The school is located on the east edge of the beautiful Flathead Valley between Columbia Mountain and the Flathead River. The old building on the campus was built in 1904, making it the oldest, standing, in-use school building in the valley. In 1968 a quonset hut was built to serve as a temporary gym. A new building was added in 1984 on the north border of the campus on land donated by Orville and Joyce Baumgartner. The newest building was later enlarged with the addition of 4 classrooms. A gym, built largely by volunteer effort, was added to the east end in 2001.
Deer Park is a tobacco free school.


text:
Record Them: The Herts company making audio documentaries of people's lives
Words & pictures: Julie Lucas
Sylvia (left) with a friend petting a carthouse at the former blacksmiths in Church Lane, Stevenage in the 1940s
Julie Lucas
In our increasingly fast-paced world, we rarely have time to sit with loved ones and hear about times gone by. Enter a St Albans company aiming to make sure those memories are not lost
'I'm not sure if it was Donk or Honk,' my mother says. She is remembering the donkey she had as a child. 'My mother used to bring him and I'd ride him home from school. He wore a hat and we would go for a picnic.'
She is in her living room in Walkern talking about her life for Record Them, a St Albans company specialising in audio histories of loved ones, and it's quite a revelation. At 79, Sylvia Richardson was born during the war, in 1940, at the grand arrival point of Brocket Hall near Welwyn, which was used as a maternity hospital at the time. Known as a 'Brocket baby' she used to be invited every so often for tea at the stately home and to see the room where she was born.
She chats animatedly about her house in Hatfield being bombed during the war. The windows blew out but she was saved from injury by the blackout screen falling on top of her cot, shielding her from the glass. She remembers her father carrying her down the stairs. She describes her brother Bobby making a homemade bomb in the garden, and her favourite house in Bishop's Stortford.
Dave sets up the recording at Sylvia's Walkern home. Putting interviewees at ease is key (photo: Julie Lucas)
Across from mum is Dave Creasey, who launched Record Them last year. A former BBC radio producer, the 27-year-old also did a stint presenting his own show in Cyprus with the British Forces Broadcasting Service, where he says he heard some fascinating stories. He describes Record Them as a Desert Island Discs for the ordinary person. As he sets up a mini recording studio around my mother, his friendly and relaxed demeanour immediately puts her, and me, at ease.
Dave came up with the idea a few years ago when a family friend lost her son.
'She said to me I can't remember the sound of his voice. She had a voicemail from him and used to listen to it, and after a while they get wiped. She said effectively on that day she lost him all over again.
'I've worked in this sort of game for a long time and we have made thousands and thousands of hours worth of audio that doesn't mean much, so thought why not do something that means something.'
The recordings are made at the interviewee's home and typically last around three hours, which is then edited down to 40 to 60 minutes. He mixes the dialogue - editing out the inevitable pauses and ums and ahhs - with sound effects, archive newsreels and music personal to the interviewee, to capture a snapshot of a loved one's life. The recording is presented on a CD and, appropriately, on a memory stick.
One of the musical pieces my mum chose is from Prokofiev's Peter and the Wolf. A popular choice, Dave says. He genuinely seems interested in what mum has to say. 'I like meeting older people because they have lived through a period that I haven't,' he explains.
Sylvia with her mum Marjorie holding writer Julie as a baby in Shephall, Stevenage in 1968
The first person he recorded was his partner's great uncle Bill, an 85-year-old Londoner who remembers the Blitz and could 'really spin a yarn'. Since then he has heard remarkable life stories from a former Foreign Office minister to an ambassador to the United Nations and a Spitfire pilot in the Second World War.
'I had a lady who was 101. She was an ambulance driver during the war and she was walking down a lane one day and there was a German Luftwaffe firing guns at her.
'Sometimes we have people who have stories about being at Dunkirk or meeting the Queen and some people have a normal life like me and you. The main point is not about the grand stories, it's more about what you want your kids to know.'
Syvia recording her memories at home (photo: Julie Lucas)
And this passing on to the next generation, and beyond, is a kind of treasure.
'They really think about things and the real nuggets you get are how that person feels about life. I think from the sound of someone's voice you can get to know who someone is. It's also a chance to ask things that maybe you wouldn't normally ask. You wouldn't go out for a lunch in a pub and say, "What were you like as a kid?". I find that audio is such an overlooked thing, people take photos or video. But unlike video, audio is a more relaxing medium which can be edited.'
He encourages his interviewees to talk openly and finds they often open up to someone they don't know - he has heard stories that even the family didn't know.
Sylvia on her wedding day to John at St Nicholas Church, Stevenage in January 1963
Has he recorded his own story? 'Yes,' he says laughing, 'to ensure I'm not forgotten!' He adds, 'It's an amazing tribute to someone. A lasting gift - can you imagine hearing your great-great-grandparents? I tell people that a recording will last longer than you or me or anyone else.'
For my mother, it made her think about things she had done long ago. Although hesitant at first she was soon chatting to Dave like he was an old friend, who every so often would ask her a question.
'Although my memory is not what it was, when I started to talk memories came flooding back to me,' she says. 'I would have loved for my mum to have done something similar.'
Sylvia on her wedding day 1963
At the end of the recording session Dave asks her some quick fire questions. What sound does she love? 'Birdsong,' she replies. What profession other than her own would she have liked to have done? 'I would love to have run a nursery as I like little children; little children are just so surprising.'
During the last half an hour my mother records some messages for family members. I feel quite emotional and have to leave the room. After listening to them later, I suggest anyone who does this to have a glass of wine and a box of tissues at the ready.
Having lost my father seven years ago, to hear his voice and hear him talk about his life again would be immeasurable. It's not until a loved one has gone that you realise how important it is to have their memories. Like images of them in your mind, their stories begin to fade. I now have something to cherish and as my mother's memory fades, which is now happening, her memories won't. u
Sharing a family album with Dave (photo: Julie Lucas)
Record Them recordings cost £899. Visit recordthem.co.uk for details.

redpajama_set_name: RedPajamaCommonCrawl


Polis, the network of European cities and regions for innovative transport solutions, and H3B Media, publishers of Thinking Highways magazine, are pleased to announce the launch of a joint venture multimedia project, Thinking Cities.
Thinking Cities is a print and online publication, a tablet app, a website, a podcast and a webinar series, focusing on shaping thought in intelligent innovation in transport for cities and regions. The magazine will be launched at the 2013 Annual Polis Conference in Brussels on 4 December.
Never before have urban and regional planning authorities had so much information and data at their disposal. Shaping integrated strategies addressing economic, social and environmental aspects requires access to and exchange of information as well as the right framework for making the best use of it. The keys to the truly Thinking City are now at the fingertips of those responsible for the design, planning and integration of our urban spaces and regions. Thinking Cities brings together the vision, the tools and the technology cities and regions need to find sustainable solutions that answer the challenges deriving from demographic development and climate change, and to successfully implement their policy agenda.
For more information on Thinking Cities please contact Dagmar Röller from Polis at DRoeller@polisnetwork.eu and Kevin Borras, editor-in-chief at kevin@h3bm.com.
A landing page for the website, http://thinkingcities.com is online now and the full site will be launched in due course, featuring details of forthcoming Thinking Cities-related events. A media pack is available upon request.


text:
La Société sportive de Nilvange ou SS Nilvange est un club de basket-ball français basé à Nilvange, aujourd'hui disparu.

Histoire

Le club a appartenu pendant 4 saisons à l'élite du championnat de France, pour un bilan de 10 victoires, 2 matchs nuls et 60 défaites en 72 matchs.

Lors de la saison 1972-1973, l'américain Bob Thate inscrira 38,9 points de moyenne sur la saison, un record en élite française. Il a également marqué 63 points contre Antibes soit la  sur un match de l'histoire du championnat de France. De plus, il est le premier joueur à avoir marqué plus de 50 points à 4 reprises. À l'issue de la saison 1972-1973, Bob Thate a rejoint le club de Caen, où il n'a plus connu la même réussite.

Lors de la saison 1971-1972 de Nationale 2, il marque 74 points contre Sochaux lors d'une victoire 139 à 101 le .

Historiquement, la SS Nilvange disposait de deux salles : une au Konacker dédiée à l'entraînement des équipes de jeunes et la mythique salle de la rue de la Moselle, baptisée récemment "Albert Bassetto", en hommage au meilleur de ses joueurs, longtemps capitaine de l'équipe première masculine.

