make pictures without people realising it or becoming self-conscious.
Later, I switched to the Nikon D800 because its 36 megapixels allowed me to make a square crop while having enough pixels to still make large prints. In fact it gave me better files because it cut out the scanning process. All the photos in this book were made with the D800.
The trade-off from switching to digital was that I lost stealth. But by then, I knew what what I was looking for, and I worked out how to be less conspicuous as a photographer, by pre-focusing and shooting from the hip.
Can you share with us your process for going out to document the coastline?
Firstly the weather or light has to be right. Then to get into the right mood, I would listen to some music. One of the songs I listened a lot to was "Sovereign Light Cafe" by Keane. I made it a point to visit a different part of the island each time. I started at Punggol and went around in a clockwise manner, so after that Lorong Halus, Pasir Ris, Changi, etc. But there are only 194 km and a lot of it is out of bounds, so over the two years, I made many repeat visits to certain sites.
So how much have you covered so far?
I don't keep track of the exact distance I have covered. But I would say I have visited most of the accessible parts of the coastline. Google Maps is a very good tool I rely on when I plan where to visit. It helped me to discover some of the more obscure parts of the coastline, such as Sungei Kadut or Coney Island.
Why are the photographs in OUR COASTLINE important to you?
The 15 photos are like a microcosm of Singapore today. While I did not intend for them to be so when I made the pictures, a lot of them can be seen as metaphors reflecting the growing pains of contemporary Singapore. On a more personal level, photographing the coastline was a way for me to sort out my feelings. When I look back at some of the more memorable photos, I can still remember how I felt when I made them.
Which part of the coastline for you was the most memorable to document?
They are all memorable, but the Marina Bay area is significant because at this moment, the area is in transition. So much construction is going on and in a year or two, it will look completely different. I will always remember the four weekends spent at the Marina Barrage, around Marina South Pier and memorising the fly-by times of the Chinook flag-carrying convoy and the fighter jets for National Day.
Has OUR COASTLINE ended, or are you going to continue shooting it? Do you have a plan to complete the full circle?
I think I will carry on until the end of the year before moving on to something else. It may sound arbitrary, but I just have a feeling that the project is near its end. Perhaps a few years from now, I can return to photographing the coastline and we will be able to compare and see how much has changed.
You have spent close to two years photographing the coastline. What do you want to say to people who feel there is nothing much to photograph in Singapore?
If you care about Singapore, and have something you really want to say about Singapore, and you spend time thinking and refining your message, your work will be unique. So really, it's not about the size of the island.
The portrait of Lim Weixiang was drawn by Flee Circus; and Leonard Goh, who will anchor all the interviews in this series, is a co-founder of Platform.


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Meet the banker and the scientist behind the UK's first...Meet the banker and the scientist behind the UK's first...Britain's first sake brewery is quickly building a reputation for itself.The brainchild of Tom Wilson and Lucy Holmes, Kanpai is set to begin...Score:1, Heat: 0, Docs:1, TopicId:865214569790611456 Published: 2017-05-18T14:02 URL: https://uk.finance.yahoo.com/news/meet-banker-scientist-behind-uks-japanese-sake-brewery-140247305.html Source id: Gb6c62
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Traveler's guide to breweries, distilleries at the...Traveler's guide to breweries, distilleries at the...These days, many of us want to know the stories behind the goods we buy, whether it's food, clothing or, yes, even alcohol. A visit to a beach...Score:1, Heat: 0, Docs:1, TopicId:865191467496513536 Published: 2017-05-18T12:34 URL: http://www.baltimoresun.com/travel/beaches/bs-fe-breweries-distilleries-beach-guide-20170510-story.html Source id: 9P8-X1
Source Title: The Baltimore Sun
Strong Family Ties Bind Upstart Indian Valley Brewing...Strong Family Ties Bind Upstart Indian Valley Brewing...The family tale behind Novato's newest brewery is fuzzy and warm in addition to being sudsy and cool.Although you can underline and boldface the...Score:1, Heat: 0, Docs:1, TopicId:865112024074199040 Published: 2017-05-17T23:55 URL: http://www.marinscope.com/novato_advance/lifestyles_entertainment/strong-family-ties-bind-upstart-indian-valley-brewing-co/article_f1e759a6-3b2f-11e7-bbbd-2b70d9534f04.html Source id: tDTG3v
Source Title: Ross Valley Reporter
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'It is only a bruise' A carefree Russian official has what seems to be a trivial accident...One of 46 new books in the bestselling Little Black Classics series, to celebrate the first ever Penguin Classic in 1946. Each book gives readers a taste of the Classics' huge range and diversity, with works from around the world and across the centuries - including fables, decadence, heartbreak, tall tales, satire, ghosts, battles and elephants.
