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Question: What Is The Kuleshov Effect Quizlet?
What is an example of montage?
Is a montage a scene?
What is the basic purpose of the continuity system?
What was the central principle of the Kuleshov effect?
What is a cutaway quizlet?
Why is montage used in film?
What is the axis of action quizlet?
What is a cutaway shot?
How does the 180 degree system influence screen direction?
What does SHOT mean in film?
Why is montage important?
What is the Kuleshov effect in film?
What does a montage mean?
Why is the Kuleshov Effect important?
What is 180 degree rule in filmmaking?
What is the ultimate goal of continuity editing?
What is Montage and its types?
What does jump cut mean?
For example, a character's whole life story could be told by showing a long succession of images, starting from baby photos and ending with a photo of the character as an old man.
This technique is also frequently set to music, creating a "musical photo montage.".
A montage is a collection of very short scenes, sometimes only a single shot each, designed to show a series of actions over time. … Often the best choice is to not even say "MONTAGE" and just let it be a series of short scenes — just a slugline and a sentence or two of description.
Generally speaking, the continuity system aims to present a scene so that the editing is "invisible" (not consciously noticed by the viewer) and the viewer is never distracted by awkward jumps between shots or by any confusion about the spatial lay-out of the scene.
What was the central principle of the Kuleshov Effect? A montage is the central principle that defines any film as an art on its own. The basic premise of this technique is that cinematic meaning is a function of the edited sequence rather than of the individual shot.
cut-away. A shot that displays totally different content from the action -typically used to cover over or bridge spots in the action. color shot. A kind of cutaway that adds to the general atmosphere but which does not provide information about the action.
Montage is a technique of film editing that combines a series of short shots or clips into one sequence, often set to music. … In a romantic comedy, a montage can show a sense of growing love or attraction between two love interests getting to know one another.
axis of action. In the continuity editing system, the imaginary line that passes from side to side through the main actors, defining the spatial relations of all the elements of the scene as being to the right or left. The camera is not supposed to cross the axis at a cut and thus reverse those spatial relations.
From Wikipedia, the free encyclopedia. In film and video, a cutaway is the interruption of a continuously filmed action by inserting a view of something else. It is usually followed by a cut back to the first shot.
How does the 180-degree system influence screen direction? It ensures consistent screen direction when shots are edited together. … The resulting shots orient the viewer as to what is happening in the scene.
In filmmaking and video production, a shot is a series of frames that runs for an uninterrupted period of time. Film shots are an essential aspect of a movie where angles, transitions and cuts are used to further express emotion, ideas and movement.
Montages enable filmmakers to communicate a large amount of information to an audience over a shorter span of time by juxtaposing different shots, compressing time through editing, or intertwining multiple storylines of a narrative.
The Kuleshov effect is a film editing (montage) effect demonstrated by Soviet filmmaker Lev Kuleshov in the 1910s and 1920s. It is a mental phenomenon by which viewers derive more meaning from the interaction of two sequential shots than from a single shot in isolation.
(Entry 1 of 2) 1 : the production of a rapid succession of images in a motion picture to illustrate an association of ideas. 2a : a literary, musical, or artistic composite (see composite entry 2 sense 1) of juxtaposed more or less heterogeneous elements.
The Kuleshov Effect informs the way modern filmmakers make movies: Pen big reactions into scripts. If you're writing a script, give your characters the chance to react to every important piece of dialogue, reinforcing their emotions, beliefs, and world views. These reactions will be invaluable in the edit.
In filmmaking, the 180-degree rule is a basic guideline regarding the on-screen spatial relationship between a character and another character or object within a scene. By keeping the camera on one side of an imaginary axis between two characters, the first character is always frame right of the second character.
What is the ultimate goal of continuity editing? To communicate the story as clearly, efficiently, and coherently as possible. What are the fundamental building blocks of continuity editing? Master shots and the 180-degree system.
Montage is a technique in film editing in which a series of short shots are edited into a sequence to condense space, time, and information. The term has been used in various contexts. It was introduced to cinema primarily by Sergei Eisenstein.
A jump cut is is an abrupt transition, typically in a sequential clip that makes the subject appear to jump from one spot to the other, without continuity. This can happen when two sequential shots of the same subject in the same scene are cut together from camera positions that vary only slightly.
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The Catholic Way of Death and Burial
What to do when there is a death in the family
"If pastoral and personal considerations allow, the period before death may be an appropriate time to plan the funeral rites with the family and even with the family member who is dying. Although planning the funeral before death should be approached with sensitivity and care, it can have the effect of helping the one who is dying and the family face the reality of death with Christian hope. It can also help relieve the family of numerous details after the death and may allow them to benefit more fully from the celebration of the funeral rites." —Order of Christian Funerals (OOCF), #17
Step 1 — Before Death
Plan a visit with your parish priest so he can offer the Sacraments to the dying family member and answer any questions the family may have about death and burial in the Catholic Church. Discuss your dying family member's wishes, taking into full consideration what is best for them and their loved ones. What kind of a funeral and burial do they want? Where will the family gather to pray for their soul in the years to come? If the person expresses a desire to be cremated, pursue their motivation. Funeral industry research shows that people often request cremation in order to save their loved ones the grief of seeing their body in death. In fact, a final encounter with the body of a loved one is extremely important to family and friends. The lack of a body during the vigil, funeral and burial often makes the reality of death more difficult to accept for those who have been left behind.
"The Catholic Church strongly prefers that the body of the deceased be present for its funeral rites since the presence of the body most clearly brings to mind the life and death of the person…The body which lies in death naturally recalls the personal story of faith, the loving family bonds, the friendships, and the words and acts of kindness of the deceased person. Indeed, the human body is inextricably associated with the human person, which acts and is experienced by others through that body."—OOCF, "Reflections on the Body, Cremation and Catholic Funeral Rites" and #411
Step 2 — At Death
Call your parish priest. He can pray with you, comfort your family and answer any questions you may have during the next few sorrowful days. After you have talked to a priest, contact a funeral director and a Catholic cemeterian. Since the arrangements for death and burial require significant choices, it is important not to hand over all responsibility for the vigil, funeral and burial to a funeral director alone. You can—and should—talk to both the rectory and cemetery office yourself, as well. After major decisions have taken place, the funeral director can confirm your plans. Unless an autopsy is expected, the funeral director will send a car to convey the body from its place of death to the funeral home.
At the parish, you will receive help planning either a Mass of Christian Burial or a Memorial Mass. The priest or parish representative will help you pick the readings and responses, and offer options for music. You may want to have a program prepared for use during the Mass or prayer service. The rectory staff can provide you with information on this, as well.
At the funeral home, the director will help you plan for the vigil, funeral and viewings. You will have to choose a casket, an important consumer option with a considerable price range. If you want him to, the director can help you order flowers and provide transportation for your family for the funeral Mass and burial at the cemetery. You will also be asked to provide information for a death notice in the newspaper. Bring as much vital statistic information as you can such as your family member's social security number, life insurance information, etc.
At the cemetery, the cemetarian will assist you in selecting a burial location. In the Catholic cemeteries of the Archdiocese, you have the option of ground burial or entombment in a mausoleum. For ground burial, memorialization is available either with upright monuments at selected locations or in-ground bronze plaques. An outer container is also needed to hold the casket. If the body has been cremated, in ground burial or entombment in a mausoleum niche are available options. These consumer choices at the cemetery are significant long-term decisions. This is why the Church recommends planning ahead, so that choices can be made without the heavy sorrow so debilitating at the time of death.
Step 3 — The Vigil
The vigil is held either at the funeral home, private residence or in the parish church. The Church provides a liturgy of the Word for the comfort of family and friends. It is also traditional to have a priest, deacon, nun, family member or friend lead the mourners in the Rosary. The vigil is a time to pray for the soul of the faithful departed and all the other souls of family members. It is a time to remember the life of the deceased and comfort the living. Depending on the family's wishes, the vigil can be held for one or two days.
"At the vigil, the Christian community keeps watch with the family in prayer to the God of mercy and finds strength in Christ's presence. It is the first occasion among the funeral rites for the solemn reading of the word of God. In this time of loss, the family and community turn to God's word as the source of faith and hope, as light and life in the face of darkness and death." —OOCF, #56
Step 4 — The Funeral
The Mass of Christian Burial with the presence of the body of the deceased in the church provides the full meaning of the Eucharistic sacrifice of Jesus Christ. Here, the family is reminded that this loved one who was baptized in Christ, and nourished at the altar, still lives on, and shares in the hope of the Resurrection of the body. The Church asks that any eulogies for the deceased take place after the Mass is ended, before the final blessing.
The Memorial Mass for the Dead takes place without the presence of the body. A Memorial service of prayers is another option with the liturgy of the Word, although the Church most highly encourages the Eucharistic celebration to pray for the dead and comfort the mourners.
"At the funeral liturgy, the community gathers with the family and friends of the deceased to give praise and thanks to God for Christ's victory over sin and death, to commend the deceased to God's tender mercy and compassion, and to seek strength in the proclamation of the paschal mystery. Through the Holy Spirit, the community is joined together in faith as one Body in Christ to reaffirm in sign and symbol, word and gesture, that each believer through baptism shares in Christ's death and resurrection and can look to the day when all the elect will be raised up and united in the kingdom of light and peace." —OOCF, #129
Step 5 — The Burial
The Church asks that the burial of the body take place after the Funeral, preferably in the sacred ground of a Catholic cemetery. Catholic cemeteries call to mind our faith in the Risen Christ as the faithful wait together in joyful hope for the resurrection of their bodies.
At the graveside, the priest or deacon leads a service of prayers, blessing the grave and praying for the soul of the deceased and all the other souls buried in the cemetery. Because the Church recognizes the great dignity of the human body, she asks that cremated remains never be scattered, but buried in the sacred ground of a cemetery. This is in keeping with the sanctity of the body and provides a place for the family to gather in prayer.
"The rite [of committal] marks the separation in this life of the mourners from the deceased, and through it the community assists them as they complete their care for the deceased and lay the body to rest. The act of committal is a stark and powerful expression of this separation. When carried out in the midst of the community of faith, the committal can help the mourners to face the end of one relationship with the deceased and to begin a new one based on prayerful remembrance, gratitude and the hope of resurrection and reunion." —OOFC, #213
Step 6 — Remembrance
The weeks and months following a death in the family are numbing and difficult. Prayer and participation in the life of the Church can provide comfort and a way of continuing the relationship with the deceased family member. Visits to the cemetery also provide comfort and an ongoing assurance of the Church's hope in the resurrection of the dead. The Catholic cemeteries of the Archdiocese celebrate monthly masses for the souls of the faithful departed buried there, as well as special prayer services and Masses such as Good Friday Stations of the Cross, Memorial Day, and the Commemorations of Our Lady of Sorrows and All Souls. Every day, family members experience the peace and comfort that comes from visiting the ground set aside and made sacred, the resting place of their loved ones.
*** Please Note : If you have experienced a death in your family, please contact a funeral home first. Generally, all funeral arrangements, including the scheduling of the Funeral Mass will be made through the funeral home with the parish. A funeral Mass or burial cannot be scheduled without the parish priest being involved in the process.
St Ann Catholic Church Fayetteville, NC
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New playground alert! Climb, run, swing, picnic, take a nature walk, play a game of pickleball and — bonus for grown-ups — even get in a workout at newly expanded Covington Community Park.
Families in the Covington area probably already know Covington Community Park for its busy soccer fields. But the athletic fields were just the first phase of this park's overhaul. Phase Two projects — the new playground and much more — opened recently and it's all amazing.
Newly opened features double the previous size of the park and include a terrific children's play area. You'll also find outdoor fitness equipment, a court for tennis or pickleball, an extended walking trail, an open-air pavilion with lawn seating and lots of picnic tables. Wow!
I took two kids there recently to check it all out.
There are two parking areas for this park; to reach the the playground be sure to use the lot off of 180th for easiest access. The first thing you will see is the big concrete semi circle arches at the entrance which look cool and make for fun photo ops.
As soon as we entered the park, my kids ran straight for the zipline. The structure features two lines, one with a disc seat and the other with an oversized seat so littles can catch a ride right next to the big kids.
Near the zip line sits the play area for 2- to 5-year-olds. Kid-sized musical instruments, including a xylophone and several different drums, were getting good use the day we visited: Nearly every kid I saw pass by paused long enough to bang out a tune or two.
Right next to the music area is the spot where new restrooms will open soon. Currently portable toilets are provided and there's a drinking fountain. When your little one needs a break, enjoy a snack or grab a book from the Little Free Library and have a seat at one of the two picnic tables which are under cover, perfect for escaping a rain shower!
Other fun for the toddler and preschooler set includes a teeter-totter, bouncy bugs and a small play structure with a tiny rock wall, slide and climbing net.
Attached to this is the play set for kids ages 5–12. This features a taller, steeper slide and a huge cargo net climbing structure plus lots of other stuff to scale. There is even a big rock for little mountaineers. The play area is on artificial turf, a definite bonus on muddy days.
A separate swingset offers four regular swings plus two gliders with a baby seat and regular swing combo, a sweet way for a caregiver or sibling and baby can swing together, facing each other.
All around the area is plenty of bench seating for parents, plus picnic pavilions. Although the park is bordered by busy streets on two sides, in the background are woods and wetlands, making for a pretty view.
Behind the playset is an open-air stage and pavilion named after Covington City Councilwoman Margaret Harto. The pavilion sports a "green roof," a roof with soil and plants growing on the surface, which helps to filter rainwater and slow down runoff during heavy rains. Kids will love the lawn seating which is newly filled in with grass and perfect for rolling down the hill or throwing a Frisbee. And don't miss the new tennis-pickleball court near the parking lot.
Wander down the paved path toward the soccer fields and you will find an outdoor gym on artificial turf. There are four pieces of non-motorized fitness equipment with which you can get a full body workout. Posted on each are instructions for use, plus a link to a YouTube video for more information.
If you would like to get in a walk, or for fledgling pedalers to practice, stroll along the paved path that goes around the area for about a quarter-mile loop. There are also some nature trails in the woods behind the pavillion, a great place to take in some nature and sneak in a little learning by reading the posted plaques with information on the flora and fauna of the forest. These are very short loops, perfect for all ages and some are ADA-accessible.
Find it: Covington Community Park is located at 17649 S.E. 240th St., Covington. Covington is a city in south King County, east of Kent and roughly 30 minutes from Tacoma, 40 minutes from Bellevue and 45 minutes from Seattle.
Parking: The park has two lots; use the lot off 180th Avenue S.E. to get closest to the playground.
Coming soon: Permanent restrooms, a concession stand and vending machines.
Plan a party: Reserve a picnic structure with a grill by calling 253-480-2487.
Snack time: Try Bruchelles's Bagel Bistro at 16340 S.E. 256th St., Suite 400, Covington, a couple of miles from the park, or there is a McDonald's with a playplace a 7-minute drive from the park at 23225 Maple Valley Hwy, Maple Valley.