Palmarès
 Champion de France de Deuxième division : 1933

Entraîneurs successifs
Pour les saisons passées en Nationale, Nationale 1
 1951-1954 : Laszlo Rebay
 1965-1966 : Robert Ostertag
 1972-1973 : Roland Erbel (Après 8 matchs (0V-8D), en désaccord avec l'équipe il est remplacé par le joueur Gérard Kulinicz mais revient après 2 matchs (0V-2D))

Joueurs célèbres ou marquants
  Jean-Claude Bonato
  Jean-Pierre Conter
  Jean-Marc Conter
  Bob Thate
  Jerry Clukey
  René Fruitier
  Frédéric Weis
  Albert Bassetto
  Yves Gachet
  Thimothée Sorel

Liens externes

Nilvange
Nilvange
Sport en Moselle

redpajama_set_name: RedPajamaWikipedia


text:
Midkine promoter-based conditionally replicative adenovirus therapy for midkine-expressing human pancreatic cancer
Eiji Toyoda1,
Ryuichiro Doi1,
Kazuhiro Kami1,
Tomohiko Mori1,
Daisuke Ito1,
Masayuki Koizumi1,
Atsushi Kida1,
Kazuyuki Nagai1,
Tatsuo Ito1,
Toshihiko Masui1,
Michihiko Wada1,
Masatoshi Tagawa2 &
Shinji Uemoto1
To develop a novel therapeutic strategy for human pancreatic cancer using a midkine promoter-based conditionally replicating adenovirus.
We examined midkine mRNA expression and midkine protein expression by seven human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, PANC-1, and Suit-2), as well as by non-cancerous pancreatic tissue and pancreatic cancers. Midkine promoter activity was measured in cancer cell lines by the dual luciferase reporter assay. Adenoviral transduction efficiency was assessed by fluorescent staining of cancer cell lines using adenovirus type 5 containing the green fluorescent protein gene (Ad5GFP). Replication of adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) was assessed by the detection of E1 protein in cancer cell lines. The cytotoxicity of Ad5MK for cancer cells was evaluated from the extent of growth inhibition after viral infection. Infection and replication were also assessed in nude mice with subcutaneous Suit-2 tumors by intratumoral injection of Ad5MK, Ad5GFP, or vehicle. E1a mRNA expression in the treated tumors and expression of the replication-specific adenoviral hexon protein were evaluated. Finally, the anti-tumor activity of Ad5MK against intraperitoneal xenografts of Suit-2 pancreatic cancer cells was examined after intraperitoneal injection of the virus.
Both midkine mRNA expression and midkine protein expression were strong in AsPC-1 and CFPAC-1 cell liens, moderate in BxPC-3, HPAC, and Suit-2 cell lines, and weak in PANC-1 and MIAPaCa-2 cell lines. Expression of midkine mRNA was significantly stronger in pancreatic cancers than in non-cancerous pancreatic tissues. The relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cell lines was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cell lines. Pancreatic cancer cell lines exhibited a heterogeneous adenoviral transduction profile. E1A expression was higher in cell lines with strong midkine expression than in cell lines with weak midkine expression. Ad5MK showed much greater cytotoxicity for midkine-expressing Suit-2 and PANC-1 cell lines than for midkine-negative MIAPaCa-2 cell lines. In the Suit-2 subcutaneous xenograft model, expression of E1A was detected in Ad5MK-treated tumors, but not in untreated and Ad5GFP-treated tumors. In the Suit-2 intraperitoneal xenograft model, the Ad5MK group survived for significantly longer than the Ad5GFP, PBS, and untreated groups.
Ad5MK has an anti-tumor effect against human pancreatic cancer cell lines that express midkine mRNA. Midkine promoter-based conditionally replicative adenovirus might be a promising new gene therapy for pancreatic cancer.
Pancreatic cancer is one of the most lethal malignant tumors, and it was estimated that approximately 200,000 people died of this cancer worldwide in the year 2000 [1]. It is the fifth leading cause of cancer death in Japan and the fourth in the United States [2]. Unfortunately, the pancreas is located at an inaccessible site within the abdomen, making the diagnosis of pancreatic cancer more difficult than that of other digestive tract cancers. Therefore, most patients with pancreatic cancer are diagnosed late after progression of their disease. Furthermore, pancreatic cancer frequently infiltrates neighboring tissues or vessels at an early stage, leading to a poor prognosis.
In the United States, the 1-year and 5-year survival rates of patients with pancreatic cancer are less than 25% and 5%, respectively [3]. At present, surgical resection provides the only chance of cure for these patients, but it has been reported that about 90% of patients do not undergo pancreatic resection and 58% are only given palliative treatment [4]. Moreover, recurrence after surgical resection is very common. We do not have any effective nonsurgical treatments for pancreatic cancer, because it shows strong resistance to the currently available chemotherapy and radiotherapy protocols [5]. In order to improve the clinical outcome, new modalities for the treatment of this disease are required.
Gene therapy using oncolytic viruses is one of the approaches that should be considered. The strategy of this therapy is to exploits the lytic property of virus replication to kill tumor cells. Recent knowledge of molecular biology makes it possible to modify viruses to target specific molecules or signal transduction pathways in cancer cells. Oncolytic viruses which are developed to be able to infect and replicate selectively in malignant tumor cells can spread and destroy malignant tumors without adverse effects in normal tissues.
To achieve tumor-selective viral replication, one approach has been the replacement of endogenous viral sequences with a tissue- or tumor-specific promoter. A number of tumor promoter genes such as α-fetoprotein [6], carcinoembryonic antigen [7], erbB-2 [8]and prostate-specific antigen [9, 10] have been used to restrict the expression of suicide genes both in vitro and in vivo.
Midkine is a heparin-binding growth factor that is induced by retinoic acid in embryonal carcinoma cells, and it may be another candidate for this purpose [11]. The biological roles of midkine are diverse and it is closely linked to neural development [12, 13] as well as to the pathogenesis of neurodegenerative diseases. At the same time, midkine is involved in the development of cancer because of its mitogenic effect [14], promotion of angiogenesis [15], anti-apoptotic activity [16], fibrinolytic activity [17], and transforming activity [18].
Midkine expression is increased in a number of malignant tumors, including esophageal, stomach, colon, hepatocellular, breast and pancreatic carcinoma, when compared with the level of expression in adjacent non-cancerous tissues [19–22]. In contrast, the expression of midkine in normal human tissues is quite limited, with moderate expression in the kidneys and weak expression in the lungs, colon, and thyroid gland [19, 20, 23].
On this basis, the midkine promoter could be a potential candidate for use in suicide gene therapy. Here, we demonstrate that an adenovirus vector encoding the essential adenoviral E1A gene under the control of 0.6 kb midkine promoter showed specific replication in midkine-expressing pancreatic cancer cell lines and not in non-midkine-expressing cells, and that Ad5MK selectively prevented tumor growth both in vitro and in vivo.
Cell culture and tumor samples
Seven human pancreatic cancer cell lines were used. AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, and PANC-1 cells were obtained from the American Type Culture Collection (Rockville, MD), and were maintained in the medium recommended by the ATCC at 37°C in a humidified atmosphere of 5% CO2. Suit-2 cells were kindly provided by Dr. Tomoda (National Kyushu Cancer Center, Fukuoka, Japan), and were cultured in DMEM (Gibco-BRL, Grand Island, NY) with 10% fetal bovine serum (FBS) (ICN Biomedicals, Aurora, Ohio). Human embryonic kidney (HEK) 293 cells were purchased from RIKEN Bioresourse Center (Tukuba, Ibaragi, Japan). Each culture medium contained 100 units/ml of penicillin and 0.1 mg/ml of streptomycin (Gibco-BRL).
Pancreatic cancer tissues were obtained from 22 patients who underwent pancreatectomy for ductal carcinoma at our Department. Other pancreatic malignancies were excluded, such as intraductal papillary mucinous adenocarcinoma, acinar cell carcinoma, and endocrine tumors. Informed consent was obtained from each patient according to our Institutional guidelines. A resected specimen was immediately examined by inspection and palpitation at the operation room. A part of malignant or normal tissues considered was cut by surgical knife and it was divided two pieces. One for the tissue samples to extract RNA was immediately frozen in liquid nitrogen and stored at -80°C, the other was fixed in 10% formalin solution to make a paraffin block and performed with HE staining to evaluate pathologically.
Rabbit polyclonal anti-midkine antibody was kindly provided by Dr. Kadomatsu (Nagoya University School of Medicine, Nagoya, Japan). The following antibodies were purchased; mouse monoclonal anti-E1A (Ad2/Ad5) antibody (clone M73 #05-599) from Upstate Biotechnology (Lake Placid, NY), goat anti-mouse IgG (#62-6500) and HRP-goat anti-mouse IgG (#81-6520) from Zymed Laboratories (South San Francisco, CA), and mouse monoclonal anti-β-actin (clone AC-15 #A-5441) from Sigma (St. Louis, MO). Anti-adenoviral hexon protein antibody was included in the Adeno-X rapid titer kit (BD Biosciences Clontech, Palo Alto, CA).
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total cellular RNA was prepared using TRIZOL Reagent (Life Technologies, Rockville, MD) and cDNA was obtained from 1 μg of total RNA by the random primer method with a First-Strand cDNA Synthesis kit (Pharmacia Biotech, North Peapack, NJ) according to the manufacturer's instructions. Five microliters of first-strand cDNA solution was subjected to the polymerase chain reaction (PCR) with synthetic oligonucleotide primers (NIPPON EGT, Toyama, Japan). For RT-PCR analysis of human adenovirus type 5 E1A, a pair of primers (5'-ATGAGACATATTATCTGCCACGG-3'/5'-TAGACAAACATGCCACAGGTCC-3') was used and PCR was done for 35 cycles at 54°C, yielding a product of 551 base pairs. The reproducibility of the technique and quality of the total RNA were confirmed by amplifying β-actin as well (primers: 5'-GGCATCGTGATGGACTCCG-3'/5'-GCTGGAAGGTGGACAGCGA-3'; product: 613 base pairs).
Quantitative RT-PCR
To assess midkine gene expression, we used quantitative real-time RT-PCR analysis based on the TaqMan fluorescence method, which employs a dual-labeled non-extendable oligonucleotide hydrolysis (TaqMan) probe in addition to the two amplification primers. The probe contains 6-carboxy-fluorescein (FAM) as a fluorescent reporter dye, and 6-carboxytetramethyl-rhodamine (TAMRA) as a quencher for its emission spectrum. During the extension phase of PCR, the probe hybridizes to the target sequence and is then cleaved by the 5' to 3' exonuclease activity of Taq polymerase. The increase in the fluorescence of the reporter is proportional to the amount of specific PCR products, providing highly accurate and reproducible quantification. The level of reporter dye fluorescence is assessed with an automated sequence detector combined with analysis software (ABI Prizm 7700 Sequence Detection System; PE Applied Biosystems, Foster City, CA). Reaction conditions were set according to the manufacturer's protocol. The following primers and TaqMan probe were used for analysis. The midkine-specific primers were 5'-CGACTGCAAGTACAAGTTTGAGAAC-3' (upstream primer) and 5'-TCTCCTGGCACTGAGCATTG-3' (downstream primer), while 5' (FAM)-AAGGCACCCTGAAGAAGGCGCG-(TAMRA) 3' was the TaqMan probe.