Leo Tolstoy was born in 1828 in the Tula province. He studied at the University of Kazan, then led a life of pleasure until 1851 when he joined an artillery regiment in the Caucasus. He established his reputation as a writer with The Sebastopol Sketches (1855-6). After a period in St Petersburg and abroad, he married, had thirteen children, managed his vast estates in the Volga Steppes and wrote War and Peace (1869) and Anna Karenina (1877). A Confession (1879-82) marked a spiritual crisis in his life, and in 1901 he was excommuincated by the Russian Holy Synod. He died in 1910, in the course of a dramatic flight from home, at the railway station of Astapovo.
More books by Leo Tolstoy
Quite a decent read, never having read Tolstoy before. I always thought his books were very heavy but this was surprisingly easy to read.
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Richard talks about the darkness lurking in his past and its connection to his role as a Nazi loot-hunter at the auction house, Sothebys.
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On National Brothers Day, Stuart Heritage reflects on fraternal dynamics, how he came to write about the 'whirlwind of aggressive single-mindedness' that is his brother Pete and how their relationship is reflected by that of brothers everywhere.


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Visit America's largest home, The Biltmore Estate, in Asheville, NC.
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Everyone needs a loan from time to time, and most of these times, the personal loan is the best choice that one can make. The reason is very simple – you don't need to tell the bank what you do with the money, you're not restricted to using the money for a strict purpose and the debt interest can be either fixed or variable.
Let's see some things that you'll need to watch out for if you want to take a personal loan from the bank.
Small Loans
It's better to take a smaller loan or exactly the sum of money that you need for your purpose. The thing is that, in general, because these personal loans are so easy to access, people are tempted to ask for more money. The next thing you'll know is that you'll pay a higher rate and you'll give back more money than you've thought. Go for smaller loans and pay them back as fast as possible.
Fixed Rate
If you choose a fix debt interest, you'll know from the start how much money you'll pay each month. Apart from this, the rate will not increase or decrease, and the total sum won't differ from what you're told.
Over Payments
Make sure that you have the possibility to pay the loan faster – two or three or more rates at once. You will have to make a written request about this, but it is worth it. The faster you pay your credit, the lower debt interest you will give back to the bank. Keep in mind that at first, you will pay more interest debt rate than the actual credit if you pay one rate per month. However, if you pay two or three rates, meaning a bigger sum of money, they'll take just the interest for that month, the rest of the money paying the actual credit (the main sum of money).
The Contract
You may know in general terms how the credit will work over time, but make sure you read the contract before signing it. You should also read what's written in fine print because usually, the banks add different clauses that are not in your best interest. Keep your eyes on the financial news and see what should and shouldn't be there – in your contract. That's important because the bank can ask for an unauthorized debt interest or other commissions that are not legal or according to your national financial policy.
In the end, the best advice would be to avoid using a credit, if it's possible. However, if you really need the money, make sure you can cover all the credit for the entire period of time, otherwise, the bank will apply enforcement over your goods and your salary, and it's not a situation where you'll want to be in.