Ginger leaf (GL) has long been used as a vegetable, tea and herbal medicine. However, its pharmacological properties are still poorly understood. Thus, we performed in vitro studies to evaluate anti-cancer properties of ginger leaf and then elucidate the potential mechanisms involved.
Cell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA.
Exposure of GL to human colorectal cancer cells (HCT116, SW480 and LoVo cells) reduced the cell viability and induced apoptosis in a dose-dependent manner. In addition, GL reduced cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. ATF3 knockdown attenuated GL-mediated apoptosis. GL increased activating transcription factor 3 (ATF3) expressions in both protein and mRNA level and activated ATF3 promoter activity, indicating transcriptional activation of ATF3 gene by GL. In addition, our data showed that GL-responsible sites might be between -318 and -85 region of the ATF3 promoter. We also observed that ERK1/2 inhibition by PD98059 attenuated GL-mediated ATF3 expression but not p38 inhibition by SB203580, indicating ERK1/2 pathway implicated in GL-induced ATF3 activation.
These findings suggest that the reduction of cell viability and apoptosis by GL may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression through ERK1/2 activation in human colorectal cancer cells.
Cancer is a major problem of public health in many other parts of the world including the United States . Among the kinds of cancers, human colorectal cancer is the third leading cause of cancer-related death in both male and females in the United States . Although surgery and chemotherapy have been the most common treatment for colorectal cancer, cancer chemoprevention with dietary factors has received attention as the most effective approach to reduce colorectal cancer-related mortality. Thus, for the last two decades, many researchers have tested and reported anti-cancer activities of natural products in dietary factors such as fruits, vegetables and teas [2, 3].
Gingers as perennial herbs belonging to the family Zingiberaceae have been widely used as spices, condiments and herbal medicine for treatment of cold, fever, headache, nausea and digestive problems . Ginger and its general compounds such as gingerols, shogaols, paradols and zingerone exert immuno-modulatory, anti-apoptotic, anti-tumourigenic, anti-inflammatory, anti-hyperglycaemic, anti-hyperlipidaemic, antioxidant and anti-emetic activities . Ginger leaves have also been used for food flavouring and traditional medicine . Past pharmacological studies of ginger were confined to rhizomes. Thus, in light of the pharmacological actions of ginger leaves, this study was performed to investigate the anti-cancer activity and elucidate the potential mechanism by which ginger leaves induces the reduction of cell viability and apoptosis in human colorectal cancer cells. Here, for the first time, we report that ginger leaves leads to transcriptional activation of activating transcription factor 3 (ATF3) which may be associated with the reduction of cell viability and induction of apoptosis in human colorectal cancer cells.
Cell culture media, Dulbecco's Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) was purchased from Lonza (Walkersville, MD, USA). The 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and SP600125 was purchased from Sigma Aldrich (St. Louis, MO, USA). SB203580 and PD98059 were purchased from Calbiochem (San Diego, CA, USA). ATF3 antibody and ATF3 siRNA were provided from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Antibodies against β-actin and poly (ADP-ribose) polymerase (PARP), and control siRNA were purchased from Cell Signaling (Bervely, MA, USA). ATF3 promoter constructs (-1420/+34, -718/+34, -514/+34, -318/+34, -147/+34 and -84/+34) were kindly provided by Dr. S-H Lee (University of Maryland College Park, MD, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.
The leaves of ginger (Zingiber officinale, voucher number: PARK1003(ANH)) were kindly provided by the Bonghwa Alpine Medicinal Plant Experiment Station, Korea. One kilogram of ginger leaf was extracted with 1000 ml of 80% methanol with shaking for 24 h. After 24 h, the methanol-soluble fraction was filtered and concentrated to approximately 20 ml volume using a vacuum evaporator and then fractionated with petroleum ether and ethyl acetate in a separating funnel. The ethyl acetate fraction was separated from the mixture, evaporated by a vacuum evaporator, and prepared aseptically and kept in a refrigerator.
Human colorectal cancer cell lines (HCT116, SW480 and LoVo), human breast cancer cell lines (MCF-7 and MDA-MB231) and human hepatocellular carcinoma (HepG-2) were purchased from Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fatal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained at 37°C under a humidified atmosphere of 5% CO2. The extracts of ginger leaf (GL) were dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 0.1% (v/v).
Cell viability was measured using MTT assay system. Briefly, cells were plated onto 96-well plated and grown overnight. The cells were treated with 0, 50, 100 and 200 μg/ml of GL for 24 and 48 h. Then, the cells were incubated with 50 μl of MTT solution (1 mg/ml) for an additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm.
Total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer's protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with primers for human ATF3 and human GAPDH as follows: human ATF3: 5′-gtttgaggattttgctaacctgac-3′, and reverse 5′-agctgcaatcttatttctttctcgt-3′; huaman GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′-ttctagacggcaggtcaggt-3′.
Transient transfections were performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers' instruction. HCT116 and SW480 cells were plated in 12-well plates at a concentration of 2 × 105 cells/well. After growth overnight, plasmid mixtures containing 0.5 μg of ATF3 promoter linked to luciferase and 0.05 μg of pRL-null vector were transfected for 24 h. The transfected cells were cultured in the absence or presence of GL for the indicated times. The cells were then harvested in 1 × luciferase lysis buffer, and luciferase activity was normalized to the pRL-null luciferase activity using a dual-luciferase assay kit (Promega, Madison, WI, USA).
The cells were plated in six-well plates and incubated overnight. HCT116 cells were transfected with control siRNA and ATF3 siRNA for 48 h at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA) according to the manufacturer's instruction. Then the cells were treated with GL (100 μg/ml) for 24 h.
Cell death was performed using Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instruction. Briefly, HCT116 and SW480 cells were seeded in 12-well plate. After 24 h, cells were treated with 0, 25, 50 and 100 μM of GL for 24 h. Cytosol was prepared using Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). Equal amounts of cytosolic extracts, immunoreagent containing anti-histone-biotin, and anti-DNA-POD were added to microplate well and incubated for 2 h under shaking. After washing, the ABTS solution was added to each well for 20 min and then the ABTS stop solution was added. The absorbance was recorded at 405 nm and 490.
After GL treatment, cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MD. USA) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000 × g for 10 min at 4°C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4°C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
Statistical analysis was performed with the Student's unpaired t-test, with statistical significance set at *, P <0.05.
To investigate whether ginger leaf affects the cell viability in human colorectal cancer cells, HCT116, SW480 and LoVo, the cells were incubated with 50, 100 and 200 μg/ml of GL for 24 and 48 h, and cell viability was measured using MTT assay. As shown in Figure 1A, GL reduced the viability of HCT116 cells by 24 and 59% at 50 μg/ml, 53 and 79% at 100 μg/ml, and 79 and 88% at 200 μg/ml at 24 and 48 h after GL treatment, respectively. We also found that the viability of SW480 cells was reduced by GL treatment for 24 and 48 h by 23 and 40% at 50 μg/ml, 42 and 57% at 100 μg/ml, and 55 and 76% at 200 μg/ml, respectively (Figure 1B). In addition, GL suppressed LoVo cell viability by 20 and 33% at 50 μg/ml, 34 and 55% at 100 μg/ml, and 59 and 80% at 200 μg/ml after GL treatment for 24 and 48 h, respectively (Figure 1C). HCT116 and SW480 cells were treated with 25, 50 and 100 μg/ml of GL for 24 h, and apoptosis was measured using Western blot against cleaved PARP and the cell death assay using ELISA-based cell death kit. As shown in Figure 1D, the cleavage of PARP was observed at 50 and 100 μg/ml in HCT116 and SW480 cells, respectively, which indicates that GL may induce apoptosis. In addition, significant increases of cell death were observed in the cells treated with GL (Figure 1E). To determine if GL shows a reduction of cell viability in other types of cancer cells, breast (MCF-7 and MDA-MD-231) and hepatocellular carcinoma (HepG-2) cells were treated with 100 μg/ml at 24 h. As a result, GL-treatment for 24 h reduced the cell viability by 36% in MCF-7, 44% in MDA-MB-231, and 30% in HepG-2 cells, respectively (Figure 1F). These data indicates that GL may have anti-cancer activity in colorectal cancer, breast cancer and hepatocellular carcinoma cells.
The effects of GL on cell viability and apoptosis in human colorectal cancer cells. HCT116 (A), SW480 (B) and LoVo cells (C) were treated with 0, 50, 100 and 200 μg/ml of GL for 24 and 48 h. Cell viability was measured using MTT assay system and expressed as % cell viability compared to the cell without GL treatment. *P < 0.05 compared to cells without GL treatment. (D) HCT116 and SW480 cells were treated with 0, 25, 50 and 100 μg/ml of GL for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against PARP. Actin was used as internal control. (E) HCT116 and SW480 cells were treated with 0, 25, 50 or 100 μM of GL for 24 h. the cytosol fraction was extracted from GL-treated cells, and the cell death was measured using the Cell Death Detection ELISAPLUS Kit, and expressed as absorbance (A405-A490). *p < 0.05 compared to cells without GL treatment. (F) MCF-7, MDA-MB-231 and HepG-2 cells were treated with 100 μg/ml of GL for 24 h. Cell viability was measured using MTT assay system and expressed as % cell viability compared to the cell without GL treatment. *P < 0.05 compared to cells without GL treatment.
Since there is growing evidence that ATF3 is associated with to cell growth arrest and apoptosis in human colorectal cancer cells , we evaluated whether GL affects ATF3 expression in HCT116 and SW480 cells. As shown in Figure 2A-D, GL-induced ATF3 expressions were observed at 50 and 100 μg/ml of GL in both protein and mRNA level. To investigate whether increase in ATF3 expression by GL treatment was mediated from transcription activation of ATF3 promoter, ATF3 promoter (-1420/+34) transfected HCT116 and SW480 cells were incubated with 25, 50 and 100 μg/ml of GL for 24 h. As shown in Figure 2E and F, GL dose-dependently activated ATF3 transcription. In time-course experiments, we found that the expression of ATF3 mRNA activation by GL started to be increased at 1 h after GL treatment in HCT116 cells (Figure 2G). To see whether ATF3 expression affect GL-mediated apoptosis, cleaved PARP was measured by Western blot in ATF3 knock downed-HCT116 cells. As shown in Figure 2H, knockdown of ATF3 by ATF3 siRNA reduced the cleavage of PARP by GL, indicating that ATF3 may be one of important genes in GL-mediated apoptosis.