The PCR parameters were 95°C for 10 min (for activation of Taq-Polymerase), followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Amplification of β-actin for quality control and normalization was done with the TaqMan β-actin Control Reagent kit (PE Applied Biosystems), which utilizes standard TaqMan probe chemistry.
Cells were lysed in RIPA buffer containing 50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride (PMSF), and 20 μg/ml gabexate mesilate, and were incubated on ice for 10 minutes. Then the lysate was sonicated for 10 sec. Total extracts were cleaned by centrifugation at 15,000 rpm for 10 min at 4°C and the supernatants were collected. The protein concentration was measured with the BCA protein assay reagent (Pierce, Rockford, IL). Lysates were resuspended in one volume of gel loading buffer, which contained 50 mM Tris-Hcl (pH 6.7), 4% SDS, 0.02% bromophenol blue, 20% glycerol, and 4% 2-mercaptoethanol, and then were heated at 95°C for 5 min. The extracted protein was subjected to Western blotting. In brief, 50 μg aliquots of protein were size-fractionated in a single dimension by SDS-PAGE (6–10% gels) and transblotted to 0.45 μm polyvinylidine difluoride membranes (IPVH304F0, Millipore, Billerica, MA) with a semi-dry electroblotting apparatus (Bio-Rad, Richmond, CA).
The blots were then washed three times in TBS with 0.1% Tween-20 (TBST) and incubated for 1 hour at room temperature in blocking buffer (Block Ace, Dainipponseiyaku, Osaka, Japan). Subsequently, the blots were incubated with an appropriate primary antibody for 1 h at room temperature or overnight at 4°C. Excess antibody was removed by washing the membrane with TBST three times for 10 min each. Then the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, followed by an addition of TBST. Reaction products were detected with the enhanced chemiluminescence system (Amersham, Buckinghamshire, United Kingdom). The membranes were treated with chemiluminescence reagents according to the manufacturer's protocol, and were exposed to X-ray films for 5–120 sec.
Dual luciferase assay
We prepared the midkne 0.6-luc vector, in which the 609-base pair genomic DNA fragment of the midkne gene was cloned into the pGL2-basic vector (Promega, Madison, WI) and the firefly luciferase gene was included without a promoter sequence[24].
The transcripitional activity of a number of pancreatic cancer cell lines was measured with this dual luciferase reporter assay system (Promega, Madison, WI). Midkine 0.6-luc and a control vector (the renilla luciferase gene fused with the HSV-TK promoter (pRL-TK, Promega, Madison, WI) at a molar ratio of 10:1) were transfected together into target cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The cells were lysed after 2 days and luciferase activity was measured according to the manufacturer's protocol. The relative firefly luciferase activity of each cell lysate was calculated from the level of luminescence.
Adenoviruses and adenoviral transduction analysis
We prepared recombinant adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) for midkne-regulated expression of E1A[24]. Type 5, E1A-deleted, replication-defective adenovirus containing the green fluorescent protein gene (Ad5GFP) was constructed using AdEasy XL Adenoviral Vector System (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Adenoviruses were propagated in HEK 293 cells, purified by two rounds of cesium chloride density centrifugation, dialyzed, and stored at -70°C. Viral titers were determined with an Adeno-X Rapid Titer Kit (BD Biosciences Clontech, Palo Alto, CA).
To assess the efficiency of adenoviral transduction in human pancreatic cancer cells, we performed fluorescent staining using Ad5GFP. Pancreatic cancer cells were seeded onto coverslips and infected with at recombinant adenovirus at various multiplicities of infection (MOIs). After 48 hours, coverslips were mounted on the glass slides with Vectashield mounting medium (Vector Labolatories, Burlingame, CA), and the cells were examined under a fluorescence microscope (Olympus, Tokyo, Japan).
Assessment of adenoviral replication
First, cells were infected with Ad5MK at various MOIs for 1 h and then the virus was removed. The infected cells were lysed in RIPA buffer to extract proteins after culture for 48 h. The proteins were subjected to SDS-PAGE and expression of E1A protein was analyzed by Western blotting. Next, cells were infected with Ad5MK at 1 MOI for 1 h and the medium was then refreshed. After the cells were cultured for 2 days, the cell lysate was prepared with three cycles of freezing and thawing.
HEK293 cells were infected with serially diluted cell lysates or tissue lysates. After 48 hr, the cells were stained with anti-adenoviral hexon protein antibody by using an Adeno-X rapid titer kit (BD Biosciences Clontech, Palo Alto, CA).
In vitro cytotoxicity test
Pancreatic cancer cells were plated into 12-well plates in triplicate at a density of 1.0 × 104 cells/well. After 24- to 36 h of culture, cells were infected with Ad5GFP or Ad5MK at various MOIs for 1 h, and the infecting medium was replaced with complete medium. After 1, 3 and 5 days, the number of viable cells was counted using a cell counter (Coulter Z1, Beckman-Coulter, Fullerton, CA).
The animal study was performed in accordance with the guidelines for animal experiments of the Institute of Laboratory Animals at Kyoto University. Six-week-old male BALB/c nude mice were purchased from CLEA Japan (Tokyo, Japan). First, the mice were subcutaneously inoculated with Suit-2 cells (2 × 106/ml) in 100 μl of Hank's balanced salt solution (HBSS) (Gibco-BRL) containing 20% matrigel (BD Biosciences, Bedford, MA). When the tumors reached about 10 mm in diameter, Ad5GFP or Ad5MK (2 × 109 PFU, 0.1 ml/mouse) was injected intratumorally. The mice were sacrificed at 3 or 7 days after adenoviral injection to extract RNA and lysates from the tumors. RT-PCR analysis of human adenovirus type 5 E1A and staining with anti-adenoviral hexon antibody were performed to assess the replication of adenoviruses in vivo.
Next, Suit-2 cells (2 × 106/ml) in 500 μl of sterile PBS were inoculated into the peritoneal cavity of BALB/c nude mice to create a peritoneal dissemination xenograft model. The mice were divided into the following 4 groups: (1) an Ad5MK group, (2) an Ad5GFP group, (3) a PBS group, and (4) an untreated group. Ad5MK (2 × 109 PFU, 0.5 ml/mouse), Ad5GFP (2 × 109 PFU, 0.5 ml/mouse) or PBS (0.5 ml/mouse) was administered intraperitoneally at 4 days after the injection of Suit-2 cells. Survival was measured from the start of treatment.
Quantitative data are presented as the mean ± SEM. Each in vitro experiment was performed independently at least three times. To compare mRNA levels in pancreatic tissue samples, Wilcoxon's rank sum test was used. Survival rates were calculated by the Kaplan-Meier method, and differences between groups were evaluated with the log-rank test and Wilcoxon's test. Statistical analysis was done by using JMP statistical software and statistical significance was considered to be present at p < 0.05.
Midkine expression by human pancreatic cancer cell lines
We examined the expression of midkine mRNA in seven human pancreatic cancer cell lines by TaqMan PCR (Table 1). Midkine mRNA expression was strong in AsPC-1 and CFPAC-1 cells, but it was weak in MIAPaCa-2 cells.
Table 1 Expression of midkine mRNA in human pancreatic cancer cells.
Next, midkine protein expression was assessed by Western blot analysis (Figure 1A). AsPC-1 and CFPAC-1 cells showed strong expression of midkine protein, whereas BxPC-3, HPAC and Suit-2 cells showed moderate expression. In contrast, expression by PANC-1 cells was weak and MIAPaCa-2 cells showed no midkine band. The extent of midkine protein expression was in parallel to that of midkine mRNA expression. In the following experiments, we therefore designated the MIAPaCa-2 cell line as midkine-negative.
Midkine protein expression by human pancreatic cancer cell lines and human pancreatic cancer tissues. (A) Midkine protein expression by human pancreatic cancer cell lines. AsPC-1 and CFPAC-1 cells showed strong expression of midkine protein, whereas BxPC-3, HPAC, and Suit-2 cells showed moderate expression. In contrast, expression by PANC-1 cells was low and MIAPaCa-2 cells showed no detectable midkine band. (B) Midkine mRNA expression in non-cancerous pancreatic tissues and human pancreatic cancers. Expression of midkine mRNA in the pancreatic cancers was significantly stronger than in the non-cancerous pancreatic tissues (p < 0.001).
Midkine expression in human pancreatic cancer
We assessed the expression of midkine mRNA in 22 pancreatic cancer samples and 18 adjacent non-cancerous pancreatic tissue samples by TaqMan PCR (Figure 1B). The midkine mRNA/β-actin mRNA ratio of pancreatic cancer and non-cancerous pancreatic tissue was 0.60 ± 0.55 and 0.22 ± 0.13, respectively. Expression of midkine mRNA was significantly stronger in pancreatic cancer than in non-cancerous tissue (p < 0.001).
Transcriptional activity of the midkine promoter in human pancreatic cancer cells
We investigated the transcriptional activity of the midkine promoter by the dual luciferase reporter assay in AsPC-1, CFPAC-1, MIAPaCa-2, PANC-1, and Suit-2 cells (Table 2). This assay showed that the relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cells was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cells. Transcriptional activity in MIAPaCa-2 cells was extremly low. The transcriptional activity in each of the cell lines was usually correlated with the expression of midkne, although CFPAC-1 cells showed low transcriptional activity and high midkne expression.
Table 2 Transcriptional activity of MK promoter in human pancreatic cancer cells.
Sensitivity of human pancreatic cancer cell lines to adenovirus infection
We examined the induction of Ad5GFP in human pancreatic cancer cells (Figure 2). Cells were infected with Ad5GFP (1, 10, or 25 MOI) at 16 to 18 h after seeding. After 48 h, GFP-expressing cells were detected by fluorescence microscopy. We found that pancreatic cancer cells exhibited a heterogeneous adenoviral transduction profile. Many of the CFPAC-1 and Suit-2 cells infected at an MOI of 25 expressed GFP, whereas AsPC-1 cells showed far lower adenoviral transduction efficiency. We also found that the number of GFP-expressing cells increased in an MOI-dependent manner.
Sensitivity of human pancreatic cancer cell lines to adenovirus infection. Pancreatic cancer cells were infected with Ad5GFP (1, 10 or 25 MOI) at 16 to 18 h after seeding. After 48 h, GFP-expressing cells were detected by fluorescence microscopy. (Original magnification ×100).
Ad5MK shows specific replication and infectivity for human pancreatic cancer cell lines
Since the adenoviral infection cycle is completed within 24 h, E1A expression by infected cells after 48 h reflects viral replication. Therefore, to determine the specificity of Ad5MK replication, we used pancreatic cancer cell lines with different levels of midkine expression and then examined E1A expression by Western blotting (Figure 3A). We found that viral replication was dependent on the MOI of infection.
Replication specificity and infectivity of Ad5MK for human pancreatic cancer cell lines. (A) E1A protein expression by pancreatic cancer cells. Pancreatic cancer cell lines were infected with Ad5MK showing different levels of midkine expression, Ad5GFP alone, or the vehicle. (B) Adenoviral hexon staining. HEK293 cells were treated with preparations of Ad5MK-infected pancreatic cancer cells. After 48 hr, HEK293 cells were stained with anti-adenoviral hexon antibody. (Original magnification ×100)