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Albornoz Palace Hotel Spoleto, Spoleto
- Free buffet breakfast, free WiFi, and free parking
Main amenities
- 96 guestrooms
- Seasonal outdoor pool
- Bar/lounge
- Fitness center
- Massage/treatment rooms
- Business center
- 24-hour front desk
- Air conditioning
- Front desk safe
- Snack bar/deli
- Laundry service
- Conference space
For families
- Cribs/infant beds (surcharge)
- Private bathroom
- Free toiletries
- Laundry facilities
- Hair dryer
- Elevator/lift
What's around
- Heart of Spoleto
- Rocca Albornoziana (0.5 mi / 0.8 km)
- Ponte delle Torre (0.7 mi / 1.1 km)
- Spoleto Roman Amphitheater (1.1 mi / 1.7 km)
- San Pietro extra Moenia (0.2 mi / 0.3 km)
- Piazza della Liberta (0.4 mi / 0.7 km)
Albornoz Palace Hotel Spoleto, Spoleto
Hotel Info: 866-539-8117from $72
- Deluxe Double Room Single Use
- Economy Double Room Single Use
- Deluxe Double or Twin Room
- Economy Double or Twin Room
- Comfort Double Room Single Use
- Comfort Double or Twin Room
- Superior Double Room Single Use
- Superior Double or Twin Room
More great choices
At a glance
Key facts
Hotel size
- This hotel has 96 rooms
- Check-in time starts at 2 PM
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- One child (3 years old and younger) stays free when occupying the parent or guardian's room, using existing bedding
- Pets allowed
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking (limited)
In the hotel
- Free buffet breakfast
- Bar/lounge
- Snack bar/deli
- Room service (during limited hours)
- Outdoor seasonal pool
- Fitness facilities
- Spa treatment room(s)
- Golfing nearby
- Rafting nearby
- Business center
- Conference space
- 24-hour front desk
- Dry cleaning/laundry service
- Laundry facilities
- Multilingual staff
- Elevator/lift
- Safe-deposit box at front desk
- Television in common areas
- Accessible bathroom
- In-room accessibility
In the room
- Air conditioning
- Minibar
- Private bathroom
- Shower only
- Bidet
- Free toiletries
- Hair dryer
- Cable TV channels
- Free WiFi
Also popular with travelers from the USA
Albornoz Palace Hotel Spoleto, Spoleto's small print
Also known as
- Albornoz Palace
- Albornoz Palace Hotel
- Albornoz Palace Hotel Spoleto
- Albornoz Palace Spoleto
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport.
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 1.50 per person, per night, up to 4 nights. This tax does not apply to children under 14 years of age.
Optional extras
Cribs/Infant beds are available for EUR 15 per day
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Is the description of this hotel not correct? Tell us


text:
Lyrics Depot is your source of lyrics to Bucket Bong by Frenzal Rhomb. Please check back for more Frenzal Rhomb lyrics.
Bucket Bong Lyrics
Artist: Frenzal Rhomb
Album: Sans Souci
The band has stopped the night is young...
This bar is closed but there'll be more to come...
She whispered softly. "I don't wanna be alone...
Can me and all my friends come back for cones?
I called ahead I felt so cheap
Just to make sure my grandma's sound asleep
I shoulda noticed that her look was kinda smug
She only wants me coz I got the drugs
My mouth was dry my knees were weak
She held my hand as we walked down the street
I didn't give a shit she got my first name wrong
She only wants me for my Bucket Bong
She only wants me for my Bucket Bong
And when we're at home I'm feeling scared
Coz there's no bucket, sink or (vessel?) anywhere
And if she wants a bucket I'll have the last one
And she'll hafta have it with me in the bath
She'll hafta have it with me in the bath
She only wants me coz I got the drugs....
Frenzal Rhomb Lyrics
Frenzal Rhomb Sans Souci Lyrics
More Frenzal Rhomb Music Lyrics:
Frenzal Rhomb - Cheer up Lyrics
Frenzal Rhomb - Cocksucker Lyrics
Frenzal Rhomb - I'm A Backwards Fucken Useless Piece Of Dogshit... And I Vote Lyrics
Frenzal Rhomb - Not So Tough Now Lyrics
Frenzal Rhomb - She's Not Happy Lyrics
Frenzal Rhomb - There's your dad Lyrics
Frenzal Rhomb - War Lyrics
Frenzal Rhomb - Wha' Happened? Lyrics


text:
Do you want to make Ohio.com your homepage?