Effects of GL on ATF3 activation in human colorectal cancer cells. (A, B) HCT116 and SW480 cells were treated with 0, 25, 50 and 100 μg/ml of GL for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against ATF3. Actin was used as internal control. For RT-PCR analysis of ATF3 gene expression, total RNA was prepared after GL treatment for 24 h in dose-course experiments (C, D) or for the indicated times in time-course experiments (G). GAPDH were used as internal control. For ATF3 promoter activity, luciferase construct containing -1420 to +34 of human ATF3 promoter region was cotransfected with pRL-null vector and the cells were treated with GL for 24 h in dose-course experiments (E, F) and luciferase activity was measured. *P <0.05 compared to cells without GL treatment. (H) ATF3 siRNA was transfected into HCT116 for 48 h and then GL was treated for 24 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against PARP. Actin was used as internal control.
To determine upstream kinase affecting GL-induced ATF3 transcriptional activation, ATF3 promoter (-1420/+34) transfected HCT116 and SW480 cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor) and SB203580 (p38 inhibitor) for 2 h and co-treated with 100 μg/ml of GL for 24 h. As shown in Figure 3A and B, ERK1/2 inhibition by PD98059 attenuated GL-mediated ATF3 promoter activation, while p38 inhibition by SB203580 did not affect ATF3 activation by GL in both HCT116 and SW480 cells. We also investigated whether JNK signaling affects GL-mediated ATF3 promoter activation and found that JNK inhibition by SP600125 did not affect ATF3 promoter activation by GL (data not shown).We investigated that GL treatment affects ERK1/2 activation. As shown in Figure 3C and D, GL enhanced phosphorylation of ERK1/2 protein. We also tested that the inhibitions of ERK1/2 by PD98059 and p38MAPK by SB203580 affect GL-mediated ATF3 protein level. In these experiments, the inhibition of ERK1/2 by PD98059 ameliorated the increase of ATF3 protein level by GL, while p38 MAPK inhibition did not affect ATF3 protein level in HCT116 cells (Figure 3E). Because GL-mediated ATF3 activation was affected by ERK1/2, we evaluated if ERK1/2 affects GL-mediated apoptosis. As a result, ERK1/2 inhibition by PD98059 attenuated GL-mediated cleavage of PARP in HCT116 cells (Figure 3F). These results indicates that GL-mediated apoptosis may result from ERK1/2-dependent ATF3 activation.
Up-stream signaling pathways affecting GL-mediated ATF3 activation. (A, B) Luciferase construct containing -1420 to +34 of human ATF3 promoter region was cotransfected with pRL-null vector. Then, the cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor) or SB203580 (p38 inhibitor) and then cotreated with 100 μg/ml of GL for 24 h. *P <0.05 compared to cells without the treatment of inhibitors. (C, D) HCT116 and SW480 cells were treated with 100 μg/ml of GL for indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-ERK1/2 and ERK1/2. Actin was used as internal control. (E) HCT116 cells were pre-treated with 20 μM of PD98059 (ERK1/2 inhibitor) or SB203580 (p38 inhibitor) and then co-treated with 100 μg/ml of EAFAD-B for 6 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against ATF3. Actin was used as internal control. (F) HCT116 cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor) for 2 h and then co-treated with 100 μM of GL for 24 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against PARP. Actin was used as internal control.
To investigate ATF3 promoter sites which can be responsible for GL-induced ATF3 activation, promoter activity was measured using different sizes of ATF3 promoter luciferase constructs (-1420/+34, -718/+34, -514/+34, -318/+34, -147/+34 and -84/+34). These constructs were transfected into SW480 cells and treated with 100 μg/ml of GL for 24 h. As shown in Figure 4A, GL treatment resulted in an increase of promoter activity. The fold induction in SW480 cells was 5.1, 5.0, 4.4, 4.2, 3.1 and 2.5 in pATF3-1420/+34, pATF3- 718/+ 34, pATF3- 514/+ 34, pATF3- 318/+ 34, pATF3-147/+ 34, and pATF3- 84/+ 34, respectively. Because GL increased ATF3 promoter activity more than 4.0-fold in SW480 cells, GL-responsible sites might be between -318 and -85 region of the ATF3 promoter. The Fushi tarazu (Ftz) and CREB is cis-acting elements in ATF3 promoter containing -147 and -85 by program (Gene Regulation, TFSEARCH, and Transcription Element System). To identify the role of each cis-acting element, each site-deleted ATF3 promoter constructs were transfected into SW480 cells and treated with 100 μg/ml of GL for 24 h. As shown in Figure 4B, GL-induced ATF3 promoter activity was significantly decreased when the CREB site was deleted. However, the deletion of Ftz sites did not affect ATF3 promoter activity by GL. These data indicated that CREB is an important region in GL-induced ATF3 expression.
Identification of ATF3 promoter sites responsible for GL-induced ATF3 activation. (A). Each indicated construct of the ATF3 promoter (0.5 μg) was co-transfected with 0.05 μg of pRL-null vector into SW480 cells, and cells were treated with 100 μg/ml of GL for 24 h. (B) Each deletion construct (0.5 μg) of the ATF3 promoter was co-transfected with 0.05 μg of pRL-null vector into SW480 cells and cells were treated with 100 μg/ml of GL for 24 h. Luciferase activity was measured. *P <0.05 compared to cells without GL treatment.
ATF3, an ATF/CREB subfamily member, contains the basic-leucine zipper (b-ZIP) DNA binding domain . ATF3 is dramatically expressed in response to several stresses in many different tissues and exerts diverse biological effects . In cancer development, ATF3 exerts pro-or anti-apoptotic activities dependent on cell or tissue context [8, 9]. ATF3 expression was suppressed in human colorectal cancer  and expression of ATF3 induced apoptosis, growth arrest of colorectal cancer cells and Ras-stimulated tumourigenesis [11–13]. On the other hand, ATF3 induces DNA synthesis and expression of cyclin D1 in hepatocellular carcinoma cells  and enhances cancer cell-initiating features in breast cancer . Although ATF3 has dual effects in cancer development, ATF3 has been regarded as a major target of cancer chemoprevention in colorectal cancer. Our data indicate that GL increased ATF3 expression in both protein and mRNA level in a time- and dose-dependent manner through the activation of ATF3 promoter. In addition, it was reported that anti-cancer agents such as indole-3-carbinol , conjugated linoleic acid , epicatechin gallate , tolfenamic acid  and PI3 kinase inhibitor  induce ATF3-dependent apoptosis in colorectal cancer cells. In our study, GL increased the PARP cleavage and reduced the viability of colorectal cancer cells, indicating that increased apoptosis and reduction of cell viability may be mediated by activation of ATF3 expression in GL-treated cells.
There is a growing body of evidence to suggest that MAPK signaling is an important pathway regulating ATF3 expression [18, 20]. Therefore, we examined whether GL-mediated ATF3 activation is associated with the activation of ERK1/2, p38 and JNK. ERK1/2 inhibition by PD98059 attenuated GL-induced activation of ATF3 promoter and ATF3 expression but not in inhibition of p38 and JNK by SB203580 and bySP600125, indicating that ERK1/2 activation may contribute to GL-induced ATF3 activation. In addition, inhibition of ERK1/2 ameliorated GL-mediated apoptosis. In Western blot analysis for phosphorylation of ERK1/2, we found that GL induced a prolonged activation of ERK1/2. Why would late phase ERK activation correlate with proliferation and apoptosis remains to be understood. However, there is one hypothesis that prolonged activation of ERK1/2 can promote accumulation of p21cip1 resulting in cell cycle arrest and apoptosis . Similarly, several anti-cancer agents have been reported to induce a prolonged activation of ERK1/2, which results in promoting apoptosis [22–25].
Interestingly, we found that GL-responsible sites for ATF3 activation might be between -318 and -85 region of the ATF3 promoter. ATF3 promoter includes various response elements such as AP-1, ATF/CRE, NF-κB, E2F and Myc/Max binding sites  and especially, EGR-1, CRE and Ftz are cis-acting elements in ATF3 promoter (-318/-85)  from which our data showed that GL-induced ATF3 promoter activity was significantly decreased when the CREB site was deleted. These data indicated that CREB is an important region in GL-induced ATF3 expression.
There is a report that ginger leaves has various bioactive compounds including quercetin, rutin, epicatechin, catechin, kaempferol, naringenin, salicylic acid, cinnamic acid, flavonoids and phenolics . Among the bioactive compounds, quercetin has been reported to induce ATF3 expression in human colorectal cancer cells, Caco-2 . Interestingly, we found that ginger leaves had more quercetin (2.124 mg/g dry weight) than ginger rhizoma (1.105 mg/g dry weight), which is similar to the previous report . According to the previous study [30, 31] quercetin can be efficiently hydrolysed and absorbed in the intestinal lumen and plasma. Therefore, it is thought that quercetin may be responsible for ATF3-mediated apoptosis by ginger leaves in human colorectal cancer cells and quercetin of GL may be absorbed in the intestinal lumen and plasma of animal.
In conclusion, ginger leaves may induce apoptosis and reduction of cell viability, followed by the increased ATF3 expression via activating ATF3 promoter in human colorectal cancer cells. In addition, there is a growing evidence that ginger leaves had higher antioxidant activity than rhizomes and flowers Eric Chan et al. . Therefore, ginger leaves has great potential to be developed into functional foods and other health products.
Gwang Hun Park, Jae Ho Park, Hun Min Song contributed equally to this work.
We thank Dr. Seong-Ho Lee (University of Maryland College Park, MD, USA) for providing ATF3 promoter (pATF3-1420/+34, pATF3- 718/+ 34, pATF3- 514/+ 34, pATF3- 318/+ 34, pATF3-147/+ 34, and pATF3- 84/+34). This work was supported by the BK21 PLUS program of Ministry of Education, the Ministry of Education, Science and Technology (2011-0025272), and by a grant from 2014 Research Fund of Andong National University (2014-0169).
JBJ directed and GHP, JHP & HMS designed the study. JHP undertook the formal identification of the leaves of ginger (Zingiber officinale). GHP, JHP, HMS, HJE, MKK, JWL, MHL, K-HC, JRL and HJC performed the experiments. GHP, JHP and HMS drafted manuscript. HJE, MKK, JWL, MHL, K-HC, JRL, HJC and JBJ corrected the manuscript. All authors read and approved the final manuscript.