Next, we examined the infectivity of Ad5MK prepared from pancreatic cell line in HEK293 cells by using an anti-adenoviral hexon antibody (Figure 3B). Stained HEK293 cells were found after infection with preparations from midkine-positive cells (PANC-1, Suit-2, AsPC-1, and CFPAC-1), whereas there were no stained cells after infection with a preparation from midkine-negative MIAPaCa-2 cells.
Specific cell killing effect of Ad5MK in vitro
We subsequently examined the ability of Ad5MK to kill pancreatic cancer cells (Figure 4). The number of viable cells was counted by using a Coulter counter. Ad5MK showed a much stronger-killing effect against Suit-2 and PANC-1 cells that have moderate and weak midkine expression, respectively, compared with its effect on midkine-negative MIAPaCa-2 cells.
In vitro cytotoxicity of Ad5MK. Pancreatic cancer cells were plated in triplicate in 12-well plates at 1.0 × 104 cells/well. After 24 to 36 h, the cells were infected with Ad5GFP or Ad5MK at various MOIs. After 1, 3, and 5 days, the number of viable cells was counted.
On the other hand, moderate cell-killing effect against CFPAC-1 cells was found in 10 MOI of Ad5MK. CFPAC-1 cells had high midkine expression and moderate efficiency of adenoviral transduction. Transcriptional activity of midkine promoter in CFPAC-1 cells was low, but it was higher compared to that of MIAPaCa-2 cells. This may be the reason why growth suppression of CFPAC-1 cells was observed by 10 MOI of Ad5MK. In contrast, Ad5MK had no effect on AsPC-1 cells as far as the designated conditions, although these cells showed strong midkine expression. This finding may have reflected low efficiency of adenoviral transduction. Ad5GFP had no influence on the growth of any of the cells in this assay compared with normal control cells (data not shown).
Adenoviral replication and anti-tumor effect of Ad5MK in vivo
We assessed adenoviral replication in vivo by using a Suit-2 subcutaneous xenograft model of pancreatic cancer. RNA and tumor lysates were obtained from the subcutaneous xenografts after injection of adenoviruses. RT-PCR analysis for human adenovirus type 5 E1A showed that E1A expression could be detected in the Ad5MK groups (Figure 5A). On the other hand, no band was seen in the untreated and Ad5GFP groups. After staining with anti-adenoviral hexon antibody, positive cells were only found in the Ad5MK groups, whereas there were no positive cells in the untreated and Ad5GFP groups (Figure 5B).
Replication of adenovirus in a Suit-2 subcutaneous xenograft model of pancreatic cancer.(A) Human adenovirus type 5 E1A expression. RNA was extracted from subcutaneous tumors after injection of adenoviruses. Expression of E1A was analyzed by RT-PCR. (B) Adenoviral hexon staining. HEK293 cells were infected with Ad5MK prepared from subcutaneous xenografts after injection of adenoviruses. After 48 hr, HEK293 cells were stained with anti-adenoviral hexon antibody. Positive cells were only found in the Ad5MK groups, whereas there were no positive cells in the untreated and Ad5GFP groups. (Original magnification ×100)
Finally, we assessed the anti-tumor effect in a Suit-2 intraperitoneal xenograft model after the intraperitoneal injection of Ad5MK (Figure 6). Following the inoculation of 2 × 106/ml Suit-2 cells into the peritoneal cavity of nude mice, 2 × 109 PFU of Ad5MK or Ad5GFP, or 500 μl of PBS, was administered intraperitoneally. In this intraperitoneal xenograft model, our preliminary study revealed that untreated mice died of peritoneal dissemination with bloody ascites after approximately 2 to 3 weeks. Almost all of the mice in the untreated, PBS, and Ad5GFP groups died within 35 days, whereas almost all of the mice in the Ad5MK group survived for more than 50 days. There was a statistically significant difference of the survival time between the Ad5MK group and the other group.
Anti-tumor effect of Ad5MK in a Suit-2 intraperitoneal xenograft model. After intraperitoneal inoculation of 2 × 106/ml Suit-2 cells in nude mice, 2 × 109 PFU of Ad5GFP or Ad5MK, or 500 μl of PBS, was administered intraperitoneally. There was a significant difference of survival time between the Ad5MK group and the other groups (p = 0.0002 Log-rank test and p = 0.0002 by Wilcoxson's test).
Conditionally replicative adenoviruses, which show tumor-specific replication and oncolysis, are a promising new treatment modality for malignancies resistant to conventional therapies [25, 26]. In this study, we demonstrated a possible strategy for pancreatic cancer based on Ad5MK replication-competent adenovirus. Midkine expression is increased in various human tumors, including gastrointestinal cancers [19–22]. We showed that most human pancreatic cancer cell lines (6 out of 7 lines tested) express midkine to some extent and that the level of midkine mRNA expression in pancreatic cancer tissues is significantly higher than in non-cancerous pancreatic tissues. These findings suggested that the midkine promoter may be a potential candidate for safe suicide gene therapy targeting pancreatic cancer.
We next examined the promoter activity of midkine in a dual luciferase reporter assay. A 2.3-kb fragment from the 5' region of the midkine gene contains the elements responsible for promoter activity [27]. Within this 2.3-kb fragment, Yoshida et al. have demonstrated the presence of a cis-element with strong promoter activity located at the -559/50 region using the CAT assay, and they also reported that its midkine promoter activity in tumor cells was comparable to that of the SV40 early promoter [28]. Therefore, we examined the promoter activity of this 0.6-kb fragment in human pancreatic cancer cells by using the dual luciferase reporter assay. We showed that the relative luciferase activity in AsPC-1, PANC-1, and Suit-2 cells was greater than that in midkne-negative MIAPaCa-2 cells. Transcriptional activity in the cell lines was generally parallel to the level of midkne expression. In CFPAC-1 cells, however, the level of midkne expression did not correspond well with the transcripitional activity of the midkne promoter. We cannot explain this discrepancy, but it has been reported that the level of endogenous midkne expression and its promoter activity are not always correlated with each other because of various factors such as negative and positive regulatory elements residing outside the region analysed [29]. Another possible explanation may be differences between cell lines with respect to regulation of the proteins involved in transcription [29].
Because the efficiency of adenoviral transfection varies between cell lines, it was essential to examine the infectivity of Ad5GFP for multiple human pancreatic cancer cell lines. Adenoviral transduction efficiency was far lower for AsPC-1 than the other cell lines. Adenoviral replication in AsPC-1 cells was lower than in any other midkine-positive cell line and no killing of AsPC-1 cells was observed at 10 MOI, although these cells showed high midkine expression and transcriptional activity. Thus, we consider that adenoviral transduction efficiency as well as transcriptional activity is critical for adenovirus-mediated gene therapy. In pancreatic cancer cells, the combined efficiency of transduction and transcription determines the actual cell-killing effect of Ad5MK.
When clinical application is considered, not only anti-tumor activity but also toxicity for normal tissues should be taken into account. In this regard, Ad5MK showed specific cytotoxicity for midkine-positive cells both in vitro and in vivo. Despite the target selectivity of Ad5MK, weak E1A expression was found in midkine-negative cells after infection with 5 MOI of Ad5MK. Type 5 adenoviruses are commonly used in viral therapy experiments, but show species-specific replication, and it has been reported that these adenoviruses do not replicate in mice or rats. There are no suitable animal models apart from the cotton rat to assess the toxicity of conditionally replicative adenoviruses, but replicative viruses have already shown at least low-level viral production and/or systemic toxicity in clinical trials[25, 30, 31]. The precise requirements for selective targeting to prevent damage to normal tissues in vivo need to be clarified in the future.
We demonstrated an anti-tumor effect of Ad5MK on midkine-positive tumor cells in vivo as well as in vitro. Our animal studies showed that mice treated with Ad5MK survived for significantly longer than the other groups. Even in the Ad5MK group, however, half of the mice died due to peritoneal dissemination after about 50 days. This may have been due to an inadequate dose of adenovirus or because of the regrowth of cancer cells with resistance to adenovirus-mediated gene therapy. In this study, we only gave a single dose of adenovirus intraperitoneally after tumor cell inoculation. In the future, we should investigate the optimum volume and number of adenovirus doses to improve the efficacy of Ad5MK treatment. Another way to improve the results could be employment of fiber-modified adenoviruses, which can enter cancer cells resistant to conventional adenoviral gene transfer in a coxsackievirus and adenovirus receptor (CAR)-independent manner.
Midkine expression was increased in pancreatic cancer cell lines and pancreatic cancer tissues. Ad5MK showed specific targeting of and cytotoxicity for midkine-positive cells both in vitro and in vivo. These results suggest that replication-competent adenoviruses based on the midkine promoter might have the potential to be used in gene therapy for pancreatic cancer.
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This study was supported by a Grant-in-Aid (#20390355) from the Ministry of Education, Culture, Sports, Science and Technology.
Department of Hepato-Biliary-Pancreatic Surgery and Transplantation, Kyoto University, Japan
Eiji Toyoda
, Ryuichiro Doi
, Kazuhiro Kami
, Tomohiko Mori
, Daisuke Ito
, Masayuki Koizumi
, Atsushi Kida
, Kazuyuki Nagai
, Tatsuo Ito
, Toshihiko Masui
, Michihiko Wada
& Shinji Uemoto
Division of Pathology, Chiba Cancer Center Research Institute, Chiba, Japan
Masatoshi Tagawa
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Correspondence to Ryuichiro Doi.
ET conceived of the study and performed experiments on adenoviruses. RD conceived of the study, and participated in its design and coordination and helped to draft the manuscript. KK conceived of the study and performed experiments on mice. TM conceived of the study and performed experiments on mice. DI conceived of the study and performed experiments on pancreatic cancer cell lines. MK conceived of the study and performed experiments on pancreatic cancer cell lines. AK conceived of the study and performed experiments on pancreatic cancer cell lines. KN conceived of the study and performed experiments on mice. TI conceived of the study and performed experiments on mice. TM conceived of the study and performed experiments on pancreatic cancer cell lines. MW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. MT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. SU conceived of the study, and participated in its design.
Toyoda, E., Doi, R., Kami, K. et al. Midkine promoter-based conditionally replicative adenovirus therapy for midkine-expressing human pancreatic cancer. J Exp Clin Cancer Res 27, 30 (2008) doi:10.1186/1756-9966-27-30
Pancreatic Cancer Cell
Pancreatic Cancer Cell Line
Human Pancreatic Cancer Cell
Pancreatic Cancer Tissue