By Jewell Cardwell
Beacon Journal staff writer
Retired cardiologist and author, Dr. Terry Gordon, talks about his work getting AEDS in schools all over Ohio during an interview at his Richfield home. Gordon, who is also the force behind the United Way's popular DOCS WHO ROCK program, is this year's recipient of the Polsky Award. (Ed Suba Jr./Akron Beacon Journal)
Terry Gordon performs as various personna, such as John Lennon with Yoko Ono, during a Docs Who Rock concert. (Photo courtesy of Terry Gordon)
Terry Gordon performs as various personna such as Lady Gaga during a Docs Who Rock concert. (Photo courtesy of Terry Gordon)
Click here to read or leave a comment(s) on this story.
When 6-year-old Blakely Kopatz first saw blood in her urine in January, her mother didn't know what to make of it.


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Civil Litigation Questions & Answers by State
- Alabama
- Alaska
- Arizona
- Arkansas
- California
- Colorado
- Connecticut
- Delaware
- District of Columbia
- Florida
- Georgia
- Hawaii
- Idaho
- Illinois
- Indiana
- Iowa
- Kansas
- Kentucky
- Louisiana
- Maine
- Maryland
- Massachusetts
- Michigan
- Minnesota
- Mississippi
- Missouri
- Montana
- Nebraska
- Nevada
- New Hampshire
- New Jersey
- New Mexico
- New York
- North Carolina
- North Dakota
- Ohio
- Oklahoma
- Oregon
- Pennsylvania
- Puerto Rico
- Rhode Island
- South Carolina
- South Dakota
- Tennessee
- Texas
- Utah
- Vermont
- Virginia
- Washington
- West Virginia
- Wisconsin
- Wyoming
Civil Litigation Questions & Answers
Q: If a person went to jail for domestic violence and battery and my residence this person's house and they have a no
Answered on May 28, 2017
Your right to remain at that property is based upon your legal status as either an owner of the property or a legal tenant. If you are not an owner or do no have a valid lease. It is my opinion that you can be evicted legally.
Q: Can a person evict me in fl living in his house if I don't have a lease agreement
Answered on May 28, 2017
Short answer is yes. You can be evicted from a house even if you do not have a signed lease. You do not have a right to live in someone else's property unless they agree for you to remain there.
Q: My mother passed awayvand left my sister and i 2 homes .now my sister is selling the homes with out my permission.
Answered on May 26, 2017
Probably not, but it depends on all the facts, and the way the properties were left. Did they pass by will or by trust? Was your sister appointed administrator or executrix of the estate? These are things you should discuss with a probate attorney..
Q: I am in a dispute with my neighbor.
Answered on May 26, 2017
That is a really hard question to answer without more details (which is probably why no one has answered it for you). But if you provide more details, I will try to give you a better answer.
Q: Is there a specific order I need to file claims if I plan to file a criminal and civil suit against someone who caused
Answered on May 26, 2017
There is no order to file but most lawyers will want criminal proceedings to conclude before civil proceedings go to trial. But due to statute of limitations you cannot wait to file either.
Q: Can the County give an infraction to a visitor, even if the visitor is from a U.S. territory?
Answered on May 25, 2017
Yes, the county can cite anyone for a traffic infraction, whether they have a driver's license or not, own the car or not, or are here from another country. The fact that they did not know what the law was is not a defense.
Q: the proceeding is my right to recover real property under my rent to own lease, her counter claim is that my lease is
Answered on May 25, 2017
If she copied and pasted your signature without your consent that does sound alot like fraud to me, but the facts you lay out sounds like she is claiming you committed fraud. Obviously there is a lot more going on here than you have written and a supreme court action concerning real estate almost always gets complicated. Written agreements always prevail in Real Estate, proving fraud is difficult, but not impossible. Best of luck.
Q: I need to produce inspection of a original lease document. I would like to object, I don't trust the other party,
Answered on May 25, 2017
It depends on the type of proceeding and the form of the demand. If you are in a court case, you are going to have to do it.
Q: Can a county become a defendant in a public nuisance action?
Answered on May 25, 2017
There may be another remedy, such as taxpayer action, but there is no private right of action for a public nuisance (with a very limited exception). The State may be able to bring such an action against a county, but not a private citizen.
Q: My ex hasn't kept her end of the shared parenting agreement. Can I get full custody for this?
Answered on May 25, 2017
You can file a motion with the court asking to change the parenting agreement. The court will consider all the circumstances, and decide if a change is in the best interest of the child. Talk to your divorce attorney or use the Find a Lawyer tab to consult an attorney about the situation.