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University of Miami Miller School of Medicine
"see above" Report Response
"My interviewer just asked me to summarize my life up to the present and explain what has led to me to choose medicine. I felt as though he really wanted to get to know me and to make the interview as relaxed as possible." Report Response
"Reread my AMCAS and secondary app, read through the Miami website" Report Response
"Everything! The students are so genuinely happy at Miami. My interviewer was very laid back and made every effort to make the interview as painless as possible. He just wanted to hear my life story and how it was that I came to choose medicine. The hospital is enormous (which in my opinion is a definite positive) but the faculty and students seemed very close knit. The curriculum changes made a few years ago seemed to have improved the program significantly and the students say they really like the way it is now structured. Dean Hinkley is about as friendly as they come! " Report Response
"absolutely nothing" Report Response
"Miami traffic is pretty bad." Report Response
"Having been through several interviews this year, I can say that Miami was without a doubt the best interview experience I've had. The interview day put Miami at the very top of my list!" Report Response


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Notre Dame douses red-hot Syracuse, more Snap Judgments
Notre Dame 67, Syracuse 58: OK, it certainly isn't going down in the annals of Irish hoops' lore as being on a par with Digger Phelps' John Shumate-led club throttling the Bill Walton-led and John Wooden-coached UCLA Bruins' 88-game win streak in South Bend nearly 38 years ago. But, considering this Notre Dame team has been playing without the program's best player since Tim Ambromaitis suffered a torn ACL during a practice the day after Thanksgiving and had dropped two Big East games over the previous six days, coach Mike Brey and his crew couldn't have asked for a niftier way to improve to 12-8.
The program's eighth victory over a top-ranked foe came against a Syracuse squad that had won only five times by margins of fewer than 10 points while zipping to a 20-0 start.
The Orange played without sophomore center Fab Melo (7.2 points, 5.7 rebounds and 3.0 blocked shots per game) -- he didn't make the trip, for which Coach Jim Boeheim refused to reveal the reason -- but it's doubtful his presence would have been enough to overcome an Irish offense that hit 50 percent from the field, including eight of 16 behind the arc.
No one yet should start questioning Boeheim's team's status as best in the Big East and a firm contender to get to the Final Four -- assuming Melo isn't off the floor for an extending period.
The result did much, much more to help Notre Dame's NCAA Tournament at-large bid possibilities than it did to alter the consensus of opinion on the Orange.
Florida State 76, Duke 73: Exactly two weeks before Leonard Hamilton's Seminoles took the Cameron Indoor Stadium floor Saturday for their ACC game against the Blue Devils, they were bopped around to the tune of 79-59 at Clemson, their third loss in four games and sixth in 10.
Since then they edged host Virginia Tech by four points, nuked North Carolina by 33 -- I repeat, 33 -- points in Tallahassee and methodically dominated visiting Maryland by 14 points.
Oh, yes: They left that Cameron Indoor Stadium floor a few hours later after Michael Snaer's transition three-pointer from the right wing, after a snap pass from Luke Loucks, zinged the nets as the buzzer sounded, jolting the Duke Crazies to near-silence and snuffing the Blue Devils' 45-game win streak in the building.
What a difference 14 days can make: The Seminoles, since that thumping in Clemson, are in a first-place tie (at 4-1) with the Blue Devils. The moral for those who follow college hoops? How about "Judge thee, not too harshly, a team a week into January"?
Missouri 89, Baylor 88: Until Michael Snaer's Jump Shot of the Week a couple of hours later in Durham, NC, the Tigers appeared to have a stranglehold on the Road Victory of the Week after knocking off the Bears in Waco.
Not to worry, though: That return trip to Columbia was just fine with an 18-1 record -- 5-1 in the Big 12 -- in tow.
How does a team hit 57 percent from the floor and score 88 points and still drop its second game in a row (the Bears were handed their first loss of the season Monday, Kansas doing the honors in Lawrence, 92-74)? By committing 19 turnovers, allowing the Tigers to shoot 54.5 percent from the field (including 23 of 34 inside the arc) and fouling enough to give Frank Haith's masters of "beating people off the dribble" the opportunity to hit 22 of 31 free throws.
The Jayhawks hit 35 of 61 from the field Monday night against Scott Drew's team.
We can guess what Drew will emphasize in practice before the Bears go to Norman for a Wednesday night game against Oklahoma.
Kansas 69, Texas 66: Senior point guard Tyshawn Taylor has been on the blunt end of a lot of criticism for his inconsistent -- erratic, if you will -- play for the Jayhawks.
But Bob Cousy Award voters, coach Bill Self and Jayhawk Nation had to be mightily impressed with Taylor's performances Saturday in Austin and Monday night in Lawrence.
If Season Length Is the Concern, There's a Fix to Expand CFP
Taylor had 22 points, five rebounds, four assists and no turnovers in 34 minutes Saturday -- six days after going for 28, two, six and five in 33 during the defeat of Baylor.
If Taylor plays anything close to that same level while teammate Thomas Robinson continues on his path to the John R. Wooden Award, the Jayhawks' ambitions will rightfully extend well beyond a Big 12 title.
Kentucky 77, Alabama 71: Coach John Calipari and the Wildcats didn't know it at the time, but the Wildcats' down-to-the wire SEC victory over the visiting Crimson Tide was going to be enough to move them to the top of the newest national ratings.
Anthony Davis hit just two of 10 shots from the field, but the likely National Freshman of the Year -- and very possible SEC Player of the Year -- impacted the game in significant other ways via his nine rebounds, four steals, four blocked shots and countless "shot alterations."
The free-throw performance -- 27 of 40 -- was of the so-so variety but just good enough to keep poll voters from having a really difficult decision to make on Monday.
Tennessee 60, Connecticut 57: The defending national champions may have more "talent," per se, than the Huskies possessed while going 11-0 in Big East and NCAA Tournament play last spring. After their second loss in a row Saturday in Knoxville -- and fourth in six games -- that theory is moot right now.
On an afternoon when one of the nation's most touted freshmen (Andre Drummond) and one of the players who led the Huskies to that national title (Alex Oriakhi) were on the floor, the best big man in the game was clearly another freshman -- mid-year high school graduate Jarnell Stokes (16 points and 12 rebounds), who made his college debut against Kentucky seven days before.
So much for enduring periods of adjustment ...
West Virginia 77, Cincinnati 74 (OT): Ladies and gentlemen meet the hottest team in the Big East right now. Coach Bob Huggins' Mountaineers can make that claim after their fifth win in six games since they lost by 19 points at Seton Hall two days before New Year's.
Is WVU senior forward Kevin Jones (26 points and 13 rebounds Saturday against Huggins' former program) the best player in the Big East? A compelling argument could be made in behalf of that suggestion.
Louisville 73, Pittsburgh 62: Just when it seemed as if the outlook for the rest of the season couldn't get any dimmer for Jamie Dixon's Panthers, they fell to 0-7 in Big East play, dropping their eighth game in a row overall with the home defeat to the Cardinals.
Rick Pitino's team has done a remarkable job in dealing with a lot of injuries to win three of seven conference games and, at 15-5 overall, be in solid NCAA Tourney at-large bid position.
The Panthers? Barring a Connecticut-like run through the Big East Tournament, they're going to be part of the NIT field. Who would have imagined that a month ago?
Murray State 82, Southern Illinois-Edwardsville 65: Raise your hands if you looked at Murray State's schedule in October and thought "Here's a team that might be unbeaten"?
The Racers are the last of the unbeatens after their 20th victory, the eighth in Ohio Valley Conference play, Saturday night.
First-year head coach Steve Prohm must be thinking, "It's got to get tougher from here, right?"
It sure will, Steve.
UNLV 80, New Mexico 63: Well, it had all the appearances of a "Mountain West showdown" -- for a half, at least, before the Rebels dispensed with the Lobos.
Coach Steve Alford's team has five games to play before getting a rematch with San Diego State (which beat the Lobos Wednesday night in Albuquerque) and they would be prudent to win at least four of them to stay in reasonable NCAA tourney at-large range.
As for the Rebels, they're playing for the best possible tournament seed -- regardless of what happens in the conference's postseason tourney on their home court.
Long Beach State 71, UC Santa Barbara 48: There appears to be a major gap between the two best teams in the Big West Conference -- at least that was the case Saturday night in Santa Barbara, when the 49ers-0">49ers built a 31-point edge with about 10 minutes left to go 7-0 in conference and 14-6 overall.
Perimeter players Casper Ware, Larry Anderson and James Ennis combined for 47 points, 11 rebounds, 12 assists with just four turnovers for Dan Monson's Long Beach squad while the Gauchos' Orlando Johnson was held to four of 15 shooting and 10 points.
The reason the Gauchos aren't panicking, though: They've beaten the 49ers in consecutive conference tourney title games, doing so last March after losing to Long Beach by margins of 16 and 18 points in the regular season.
Start 'Em, Sit 'Em Wild-Card Weekend
Ride Tom Brady and Rob Gronkowski to fantasy success in the first round of the playoffs.
By Michael Fabiano
Melky Cabrera Announces Retirement from MLB After 15 Seasons
Cabrera last played on a MLB field in 2019 with the Pirates before going on to spend two offseasons playing in the Dominican Winter League.
Report: CJ McCollum Will Return to Portland's Lineup Against Magic
McCollum recovered from his lung injury on Dec. 23.
'Formula 1: Drive to Survive' Producers to Make Tennis Docuseries
Netflix announced that Box to Box Films will produce a professional tennis documentary series, and filming has started at the Australian Open.
Report: Athletes in Beijing Games Urged to Use Burner Phones
Countries anticipate China to implement strong surveillance during the Winter Games.
Smith Sr. won a national title and was an All-American for OSU.
McElroy Challenges Thibodeaux to IQ, Wonderlic Tests
The former Alabama quarterback took exception to Thibodeaux's shots at the value of a UA education.
What All-Pro Votes Mean for Rodgers, Brady MVP Race
The breakdown of Friday's 2021 All-Pro votes give a significant hint at which quarterback could take home MVP honors.