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I have been a Phase One user since 2007 when I bought my first digital back (a P20+) for a Hasselblad V series camera. I had initially been lent a P20 back for a trip to the Isles of Harris and Lewis off the West coast of Scotland and loved the experience of converting this old manual camera into a 21st century tool. But to be totally honest I had no intention of investing in more kit (limited funds and not a lack of interest being the reason). However, when I returned from Scotland I worked on the images, produced some large prints and I was blown away by the tonal range and the detail. I had expected the quality from this 16Mp back to be on a par with my DSLR of the time, the Canon 1Ds MkII, but it far exceeded anything I'd got from the Canon. I showed the prints to some pro photographer friends of mine and they agreed – the images from the Phase One back were outstanding. I was hooked!
I've since sold all my DSLR gear and have gone completely mirrorless. I've subsequently upgraded to a Phase One P45+ back and an Alpa TC technical camera. The Alpa is an incredibly well made modular system where the basic 'body' can be married with a range of lenses, backs (digital & film) and other accessories to produce a customised camera made to a precision standard.
I use this outfit alongside a Micro Four Thirds (MFTs) system based on Olympus OMDs and the range of Olympus prime and pro zoom lenses. I find these two systems be a perfect complement to each other. If I need extreme wide angle or long telephoto lenses then the Olympus gives me those options; if the weather is particularly inclement then the sealed cameras and bodies allow me to work in conditions where I would be more reluctant to use the Alpa; if I need to work handheld then the Olympus comes out of the bag. And if keeping weight to an absolute minimum is essential then that is much easier with the MFTs system.
Both the Alpa TC and the P45+ were significant investments and I frequently get asked, 'Why? What do you get from this combination that you can't get from a much cheaper, high end DSLR?'.
Firstly – it's about image quality. The large, high dynamic range files can be significantly cropped, pushed quite hard in post processing and still retain a unique quality that I've yet to get from any DSLR file. For the type of photography I do (primarily landscape photography with the camera firmly mounted on a tripod) a DSLR offers no advantages. And the level of fine detail possible in huge prints from the large pixels on the medium format sized sensor is still mind blowing after all these years.
Secondly the small size and relatively light weight of the Alpa TC, Schneider lenses and Phase One back combo makes it much easier for me to get my kit on a plane as hand luggage and then to carry it around all day once on location.
But equally as important to me is the way of working that this combination encourages. It's a slow, methodical process to set the camera up as there's no viewfinder (and no live view with the P series backs), no inbuilt metering or autofocus. It encourages a thoughtful, almost meditative approach to landscape photography that really suits the way I like to work and I have found this leads me to produce images that I find far more satisfying.
In November 2014 Phase One launched their A-series camera. Basically this is an Alpa TC coupled with an IQ back. Each individual camera and back come set up to ensure the highest possible quality output from each unique combination. Each digital back also has built in factory set lens calibrations for three Rodenstock lenses – the Alpagon f5.6 23mm, the Alpar f4 35mm and Alpagon f5.6 75mm. It's effectively bespoke camera manufacture taken to the nth degree.
Clip Warning – this masks the clipped highlights in a different colour to ensure all the detail is captured with no 'blown' highlights.
Importantly both the Exposure Zone Tool and the Clip Warning are based on the RAW data and not a processed preview.
The A-series comes supplied with camera, lens (or lenses depending on configuration), an IQ back and an extensive array of accessories e.g. batteries for the back, charger, card & card reader, leather straps, cable release, lens cloths etc [See Photo Above]. In true Phase One fashion the attention to detail in what is included and the packing of everything in a solid Pelicase leaves nothing to be desired.
As an existing Phase One/Alpa user I was asked by Phase One to try out the A-series and to give my unbiased view on how it performed in use. This is my review based on a few months of use in both the UK and the USA (on a 3 week trip to Colorado). It's very much written from a users perspective i.e. it's not a technical review and I won't get into pixel peeping. I'm interested in cameras & lenses as real world tools not as subjects in scientific experiments.
I knew the camera well from my own use over the last 4 years and the basic operation of the IQ back took only a few minutes to familiarise myself with.
-a smartphone holder to attach a device to the top of the camera (this is used in conjunction with Capture Pilot, synced with Live View on the IQ back, so the image can be previewed with accuracy ).
I was keen to see how the A-series would perform in the field – would I really find a lot of difference compared to my existing set up? To find out I ventured into the Yorkshire Dales – a location I know well as the Dales are close to where I live. Choosing a familiar location meant I could concentrate on using the system rather than hunting for shots.
The first and very significant difference was the ease of use due to the IQ back's functionality (e.g. intuitive menu system, touchscreen capabilities) and the effective live view offered by the CMOS chip. As much as I enjoy the process of setting up my existing system there are times (in rapidly changing light & weather conditions for example) when I need to work extremely quickly and my Alpa TC/P45+ combination is not ideal for this. In contrast, using the A-series I was able to get the camera from my backpack, onto the tripod and ready to shoot in a fraction of the time. In fact, not much slower than using my Olympus cameras.
Most significant in achieving this was the live view capability. Switching live view on meant I could compose the shot with precision and as quickly as using a DSLR. This was so much faster than the iterative process I normally employ i.e. use my iPhone & Alpa app to get the basic composition, take a shot, review it, move the camera to refine the composition, take another shot etc etc. I was also able to use live view to zoom in on the image to check focusing and depth of field (although I found the supplied Alpa High Precision Focussing Ring to be so accurate that I could rely on this and just double check for accuracy when reviewing the image in playback mode).
In general the whole picture making process was enhanced by the impressive quality of the 3.2″ retina touchscreen (which also has the capability to zoom in to 100% with a simple tap on the screen).
Speed of operation was also improved by a relatively small (but also expensive) accessory – the Alpa synch cable; no more missed shots because I've forgotten to wake up the digital back!
One of the key criteria for me when choosing a camera is how well it performs at long exposures. Long exposure images appeal to me because they can capture the dynamic nature of the landscape – static elements (like trees, buildings, fence posts, rocks for example) can be contrasted against moving features like water or windblown clouds & foliage during an exposure of several seconds or minutes. Used appropriately the technique can create ethereal, moody images that transform a relatively mundane seen into an evocative photograph.
I'm pleased to say that the IQ back I was sent by Phase One was capable of doing a one hour exposure (I think the longest I've shot digitally is around 10 minutes) and in my tests it performed admirably. Noise was never an issue and the full tonal range of the scene was captured on the sensor. A great tool for the long exposure enthusiast.
Overall, image quality exceeded my expectations. I'm very used to the incredible standard of photographs that my Alpa and Phase One back can produce. With the precision matching of lens, camera and back of the A-series this leads to an extremely detailed image out of the camera with a quality that even the untrained eye can spot when the image is enlarged to 100% on the computer screen. And one noticeable improvement over my P45+ back is the high ISO performance of the IQ back. I didn't push it to its limit but I'm assured by Phase One that image quality up to ISO6400 is on a par with the best any DSLR can offer.
I don't think anyone interested in producing the highest quality files will be disappointed with the Phase One A-series. Every single aspect of the camera is engineered to the nth degree with image quality in mind. Not speed or convenience. Not price or versatility. Image quality and only image quality.
Of course that comes at a cost. The set up I was loaned by Phase One sells for around $47k – that's one hell of an investment for anyone. But many working pros can justify the expense in terms of business benefits and the ability to offset the cost against tax. And for the wealthy enthusiast looking to take total control of their image making and to produce photographs with a wonderful level of tonality and detail the A-series will undoubtedly reward.
I did ask Phase One if the price could be reduced by cutting down on the accessories in the kit or offering them as optional extras. For example, with an effective live view I didn't use the smartphone holder at all and the rosewood handle with the leather strap was something I could do without as I always use the Alpa on a tripod. But I was assured by the company that the accessories were effectively added into the kit cost free to offer a complete and comprehensive package with guaranteed quality control to any purchaser. And the whole kit is covered by a full 5 year guarantee (and this probably says a lot about their confidence in the quality of the whole system).
Of course it's not a camera designed to be a one stop solution. If sports photography is your interest or you are a fashion photographer then there are probably better options out there for you. But if your interests are in fine art, landscape or architectural photography then, in my experience, there's no better tool for the job.
In conclusion I really enjoyed the more fluid operating process brought about by the refinements of the A-series. Rarely in photography are there 'win/wins' (often some sort of compromise is involved) but with the A-series I felt I got the best of both worlds – the speed of operation of a DSLR combined with the methodical way of working and of course the fantastic image quality from a high end digital back.
A much photographed location and for good reason. The graphic design of the walls and barns make for a good image – I composed this shot to make the most of the zig-zagging lines of the walls and the curving lines of the barns through the frame. But to lift it above the ordinary something different, special was required. On this particular occasion I was gifted some wonderful, dappled light creating patterns of light & shade through the image. I waited until all the barn roofs were lit before firing the shutter.
I was luck to get early access to this bridge – arriving at 5.30am well before the park opened to the public. This enabled me to get a shot of the bridge without the distraction (and the vibration) of other people walking across. I decided to go for a simple graphic composition, getting down low to make the most of the receding lines and the texture on the wooden planks.
A pre-dawn start was rewarded by some fantastic early morning light, side lighting the rocks and accentuating their warm colours against the sky. I fitted two ND grad filters (0.9 and 0.6 soft) to balance out the exposure for the sky and the foreground in shade. Later I used the colour selection tool in Capture One v9 to create a mask for the sky which enabled me to bring back all the detail.
I also produced a B&W version (sometimes you get lucky and get two shots for the price of one!). I love the textures on the rocks in this version and the contrast between light & shade. The removal of colour also accentuates the timeless (Jurassic Park?) feel I was after.
A very typical Yorkshire Dales scene taken early in the summer (hence the rich greens in the colour version). It's the dappled light taking the viewers eye down the dale that makes the photograph for me. I waited for the foreground barn to be kissed by the light, set against the shaded far side of the dale to give the shot a three dimensional feel.
After an early start to catch the dawn light the fog rolled in (I was told this was unusual in Colorado in July) totally surrounding this barn which, as an Englishman, I felt was so typically an American structure. Timing was crucial – I wanted the barn enveloped in mist but enough warm, early morning light to be creeping through to illuminate it. I set the camera on my tripod and waited for around an hour as the light changed and the fog came & went. I find the A-series encourages a more patient, methodical approach (which is why I love using it).
This railway viaduct, built in the 19th century is located in a remote, harsh environment in the Yorkshire Dales. It's a location that bears the brunt of the unforgiving weather and one can only imagine what it must have felt like to work there, erecting the viaduct in these conditions. I photographed it on a day when bands of rain were constantly sweeping in followed by bursts of light and then more rain. I hope the image captures how it felt to be there on that day.
I composed the shot using the line of the wall and the viaduct to move the viewer through the frame. Getting adequate foreground to background depth of field was important so the Live View feature of the IQ250 and the precise focussing scale on the lens were invaluable to enable me to check the accuracy of my settings.
On the drive to Bishops Castle we spotted this old farmstead at the side of the road nestled next to a clump of trees and I had to stop to take a shot (sad I know but it made me think of 'The Little House on the Prairie'!). The soft light, the colours, the textures on the wooden buildings and the rain clouds passing over all combined to give me the shot I was after. It's an image that benefits from being printed very large so I was grateful to be able to shoot it on the A-series with the IQ250 knowing that I would have the detail and the resolution necessary to make that huge print.
I had seen the potential to take this photograph, set the camera up and then the heavens opened. I beat a hasty retreat with the camera back to the shelter of our vehicle. Several minutes later I ventured out again. The approaching clouds told me another heavy burst of rain was on its way so I had to work fast to get my shot. I was relieved to have the A-Series/IQ back with its Live View feature as this makes setting the camera up a relatively speedy process when circumstances necessitate. I managed to fire off a couple of frames before the rain returned.
I loved the textures of the rocks in the foreground and the lines leading to the two large rocks so I composed in vertical format to make the most of those characteristics. One of the advantages of the A-series is the speed in which the back can be switched from horizontal to portrait format requiring only the unlocking of two levers. I waited until the two large, darker rocks were set against the white clouds behind before taking my shot.
I have a philosophy to my landscape photography that is usually more true in the UK than in the USA – if I haven't got wet then I probably haven't got a good photograph. This is because I like to work on the edge of weather systems – when rain is moving in or away dramatic light and atmospheric conditions usually are the result.
Looking out of our hotel window early one evening I saw the storm clouds developing over the mountains. I grabbed my bag & tripod, jumped in the car and headed towards the approaching storm. This is another occasion where the A-series and IQ back enabled me to work quickly enough to get my shot and back to the vehicle just before the rains reached me. On seeing the resulting image I was glad that I had made the effort.
This was one of my first photographs taken with the A-series and I took it primarily as a test to see how precise I could be in fine tuning my composition using Live View and then checking with the zoom facility of the IQ back. Compositionally I felt it was important to keep the lines of both railings below the horizon and also prevent the top of the right hand side railing from cutting across the bottom of the light at the end of the pier. I'm pleased to say the process was made a whole lot easier using this combination than with my P45+ back.
I always think this tree stump and its spreading roots have a prehistoric feel to them. The weather conditions on this day suited this view – dark scudding clouds with small patches of light illuminating the landscape in the distance both helped to recreate a sense of the elemental nature of the scene. I fitted a 10 stop neutral density filter to give me the long exposure I wanted to record the windblown clouds.
Quite often I take exposures of 30-60 seconds or longer and the wonderful feature of the IQ250 back is that it can easily handle exposures of this length and retain superb image quality (it's maximum exposure length is one hour but I've not pushed it that far – yet!).