Q: Subject Matter Jurisdiction (personal)
Answered on May 25, 2017
No, appearance in court without contesting personal jurisdiction is consent and there's no "duped" exception.
Q: Can a parent sue a school district for retaliatory actions when the parent has exposed violations of law?
Answered on May 24, 2017
The school district may be in violation of your daughter's civil rights to an equal educational opportunity for failing to provide for all her educational needs. This is a highly specialized area of law, and you need counsel with experience handling education law issues. In and around the bay area you should be able to find highly capable lawyers or law firms with the expertise to represent your child in such matters. No matter how much you have learned on your own, you will never know as much...
Q: I have a judgement against someone called a warrant in debt. It was issued in 2003. Can I still collect?
Answered on May 24, 2017
The answer is "it depends." In Virginia, a judgment on a warrant in debt is good for 10 years in the General District Court. If you recorded a certified copy of the judgment abstract for a small fee of $10.00 in a Circuit Court in Virginia, the judgment's life will be 20 years in the Circuit Court.
Q: How do I retrieve a vehicle from a mechanic that will not return calls and was prepaid 6 months ago?
Answered on May 24, 2017
File a lawsuit- in Tennessee, you can do this in "General Sessions Court" the court clerk may have a form you can use- it would be called something similar to " action to recover personal property" or if they don't have that form, use a "civil warrant" and just describe what you want ( identify the vehicle, make, model, year etc). There will be a fee to be paid at the clerk office when you file and you will need the address of the mechanic so the sheriff can deliver the court papers to him. if...
Q: If I break my lease and pay the lease termination fee, when is that fee due? In Oregon.
Answered on May 23, 2017
Leases are contracts so the terms of you contract should govern what the consequences are of breaking your lease. If the contract doesn't specify you and your leasing agent should agree on something, but within 30 days would be reasonable, although not necessarily a rule. What you don't want is to drag it out so your leasing agent has to file a law suit which will show up on public records and could hurt your credit even if you eventually pay the fee in full.
Q: Seeking legal advice on how to protect myself. Got involved with a woman at work and she is threatening to "get revenge"
Answered on May 22, 2017
You may want to talk to either your supervisor or someone in HR to explain the situation and to advise that you are now being harassed. You need to consult with someone at work in whom you feel comfortable confiding. But you also need to protect yourself with human resources. Honesty is the best policy. You need to get right both at home and at work. Now you know why work relationships are discouraged.
Q: Pujury took place at hearing, how do I address that issue in closing arguments?
Answered on May 22, 2017
Why don't you have a lawyer representing you? Regardless, unless the evidence is self-authenticating you may not be able to introduce it into the record because you didn't raise/intro. it during hearing and Closing Argument is not the proper part of the process to bring in evidence. You could raise the issue in Closing, but the evidence is trickier.
Q: Can law enforcement pursuit a search warrant on a residence if nobody was at residence at time of search and seizure
Answered on May 21, 2017
Yes. There is no requirement for anyone to be at home, and is often preferred by law enforcement for safety. A search warrant is only for a location that has been allowed by judicial review and probable cause.
Q: I noticed my tax lady has omitted and reported smaller amount that what I have actually paid. what can i do?
Answered on May 20, 2017
Depending on the type of tax returns she prepares for you; the tax preparation fee may not have a material impact on the tax liability. Ask her to determine the tax impact of all the items left out, and amend the returns accordingly. On a separate note; why would you work with someone you described? The information presented herein is for general purposes only. It is not intended to, and may not be construed as legal, tax or accounting advice. Neither is it intended for solicitation purposes....
Q: a relative went to psychiatrist in FL. She decided to admit him to the hospital. does he have any rights to be released?
Answered on May 19, 2017
There must be some fear on the part of the Psychiatrist that the person is a danger to himself or others. You may want to speak with a guardianship attorney in your area, or rather where your relative is in the facility to determine what the next step might be. the attorney might be able to find out more details from the facility.
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Rustic cooking utensils
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@shahrouzt: My dad recently fell for one of those Nigerian prince scam emails. I feel bad for him but I really needed the money.
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