Stop trying to hack early-stage startup growth.
Get ready for real world, practical advice on bootstrapping a software business.
David Hauser has been there and done it and he isn't brainwashed by some of the Silicon Valley methods for hacking growth.
David bootstrapped Grasshopper from $0 to $30M in revenue before selling it & founded Chargify, made it profitable then raised money through Mark Cuban.
3:50 - Tell us about Grasshopper and Chargify. High level: what do they do?
5:15 - Where did the idea for Grasshopper come from?
7:10 - How did David start selling? Who were the first people he reached out to?
9:30 - What were you charging?
16:30 - It's a whole different experience managing 6-10 employees. How did things shift? What was the shift like?
17:30 - Growth hid some of the problems that the company was experiencing. When did they start surfacing?
23:15 - So Chris learned about A/B Testing from the Obama campaign?
23:20 - How was David funding it all?
26:00 - Why did David decide to bootstrap, early on?
28:30 - When did Chargify come into play?
30:55 - What were some of the other ideas that Grasshopper tried, when growth started to slow?
31:50 - What were the biggest lessons that David learned after investing and losing $1M in Grasshopper Labs? If he were doing it over, what would he do differently?
35:00 - Looking back on all of this, what were some of the biggest lessons that you learned? Let's start with $0 - $250k / year.
David has always believed in creating a sales process that can close sales online. He didn't want to talk to potential customers on the phone to discuss proposals and contracts. That's wasted time.
At launch, the process allowed them to acquire customers through paid advertisements and build the product at the same time. This sales process also proved highly scalable.
David built a culture of testing and optimized everything.
They were profitably selling the product within three months – and they were profitable every month. Within 18 months, Grasshopper exceeded $1M in revenue.
As the company grew and David hired more people, the company needed better structure and communication – but constant business growth obscured those problems.
The problems didn't start to surface themselves until Grasshopper reached $10M in revenue.
Multiple layers of management emerged.
Performance management, evaluation and compensation became more complex.
They needed different people. They needed to shift from hiring Doers to hiring Strategic Thinkers.
Raising investments for Grasshopper didn't make sense to David.
In 2010, there were fewer people willing to invest money.
Investors wanted to know David's plans for selling and exiting the company, and he was focused on building a great company that he loved working at.
Later on, instead of taking a large investment, they chose to go into debt to substantially expand their marketing budget – from $2M to $14M. Thanks to a culture of testing, they knew exactly how much money each dollar spent would earn. They paid off the debt faster than expected.
The idea isn't that important. It might change.
You need passion to serve a specific market.
Your goal should be to get traffic & sell something.
For David, this stage was all about hiring.
They never hired people for a function that David or his partner had not spent time doing.