Fr Peter McVerry SJ has been working with Dublin's young homeless for more than 30 years. During this time he has campaigned tirelessly for the rights of these young people.
In 1979, Fr McVerry opened a hostel to address the urgent need for accommodation for young homeless people. Four years later he set up a charity called The Arrupe Society, to provide further housing and support. Renamed in 2005 as Peter McVerry Trust, the charity has progressed from providing a three bedroom flat in Ballymun to today's wide range of services catering for the diverse needs of young homeless people. Some services include an open access service, supported accommodation, drug services, under 18s hostels and apartments across the city to offer longer-term housing for those ready to live independently .
The Meaning is in the Shadows is a collection of writings by Fr McVerry, which reflect on his experiences working in Dublin's inner city. He questions the structures that affect the lives of those on the margins and makes radical suggestions for change.
Jesus:Social Revolutionary? is the new book written by Fr McVerry, challenging Christian churches to re-examine their priorities, saying that social justice should be at the heart of all that the Christian churches preach and do.
Both books are published by Veritas, and can be ordered online at www.veritas.ie.


This fall, Chip Kalback joined the folks at Motive to photograph three individuals who celebrate the 'Farmer In All Of Us' for Ram Trucks.
They met an 18-year-old farmer who's taking over her 200-year-old family farm in Kentucky, a recent Cornell grad who farms in Upstate New York, and a rancher in Wyoming who oversees 2,400 acres of land.
Photographer Ray Collins captures the magic that happens at the intersection of water and light. Each shot in this film was created from a single one of Ray's original photos. The stills are transformed into cinemagraphs — a hybrid between photo and video — creating an infinite loop that makes a single moment last forever.
When outfitter Eddie Bauer approached the NBC "This is Us" star and asked her where in the world she'd like to go on a sponsored expedition, Mandy Moore remembered seeing the IMAX movie Kilimanjaro: To the Roof of Africa along with her reaction to seeing it at 18 years old: I'm going to do that one day.
Jamie Kripke recently went to the Air Show in Dayton, Ohio to capture a series of photos to add to his collection of personal work. What follows is a journal entry about the experience in Jamie's words, as well as some of the shots taken long the way. View the whole Air Show project here.