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Annual and Sustainability Report 2018
Employees, Society
Production, Sales, Finance
Annual & Sustainability Report 2018
Works drivers
Press releases & Newsletter
911 GT3 R: Black magic
The new 911 GT3 R race car goes to Sebring for a final shakedown. A 30-hour endurance test on the former airfield in Florida confirms the car is ready to go for the 2016 season.
The 911 GT3 R is dressed completely in black. Not because Porsche has an official dress code for its new customer GT race cars, but for very practical reasons. "All of the carbon-fiber parts start off black and aren't painted until after the test phase," project director Sascha Pilz explains.
This black beast descended on Sebring International Raceway toward the end of summer, accompanied by a number of support trucks. The 911 GT3 R has already survived multiple ordeals, including component tests and test drives on various European racetracks and a heat trial in Italy. Now, on this iconic raceway in central Florida, the car faces its final exam: a 30-hour endurance run, split into three 10-hour segments due to safety regulations at the site. The testing team described their plans as follows: "During the endurance run we will only be returning to the pit to refuel, change the tires, and switch drivers." This makes perfect sense, as the new race car will be competing in several 24-hour contests over the 2016 season and the idea is for it to win.
Data, data, and even more data
At the moment, the 911 GT3 R is still reclining on a platform under a tent in the pit. Without its hood and wheels, it looks more like a test lab on wheels than a race car. The bicep-like bars of its roll cage are nearly obscured by a colorful mass of wires. What might look like a heap of confetti, however, is a carefully connected assemblage to an array of electronic control modules. After all, the 911 GT3 R is not only supposed to complete its 10-hour tests, but also generate data, data, and even more data on a constant basis. Even its transverse links have been furnished with strain gauges, to gather information on stress levels and the associated effects.
Situated in swampland about 130 kilometers south of Orlando, Sebring evokes a romantic and specifically American air of racing. Originally an army airfield, it was converted into a challenging raceway in the late 1940s. The front straight, which was one of the runways, used to be separated from the bleachers by bales of hay. The course has been changed a number of times since, but still retains its original flair. The hay, by the way, has been replaced by concrete barriers.
The surface and curves are especially hard on tires and brakes
The 12 Hours of Sebring has been one of the highlights of the racing season since 1952. And Porsche is the greatest name in its annals. Back in the early days, small 550 Spyders competed against gigantic front-engine roadsters from Cunningham, Maserati, and Ferrari. They set the tone for what would become a tradition: with 18 overall victories, sports cars from Zuffenhausen are by far the most successful make at Sebring. The most recent victory came in 2008, with Timo Bernhard, Romain Dumas, and Emmanuel Collard at the wheel of an RS Spyder. So the 911 GT3 R could enjoy a home-field advantage, although the track remains as challenging as ever. Its combination of asphalt and concrete surfaces is considered especially rough on the suspension and brakes. And weather conditions in the late summer here about 60 kilometers from the Atlantic coast can bring their own cause for concern, such as a tornado or torrential downpour along with the subtropical heat.
Developing 368kW (500hp, combined fuel consumption 12.7 l/100 km; CO2 emissions: 296 g/km), the four-liter boxer engine of the 911 GT3 R is based on the current GT3 RS, i.e. on the fiercest naturally aspirated GT engine yet to be derived for the 911. The body shell starts off on the assembly line for roadgoing 911s at the plant in Stuttgart-Zuffenhausen, but is soon removed. After the roll cage is mounted, comprehensive finishing work is done by the motorsports department at the Weissach Development Center. The new powertrain with direct fuel injection is based on the 9A1 generation of the street-legal RS, and is also finished on the GT engine line in Zuffenhausen.
With the hefty addition of double-clutching when shifting down
New bearings for the forged crankshaft, new tuning for the intake camshaft, and an engine control unit designed for racing increase torque even further. The rev limiter doesn't kick in until 9,500 rpm. The sequential six-speed gearbox, developed by Porsche and made by Ricardo, the English specialist in racing transmissions, is operated by toggle switches behind the steering wheel. The clutch, too, differs from that in the street-legal RS: it is made of carbon and plays only a cameo role. It is only used to move off at the start or after a pit stop. Too much time would be spent on engaging and disengaging it during gearchanges. The electronic engine system handles this faster and more efficiently, with the hefty addition of double-clutching when shifting down.
Despite heat convection and haze, the lines on the long start-finish straight on the former army airfield are sharp and clear. In its high-tech carbon-fiber outfit, the 911 GT3 R looks like an earthbound stealth bomber training for an extreme mission at zero altitude. Its unadorned carbon-fiber surface swallows all of its curves, edges, inlets, and arches. The only lines that remain form the silhouette of a Porsche 911 – clear, unambiguous, inimitable.
Quick-release fasteners connect the front sill to the wheel arches
Hovering above it all is the rear wing, an imposing aerodynamic element sitting at chest height or above. It appears to be even broader than the brutish looking wheel housings. A closer look, however, reveals the wing's precisely designed shape, not unlike that of the street-legal RS.
The sound of the 911 GT3 R is every bit that of the gentleman racer. The exhaust system's two tailpipes emit high-end tones in place of the unchecked volume of old. Clear and controlled, the GT3 R does not shriek at high engine speeds. At a distance all you hear is an audible but not overpowering race-bred boxer sound. When down-shifting at the end of the front straight, however, the engine throws off all its discretion. "Rrmm, rrmm, rrmm" – its unimitable soundtrack still fills the air long after the 911 GT3 R has disappeared around Turn One.
The temperatures in the cockpit approach those of a sauna
On the first day of the test, all of the systems are checked. The car leaves the pit, completes a single lap, and returns. Laptops are connected, data is downloaded, and the car goes out again. Of the six drivers scheduled for the week, Jörg Bergmeister and Wolf Henzler are taking turns. Each day's data is sent to the Weissach Development Center that same night. One important finding: the temperatures in the cockpit approach those of a sauna.
The next day's agenda calls for stints covering the range of a full tank of gas. Modern pit structures are viewed as a sign of weakness in Sebring, so all the action takes place out in the open along the pit wall. The performance and powertrain engineers check their laptops at a mobile bridge. To keep operating costs down, brakes in the GT3 class have conventional steel rotors instead of carbon-fiber composite versions. And the Tudor United SportsCar Championship (TUSC) allows just one type of Continental tire, so drivers and mechanics are taking the opportunity to become more familiar with it. Wolf Henzler, Jörg Bergmeister, and Bryan Sellers each drive for about half an hour before the "pit, pit, pit" command on the radio orders them back to the crew.
Pit stops during the 30-hour endurance test acquire a high-precision routine
The sequence of events during the pit stops acquires a nearly casual routine: braking right before the lollipop, jacking up the car, changing the tires, refueling, switching drivers. The brake technicians check the temperature of the rotors while the mechanics examine the wear on the tires. One mechanic wearing a helmet and fire-resistant overalls climbs onto the hood to insert the hose into the tank for rapid refueling. A new driver, the same car. Although the telemetric systems record every piece of data the 911 GT3 R transmits, some crew members automatically press their stopwatches. That comes from a long tradition. What makes test drives so valuable is not just the data recorded by the mechanical systems, but also the insights and impressions gained by the crew.
The drivers report a few irregularities under braking at the end of the front straight. Discovering this type of thing is precisely the reason why tests are held under extreme conditions in Sebring. The raceway is a challenge to both the materials and the calibration. But although brake performance declines – ever later pressure points, delayed response – the lap times remain consistent. Sellers, Henzler, and Bergmeister use their talent to compensate, and provide detailed analyses when back in the pit. American driver Bryan Sellers, who will be paired with German driver Wolf Henzler in the TUSC GT series, concludes his monologue about the brakes with a grin and says "tell me if I'm talking too much."
The 911 GT3 R will be able to compete as a customer race car in all 2016 series that follow FIA regulations as well as the TUSC. That includes the popular and very challenging 24-hour races in Daytona, Spa-Francorchamps, Dubai, and on the Nürburgring. Only the add-on parts will keep the carbon look, however, because the car bodies will be delivered in white.
Text first published in the Porsche customer magazine Christophorus, No. 374
By Eckhard Eybl // Photos by Victor Jon Goico
Consumption data
911 GT3 RS (TYPe 991): Combined fuel consumption: 12.7 l/100 km; CO2 emissions: 296 g/km
30-hour endurance test in Sebring
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Motorsports 06/23/2019
Pro-Am class win at the 24-hour marathon
Nürburgring 24 Hours: The Porsche 911 GT3 R No. 12 vehicle has won its class in the Pro-Am category.
Winning weeks for Porsche customer racing vehicles
The Porsche customer teams have celebrated successes in the USA, Germany, Malaysia and France.
IntGTC: 911 GT3 R finishes eight-hour race in third
The new Porsche 911 GT3 R scored a place on the podium at its first outing in the Intercontinental GT Challenge.
Porsche 911 GT3 R goes racing in Asia
The motorsport season 2016 is on the finish straight.
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On a recent staycation, we went to the Duke Kahanamoku lagoon, and the kids were eager to try out a pedal boat after seeing other kids having fun with them on the water.
Waikiki Beach Activities has a rental station at the lagoon, offering not only pedal boats, but also stand up paddle boards, kayaks, rafts, boogie boards, and more.
The kids and my husband decided on a 1-hour pedal boat rental (it was under $40 with a kama`aina discount), and they had fun pedaling around the lagoon.
The pedal boat seats 4, and each of the kids took turns in the front, helping (or pretending to help) my husband pedal the boat.
The 1-hour rental was just enough - as my husband got a little tired pedaling the boat, since my kids had more fun riding in the back than pedaling.
But they all had fun, and the hour went by quickly!
For more information on rates and rentals, click here.


text:
BBC Home > BBC News > Science & Environment
UK 'must act' on plane emissions
17 October 06 13:08 GMT
Britain will not be able to meet its goals on climate change without curbing the demand for air travel, according to an Oxford University report.
The government is aiming for a 60% cut in carbon dioxide emissions by 2050.
But the report's authors say the UK is becoming "air dependent" and government policies on increasing air travel contradict that stated aim.
However, the British Air Transport Association said targeting UK air travel alone would have little impact.
The government has said it wants aviation included in the next round of the EU Emissions Trading Scheme.
Carbon dioxide emissions from aviation doubled during the 1990s while those from the rest of the economy fell.
Currently, aircraft produce about 5.5% of UK emissions.
The report says that without new policies, they will account for about a quarter of the national total in 2050.
Numbers up
The report, entitled Predict and Decide: Aviation, Climate Change and Policy, comes as a number of UK airports are investing in new facilities to boost passenger numbers.
The Oxford team notes that airport expansion was backed in the recent Aviation White Paper, which aims to increase the number of passenger movements in UK airports from about 200m per year now to about 470m by 2030.
"The government has to confront the contradictions in its policies," said project leader Dr Brenda Boardman, from Oxford University's Environmental Change Institute.
"Unless the rate of growth in flights is curbed, the UK cannot fulfil its commitments on climate change."
A Department for Transport spokesman told the BBC News website: "Tackling the climate change impacts of aviation needs international action. "This is why we believe that the best approach is to include aviation within the EU ETS [Emissions Trading Scheme] as soon as possible."
Roger Wiltshire, of the British Air Transport Association, said the latest findings were "nonsense".
"Only 5.5% of the UK carbon is down to air travel. What happens to the other 94.5%? is what we ask." he told BBC News 24.
He said the most important thing was to "have an international solution to what is a global problem".
"If you close down all the airports in the UK tomorrow, you'd save a fraction of a per cent ... of global climate impact and that would have no impact. What it would do, unfortunately, is to really affect the UK economy and UK travel habits."
But the Oxford team backed the conclusions of the Environmental Audit Committee, which earlier this year supported higher taxes to cut emissions.
"Raising air passenger duty would help to counter reductions in fares, which are estimated to have been responsible for at least 40% of recent aviation growth," said Dr Sally Cairns, from University College London, one of the report's commissioned authors.
It concluded that taxation through passenger duty would be easier and quicker than focusing on the ETS.
Clean habits
Paul Charles, a spokesman for Virgin Atlantic, said his airline had made suggestions to tackle the problem.
"We've suggested starting grids at airports, so that planes don't have to run their engines for half an hour all the way to the runway while they're queuing up. That will cut millions of tonnes of CO2 emissions," he told the BBC.
More efficient procedures before planes come into land and more efficient air traffic control would make a dramatic difference, he added.
Britain, the report makes clear, is a vital player in attempts to reduce aviation emissions - one-fifth of all international passengers are on flights which arrive at or leave from UK airports.
And it says Britain is developing an air dependent culture. If nothing is done, it says, our expectations will include frequent flying for business and pleasure, second homes abroad, and more geographically distant networks of friends and family.
But the authors believe we are ready for change, with recent opinion polls suggesting a majority of people are in favour of airlines paying higher taxes to reflect emissions, even if this results in higher fares.
And although restricting flights might be bad for airports and airlines, other sectors of the economy such as domestic tourism would benefit, the report concludes.
Related to this story:
Special report: CLIMATE CHANGE
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text:
Burli ist ein Lied der österreichischen Popband Erste Allgemeine Verunsicherung. Es ist Lied Nummer acht auf dem 1987 erschienenen Album Liebe, Tod & Teufel.