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It is the middle day of a three-day holiday weekend here and the weather could not be more perfect. Cool at night, warm and sunny during the day, but not too warm, comfortable.
No chicken video today. They are free ranging in the chicken garden and really, how much video of them scratching in the leaves can one really want? They do make wonderful chicken noises now though, no more peeping. They have happy sounds and distressed sounds and angry sounds. Their happy sounds are soothing and pleasant to listen to, the chicken equivalent of a cat's purr.
Picked the first peas from the garden today. It is not a banner pea year, unfortunately. This makes me sad because I love peas so very much. It just got too hot in early spring and at least half the plants are stunted, some of them have died. Only a few are truly vigorous. I will really be appreciating these peas since there won't be many more! I am going to try and plant some peas in August and see if I can't get some in October before the freeze. Fingers crossed!
It is a banner year for the black raspberries. We've already frozen about two pints and Bookman made a third pint into muffins today ( I know I owe a certain someone a muffin recipe—coming soon!). There are still lots out there too. The red raspberries will be starting to get ripe probably in a week or so. And it appears that the blackberry I have given up on and was planning on pulling out might have decided to live and produce some fruits. There are little green berries on it. We'll see how that goes.
It is still too early to tell but it looks like it might end up being a good year for beans. All of the beans I have planted are looking really good and growing into strong plants. They will start blooming in another couple weeks and then I'll know for sure, but so far, so good.
The zucchini, pumpkins and cantaloupe are also all looking strong. We planted lots of zucchini because last year we made sweet zucchini relish and Bookman loved it so much he wants to make and can jars and jars of it. The stuff is great on sandwiches and in salads too. We use it like sweet cucumber relish and it is perfect for when the zucchini grow a bit too large.
Several weeks ago Bookman found an old seed packet of broccoli and asked what he should do with it. I said whatever he wanted to and he tossed them into an untended area of the garden by the chicken coop where we used to have a red twig dogwood growing. I noticed earlier in the week that there are a good many broccoli sprouts that are already putting out second and third leaves! Of course the two years we have actually tried to grow broccoli on purpose, we got nothing, not even a sprout. And now old seed randomly sprinkled has grown up strong and healthy!
This seems to be the year of garden irony since none of the lettuce we planted came up but arugula we did not deliberately plant is growing very well all over the garden. I have a packet of winter lettuce mix that is two years old. The end of August I am going to randomly sprinkle it somewhere and see what happens. Of course, irony will take over and my deliberate "random" sprinkle will do nothing.
In a pollinator patch by the clothesline pole I planted a wild bergamot last year along with prairie smoke and butterfly weed to go with the already well-established anise hyssop. Prairie smoke is a delicate spring flower and it did fairly well. The butterfly weed may be doing well but I can't tell for certain because it has been consumed by the wild bergamot that is attempting to take over. I have bergamot planted in the the front yard and it has been well-behaved and fairly contained. It stays kind of short and has not spread anywhere. The pollinator patch by the veg garden must be the ideal location for the bergamot because it has turned into a monster! It is tall and sturdy and spreading wide. It is just beginning to bloom and will very shortly be covered in pale purple spidery flowers. I love the smell of the bergamot in the front yard so I hope this backyard monster will smell as good or better.
I went out on a long bike ride yesterday with my trusty Astrid. No accidents this time! The scabs on my chin and lip from last week's mishap are gone and I have bright pink skin that does not match the rest of my skin yet. My teeth are feeling much better too. They are still super sensitive but every day just a little less so. It has been difficult to not be able to bite things and I expect it will be even harder once my teeth are no longer sensitive. Eight weeks is going to feel like a long time. I love sandwiches and I usually have one at work for lunch. They are quick and easy to make and so very portable. Bookman has been hard pressed in the creativity department for my lunches and resorted to lots of noodles last week. I told him Thursday that I was tired of noodles and he is going to have to put his thinking cap on. We shall see what he manages this week.
It was a perfect day for a bike ride yesterday. I was out just after 7 in the morning and was glad for the early start since by the time I turned back for home the trails and roads were getting really busy with people out and about for the holiday weekend.
I rode out on the same trail I did last weekend but then detoured onto a county road and headed to the town of Stillwater on the Minnesota-Wisconsin border along the St. Croix River. The county road turned out to be a lovely ride. It was good, smooth pavement and had some great rolling hills. From the number of cyclists going by on the other side of the road it appears it is a favorite riding area for folks who live nearby.
From Stillwater I picked up a trail that connected me back to the one I had ridden out on and spit me back out onto the rutted and potholed streets of St. Paul just north of the state capital. I thought Minneapolis streets were bad! There were many long stretches of pavement that were bumpier to ride on than gravel. At least gravel is a constant vibration, but the streets are just jarring bump after bump after bump and do not make for pleasant riding at all.
I made it home safe and sound and super hungry because I didn't pack enough food for 70 miles/112 km. Hummus never tasted so good!
The Tour de France got off to an exciting start yesterday. Normally I don't follow it that closely but this year I am very excited for it mostly I think because I have a few ways to follow it other than brief reports on the radio. Even Bookman is finding it exciting to watch.
Oh, and we got our "new" car Thursday night. A white exterior/grey interior 2013 Honda Fit with just over 7,000 miles on it. We were expecting the whole start to finish of the transaction to take maybe an hour. Hahahahaha! A little over two hours later they finally gave us the keys and we drove home. It was after 9, past our bedtime, and we were so tired from the ordeal that we didn't stay up reading for even a few minutes.
The car is a hatchback and the seats fold down making the back quite roomy. Bookman and I both have Independence Day off and he has consented to go on a bike ride with me. We are going to drive out to a trail and see how the bikes fit in the back of the car. Hopefully they do well since the bike rack we had for the old car will not work on a hatchback and I really don't want to have to buy a new rack.
I am making plans for several autumn rides and races. More on those soon!
Happy Independence Day to everyone reading in the U.S.!
Having spent too many months on what I call "the porridge diet," I can offer a few tips for lunches: any sandwich with a soft spread, if spread on soft bread, can be torn (rather than bitten) with the front teeth and then chewed with the back. I like tomato sandwiches, and pimento cheese. Hard-boiled eggs are fairly soft. Kasha can be seasoned with salt and pepper and it's quite different from the usual round of quinoa, couscous, or grits. I'm a big fan of black-eyed peas, purple-hull peas, and crowder peas, all of which need to be cooked soft (or bought in a can, seasoned by Bush's) to be good. Bananas and peaches are good, although I'm guessing you can eat any fruit, as long as it's cut up. Ice cream, yogurt and pudding are all great, of course, if you can eat dairy.
Walker came home with a really wonderful food from Russia. It's called Halva, and it's a sweet sunflower seed paste. You pinch off a corner and eat it, often with tea. Not a lot of chewing required. (I looked up the spelling and discovered that it's often made with sesame paste too. We're loving the sunflower.
Looks like you should be bringing your harvest to market, Stefanie. So many variety of produce. Do you have further plans after this? Maybe expand into a farm? I can imagine the amount of work that goes into this prolific venture. It's definitely more than just backyard gardening. And there are the chickens too. You're one busy couple indeed!
Happy 4th of July to you! Glad that you are able to go biking again and had such a nice trip. Enjoy the trip with Bookman today. Can you understand the Tour De France? All those yellow jerseys and kings of the mountain tend to leave me flummoxed. England being beaten by Iceland in the soccer…well that was the sporting highlight of the year!
Ian, thanks! The Tour used to be completely mysterious to me but as I have been cycling more and reading more about racing it is all beginning to make sense in a strange sort of way. England beaten by Iceland? Wow, that must have been quite the upset!
BookerTalk, oh I am so relieved to know it isn't only me! And wild flowers, I have not been able to grow those either in nicely prepared beds.
Independence Day is also my mum's birthday so always special here! Hope you and Bookman had a lovely day and he enjoyed his bike ride. Love your garden irony!
My brother and family are here this weekend – partly for said Mum's birthday – and he told us that their last chook died. A dog I think got it. Very sad.
whisperinggums, happy birthday to your mum! I hope everyone enjoyed celebrating. How sad about your brother's last chicken. Will he be getting more? The bergamot is a native plant and the variety is actually called "wild bergamot." In the US they sell packets of "wildflowers" but they aren't necessarily native even though I'd wager most people assume they are. Ha, it is a bit of a contradiction to grow wild flowers in a cultivated garden!
Now I'm completely confused about what wildflowers mean!
No, I'm not sure they will get more chooks, at least not at present. They do have a coop but I think they need something like Barton Cottage really to be successful!
Now, there's a question and a half! Probably being called a wildflower when it's not!
I don't know why, but I never have much success with peas. When they do grow, I never get any to table because the kids snack on them all straight in the garden (which is great, I love their enthusiasm for fresh peas). You should save seed from those few vigorous plants, for next year. Maybe those few liked your conditions and you can capitalize on that.
cirtnecce, thanks! I'll find the zucchini relish recipe and send that one along too! Bookman is terrible when it comes to recipes, he rarely follows them! It's great because he really comes up with some good stuff but then he doesn't write it down so it becomes impossible to replicate. And any vegan recipes you want to send my way I will not say no!
It is too bad you live so far away!
Your wild bergamot is lovely! I planted some monarda, but it's just doing okay. It isn't a banner year for anything in my little garden, unfortunately. We had too much rain in the beginning of the summer, variable temperatures, and then too little rain. Hopefully, the rest of the reason will be better!
AMB, thanks! I've never had much luck with other varieties of monarda, for years I wanted a red one very badly and it would always die. But the bergamot finds my garden conditions to be perfect and it is currently buzzing with bees. Climate change is making the weather less consistent which presents a real challenge. I hope the rest of your season turns out great!
Thanks! The garden is still pretty, but not nearly as lush as it was last year. I feel sorry for the bees and hummingbirds. They don't have as much to feast on this year. My monarda is "fireball," which is red. It's struggling a little bit, but maybe it'll turn out to be okay.
hopefully next year will make up for this one. Hope your monarda does ok. The red one I tried was "Jacob Kline." Maybe the red ones are more finicky that the others?
So do you have a key to your garden–have you drawn a plan so you know what is where and how much you planted? Or is that a level of organization that is just a little too much? I like the idea of randomness! I should hire you out for a little advise of what to do in my very shady backyard. I have never been able to grow anything except lots of weeds. And dirt! I would love to have a huge patio are made of bricks and then maybe wildflowers or wild grasses on the edges, though with the number of 'critters' I seem to be housing back there, I wonder if it would be an almost dangerous ecosystem–I have at least one groundhog, a family of racoons (mama and three babies, which I must admit were really cute at first, but they won't be for long…) and loads of squirrels. I am not a good gardener myself, though I love being in them. Sounds like you had a really nice weekend–the weather was okay here as well–rainy but it was nice to have a break from the heat. Alas it is not going to last as the heat and humidity are back and it is going to be a steamy and maybe stormy week. How many more weeks to go until you can use your front teeth again? You'll have to plan a celebration!
Please just PLEASE tell me how I can make all these green tomatoes turn RED! I have picked SO MANY green ones to try and leave 'energy' for the rest to turn red and all they do is make more green ones! I think hate tomatoes. My cucumbers have been happily delightful, though.