Das Lied handelt von einem Jungen namens Anton, genannt "Burli", der nach einem GAU eines Kernkraftwerks zur Welt kam und zahlreiche Mutationen aufweist. So heißt es im Lied: Der Burli hot links und rechts drei Uhrli, am Kopf hot er a Schwammerl, zwölf Zechn auf die Fiaß … In weiterer Folge wird die Lebensgeschichte von Burli von der Geburt über das Erwachsenwerden bis hin zur ersten Liebe beschrieben. Die Geschichte findet schließlich ein Happy End, als Burli seine (durch die ionisierende Strahlung ebenfalls mutierte) Nachbarin Amalia kennenlernt und heiratet. Der Text deutet dabei an, dass Burli auch mehr als ein Geschlechtsteil hat.

Hintergrund für das Lied war die Reaktorkatastrophe von Tschernobyl, was sowohl durch den Text als auch durch das Erscheinungsdatum ersichtlich wird. Ein Charakteristikum der EAV ist es, Lieder zu aktuellen Ereignissen zu schreiben.

Auf dem 2015 erschienenen EAV-Album Werwolf-Attacke – Monsterball ist überall wird das Lied in Mrs. Fuckushima weiter geschrieben. Darin verlässt Burli seine Amalia für das "Strahle-Girl Fukush-Irmi". Hintergrund hierfür ist die Nuklearkatastrophe von Fukushima.

Skandal um Burli
Zum Skandal um das Lied kam es schließlich im Februar 1988, als der Radiosender Bayern 3 den Liedtext als gegenüber Behinderten beleidigend einschätzte. In der Folge wurde das Lied von manchen Radioprogrammen in der Bundesrepublik nicht mehr gespielt und war entsprechend der einzige EAV-Hit in den 1980er Jahren, der nicht in die deutschen Airplay-Charts kam.

Für einen weiteren Skandal sorgte die Single von Burli, da sie außerdem noch ein Lied namens Kurti bzw. Wann man gehn muss enthielt, welches die Vorwürfe gegen den österreichischen Bundespräsidenten Kurt Waldheim wegen seiner Aktivitäten in der Zeit des Nationalsozialismus zum Thema hatte.

Weblinks
 Liedtext auf der Webseite der EAV

Einzelnachweise

Lied 1987
Popsong
Erste Allgemeine Verunsicherung

redpajama_set_name: RedPajamaWikipedia


Marie is a single mother; Simon has been separated from his wife for five years; Paul has been divorced for a few years; Chantale is an 18-year-old who could not get along with her parents, left school and the city of Chicoutimi, where she was born, and moved to Montreal to look for work. All of them recently filed an application for financial assistance with the Ministère de l'Emploi et de la Solidarité sociale. In response, each of them received a letter from the Ministère requesting proof that they will institute proceedings for support. Marie must institute proceedings for support against the father of her child, Simon and Paul must ask for support from their former spouses, and Chantale must ask for a parental contribution from her parents. "Things are calm right now. Why stir up trouble?" they ask their lawyer.
Married or civil union spouses, and relatives in the direct line in the first degree, owe each other support (article 585 of the Civil Code of Québec).
Marie has looked after her child on her own since he was born. However, the birth certificate contains the name of the biological father. Thus, the child has rights he can assert against his biological father and, in particular, the right to claim support from him.
Given that Simon is not divorced, his wife owes him support.
As for Paul, he has a divorce judgment that does not mention anything with respect to the support owed between the spouses. Therefore, his lawyer will have to analyze the file and the situation of the parties in order to assess the likelihood of success of proceedings for support.
Until recently, Chantale was living with her parents in Chicoutimi. She left school after getting her high school diploma and has never worked full time. Pursuant to section 57 of the Individual and Family Assistance Act, she will be deemed to be receiving a parental contribution if she does not institute proceedings against her parents. Usually, the parental contribution is calculated by taking into account the situation of the parents and the income obtained pursuant to the application of sections 152, 153 and 154 of the Individual and Family Assistance Regulation.2 The amount determined as a parental contribution is a resource that will be taken into account in computing the last resort financial assistance benefit to be paid to Chantale. If Chantale's parents do not cooperate with her, she will have to sue them for support.
1 Individual and Family Assistance Act, R.S.Q, c. A-13.1.1.
2 R.S.Q., c. A-13.1.1, r. 1.


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Bitfield flags for supported features.
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Simulation of a backend storage in memory.
Proxy backend that mirrors writes to several internal backends.
Release a scoped lock and set any errors in the attatched StatusValue object.


Driffield Hockey U16A team travelled to Thirsk to play in their 3 game tournament.
from the Thirsk attacks. The Driffield Goalkeeper, Louisa Chacksfield, having to make some great saves to keep Thirsk off the score sheet.
defend well and although frustrated with the 0-0 final score, could be proud of their first game.
of the goal. Other goal scorers were Abigail Clark and Keira Dinsdale-Young with the final result of 4-0 well deserved.
umpire had not realised the ball had crossed the line) and resulted in a final score of 3-0.
Goal scorers in this match were Abigail Clark, Keira Dinsdale-Young and Emily Williamson.
Snaith for his never ending 'encouragement' from the touchline and to Mieke Davison for umpiring.


AP Brake Rotors There are 19 products.
AP Racing Replacement Racing Brake Rotors - DiscoveryParts AP Racing is world class braking for any track in the world. The more demanding your application, the more you want AP Racing. Precision and quality meet with every AP Racing Brake kit.


Your correct with the tunnel, but not correct with the location. So you are all narrowing it down further, not Scotland, and not in Wales.
Here is the full image, there is a clue in the photo that will could help narrow down the location a bit further. But i guess you would need to be a little bit of a railway geek to spot it!
Well, it appears to be dual gauge lines in both direction, so it will have to be on a main line, where trains of standard and non-standard (narrow) gauge were able to travel.
Unfortunately I haven't come up with a search word to cover all that.
Dual gauge tunnel England came up with a lot of model railway stuff.
You are misinterpreting what you can see Ombugge.
Both the photo and your comments imply that it is a real railway tunnel, but is the structure itself a folly? Seems to be a garden just behind, sort of lawn with a bit of trimmed hedge.
I believe that the third rail supplies electric current to the trains.
Could it be in the outskirts of London ?
The small building you see in the middle is inhabited, but you are correct about the rest of the structure Ivy, it is a fine example of a typical Victorian grand facade.
Well spotted Nichu, the third rails do indeed supply the electric current to the trains, 750 DC to be precise, so not a wise idea to step on it!
You have also used that information to narrow down the location, you are indeed in the correct part of the country - south east, but a little further out from London than the outskirts. From the map below you can see that the South East of England accounts for 95% of all third rail electrification in the country. Thus my earlier hint, "there is a clue in the photo that will could help narrow down the location a bit further".
So we now know that the structure is located in the South East of England somewhere. Another clue for you all, Why did they build such a grand facade for this tunnel in-particular? It's not the only tunnel on the line, so why go to the lengths of building such a grand portal?
Based on the map, it must be somewhere south of London.
Have no idea why this great portal was built.
Unless it is related to an accident in 1861.
This tunnel was built in the 1840's, and the facade was also built at that time, so no, the reason for the grand facade does not relate to an accident in 1861.
But i do think you know the name of this tunnel Nichu, if so, it's time to tell!
Last edited by Steve.B; July 13th, 2010, 02:06.
I think this is the South Eastern Main Line, which has a third track for power supply.
That means either Abbotscliff Tunnel or Martello Tunnel, I believe, although I haven't been able to find any matching photo.
It is close to my Father-in-Law's birthplace.
Last edited by ombugge; April 16th, 2011, 16:06.
An old Clayton Tunnel Postcard.
Yes, Clayton Tunnel it is, and your location is spot on Ombugge. Situated about 6 miles outside of Brighton on the London to Brighton mainline. Clayton Tunnel, at 1.25 miles in length, it is the longest Tunnel on the line.
When the Tunnel was built back in the 1840's, the farmer that owned the land would not grant access to the tunnel unless an edifice was built at the entrance, so the railway company were forced to build the grand facade that you see today. The small cottage above the Tunnel was originally used to house railway workers, but these days it is privately owned.
Nichu came very close to identifying the Tunnel when he mentioned a crash that occurred in 1861. Though that crash occurred at the other end of the Tunnel, the southern portal, which is just a plain standard brick affair.
The Clayton Tunnel rail crash, which took place in 1861, five miles from Brighton on the south coast of England, United Kingdom, was the worst accident to occur up to that time on the British railway system. An excursion train crashed into another which had stopped in the tunnel, on Sunday 25 August 1861, killing 23 and injuring 176 passengers.
So over to you again Ombugge!
OK, my turn to come up with a challenge again.
It is getting harder to find any own photos that would challenge the consummate puzzle solvers on this forum, but here is one that MAY do the trick.
Not my own photo though.
Last edited by ombugge; April 16th, 2011, 16:07.
What on earth was Lookchin doing up there when he was so young??
Sorry, slightly 'OT', but i was a little surprised to discover that Lookchin was out on such an adventure at that young age!
Are we in Canada with this one?
Definitely NOT Canada, this place is in Indonesia!
I have not narrowed it right down yet, but i believe this is somewhere around the Jaya Peaks. Shall endeavor to nail it completely!