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What Are The Best Comic-Themed Online Slots?
by Giovanni Aria on March 23, 2021 What Are The Best Comic-Themed Online Slots?2021-03-23T06:27:19-07:00 - News
Comics are a popular form of entertainment and have helped inspire and influence some of the biggest media franchises, like the Marvel and DC film series and plenty of independent films and television shows. Alongside films, comics have also helped inspire games, including casino slots, and we thought we'd take a look at some of the best comic-themed slot games available.
Playtech's DC Comics Jackpot Slots
Playtech arguably offers the most well-known comics-themed slot series. The developer runs several jackpot slots themed on various DC Comics superheroes, including Batman, Superman, Wonder Woman, and more. All of these slots are cartoonish in appearance, based on the actual comics rather than the DC film series, and most feature connected jackpots through which you can earn some big cash prizes.
NetEnt's Jack Hammer Games
NetEnt also released two comic-themed slots, although neither of the games is based on actual comic titles. Jack Hammer released in 2011 and follows crime-solving detective Jack Hammer in an animated slot filled with noir-themed visuals and special features like respins, free spins, and wild symbols too, but no jackpots.
A sequel, entitled Jack Hammer 2, launched in 2013 and continues the story of Jack Hammer. The sequel includes most of the same special features from the original game but benefits from improved visuals, making the game even more fun to play.
Other than Playtech, NetEnt games are available at most online casinos and their sister sites. In most cases, you can browse the operator's games selection directly on the homepage and before registering an account.
Playtech's Marvel Comics Slots
Around the late 2000s and early 2010s, Playtech had a licensing agreement with Marvel and released several popular video slots based on Marvel Comics, including The Avengers, The Incredible Hulk, Iron Man, The Punisher, X-Men, The Fantastic Four, Ghost Rider, The Mighty Thor, Daredevil, Captain America, Spider-Man, Elektra, and more.
All of these slots use semi-realistic but animated visuals and come with various special features, including free spins, wild symbols, and scatter symbols too. There are no groundbreaking visuals here and no jackpots, but the games were generally quite fun to play. Unfortunately, these games are no longer available to play, potentially due to licensing agreements.
Playtech's New DC Comics Film Slots
In addition to its animated DC Comics slots and Marvel Comics slots, Playtech has over the last few years released several video slots based on the new DC Extended Universe film franchise. These games are all based on the DC films, including Aquaman, Suicide Squad, Batman V Superman, Justice League, Man Of Steel, and more.
These slots' visuals are all based on their respective movie, and the symbols are based on the characters from each movie too. Like their animated counterparts, these DC Comics film slots are part of a jackpot network, providing you with a chance to win large cash prizes.
There are numerous other comic-themed video slots available for you to play at online casinos, but we believe that the slots we've mentioned above are some of the best. If you can, we suggest you try playing them – we're sure you'll enjoy them!
https://www.firstcomicsnews.com/what-are-the-best-comic-themed-online-slots/https://www.firstcomicsnews.com/wp-content/uploads/2021/03/Superhero-slots-600x316.pnghttps://www.firstcomicsnews.com/wp-content/uploads/2021/03/Superhero-slots-150x79.png 2021-03-23T06:27:19-07:00 Giovanni AriaNews
Comics are a popular form of entertainment and have helped inspire and influence some of the biggest media franchises, like the Marvel and DC film series and plenty of independent films and television shows. Alongside films, comics have also helped inspire games, including casino slots, and we thought we'd...
Giovanni AriaGiovanni.Aria@firstcomicsnews.comAuthorGeo, is the Guido of Greatness, the King of Comics and the Toa of Techies. Its not cool until Geo certifies it cool. He likes everything from Archie to WWE and everything in-between, as long as its funny, edgey, or over the top exciting.First Comics News
About Giovanni Aria
Geo, is the Guido of Greatness, the King of Comics and the Toa of Techies. Its not cool until Geo certifies it cool. He likes everything from Archie to WWE and everything in-between, as long as its funny, edgey, or over the top exciting.
View all posts by Giovanni Aria »


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A somewhat entertaining dispute has erupted between the American website The National Interest and the Russian press corps.
The National Interest has made a list of the 5 Worst Aircraft Carriers Ever Put to Sea, and the Russian Admiral Kuznetsov is on that list. That does not go over well in Russia.
For example Lenta.ru has collected statements from a range of Russian military experts who disregard the American website as a propaganda tool in an anti-Russian campaign.
The Russian experts also complain that the American journal includes Admiral Kuznetsov in a list of "aircraft carriers" because actually it is an "aircraft-carrying missile cruiser". Those things are apparently not comparable at all.
One of the Russian experts explains that Admiral Kuznetsov held up very well during the operations in Syria (despite losing two aircraft), and another claims that the Russian ship brings more strategic weight to the battlefield than a modern American carrier which is little more than a floating airport.
The other four ships on the list are the British HMS Eagle from 1924, the French Bearn from 1927, the Japanese HIJMS Kaga from 1928, and the American USS Ranger from 1934.
The Russian media seems convinced that the list is prioritized so Admiral Kuznetsov is named the worst aircraft carrier ever. As far as I can tell, it is just in chronological order.


Belize is a nation on the eastern coast of Central America, with Caribbean Sea shorelines to the east and dense jungle to the west. Offshore, the massive Belize Barrier Reef, dotted with hundreds of low-lying islands called cayes, hosts rich marine life. Belizes jungle areas are home to Mayan ruins like Caracol, renowned for its towering pyramid; lagoon-side Lamanai; and Altun Ha, just outside Belize City.
Ambergris Caye, Belize's largest island, is known for its water sports, scuba diving and Caribbean beaches. Off the eastern shore, the Belize Barrier Reef features famed snorkeling and dive sites such as the Hol Chan Marine Reserve, with an abundance of sea life, and the 124m-deep Great Blue Hole. The town of San Pedro is the island's gateway and center for sailing and reef exploration.