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10 Steps for Building a Crisis Management Communication Strategy
June 23, 2020 / Chesley Brown
Small changes have a big impact.
As a public health crisis of worldwide proportions comes into focus, it is imperative for individuals, organizations, and nations to develop effective resilience strategies, including communications and outreach as part of any comprehensive crisis management plan.
Chesley Brown has responded to the COVID-19 crisis by making our Resource Center available to the public. It provides professional advice, enterprise tips, workflow suggestions, and updates on handling the coronavirus crisis and more. I highly recommend you check it out.
In this section, we present 10 recommendations for corporations to understand the crisis landscape, prepare internal groups with a messaging strategy, and suggestions for interacting with more than a few stakeholders. They are drawn from our very own experience, and that of our vast network of global resources on crisis management.
DOWNLOAD OUR FREE 7 STEP PLAN
Step 1: Study the nature of the crisis.
A crisis of any size, scope or dimension can strike at any time creating chaos and sowing confusion. In order to mitigate or eliminate the impact a crisis may have it helps to map out the landscape. Some crises are internally caused (eg. defective products), others are external in nature (eg. the spread of infectious disease). Some will have a local impact, others are global in scale.
Some crises have an effect on specific communities or demographics, others will target the general public at large. Each of those dimensions should inform and influence your objectives, team composition, and available options. What the cause, its important to include a variety of contingencies with in your crisis management plan.
Step 2: Map of the information landscape.
Simply testing a disaster plan with senior board members substantially reduces both the number of crises as well as the negative consequences when they do arise — 21% of enterprises that include board members in their crisis management plans reported a decrease in the number of crisis situations they faced, compared to only 2% of the control group. This has the added benefit of creating an open framework for dialog as well as buy-in from each of the stakeholders.
Unfortunately, similarly to breaking news in a time of disaster, there can be an alarming volume of fake news, misinformation, hate speech, and unsubstantiated rumours. Crisis analysis begins by separating authoritative sources from dubious sources. Local, national, and international authorities should be the primary sources for important information.
By leveraging the principles of Knowledge Management, you may be able to better classify content material into facts, records, and know-how. With information breaking all the time, it is critical to update response tips and procedures to maintain accuracy and relevance in your crisis management plan.
Statistics should play a vital role in prioritizing action. This will also allow you to plan future action based on projections. Designing and visualizing information into an understandable way is a powerful method for communicating eventualities with a wide variety of audiences. This could consist of image design, interactive charts and infographics, or press clippings.
Step 3: Understanding the media.
Every business should be in charge of its own digital footprint, and also understand how to engage with the media. Major traditional media typically have wide ranging influence and authority, but have also been known to make mistakes; hence "media literacy" is an important competence.
Social media is a rich platform for the "people's voice" and also another important channel for communicating. Social media monitoring and outreach help you keep a finger on the pulse of your audience, while minimizing the barriers to direct communication.
Step 4: Build a crisis management team.
Though plans may change, it is essential to have a crisis communication team on location to adjust and plan messaging, online assets, and other activities. The organizational leader and spokespersons have to be the center of this group, but organization-wide support and inputs are also needed. It therefore makes sense to utilize a cross-functional team to build and vet your communication strategy.
Communication to inner and outside stakeholders ought to be at regular intervals (eg. every morning or evening), along with additional emergency bulletins as relevant. Sensing, sensemaking, and articulation should be executed in a systematic manner.
Communication channels for strategic communication have to be prioritized in your crisis management plan. The ultimate goal is to transition from responsive communication to proactive engagement.
Step 5: Use the Five Fundamentals of Crisis Communication.
Have a plan.
A crisis communications plan can help ensure accurate records immediately get to decision makers, accelerating response times and keeping the organization's decisions firmly grounded within its vision and ideals. The reason awful crises get genuinely awful is they are allowed to fester due to the agency's reluctance to admit that a crisis is happening. An effective crisis management plan protects against that. It would be misguided to assume all organizations have been diligent in proactively crafting their crisis management plans and communication strategies for a variety of situations, but the truth is most organizations set about creating their plans under one of three situations:
Immediately following a crisis (whoops)
After a near-miss (that was close!)
After a similar organization has experienced a crisis (Better prepare)
The point is, don't create your plan under duress. You'll only be faced with bad decisions. In these situations you will rarely be afforded a chance to catch your breath, let alone think objectively.
Know the facts first.
In order to make the best decisions you must have objective information freely available. There are few things that are more dangerous to an organization's reputation than publishing information during a crisis that turns out to be wrong. Once trust is gone, the entire organization is in serious peril.
Own it.
With very few exceptions, it is better for a company to report its own bad news. This offers you an opportunity to 'frame' the story and provide the right context. This inevitably becomes a tussle between public relations and legal counsel. There are strategies to work through these tensions, especially if organizational leadership can be convinced that the whole story will eventually come out, which it will.
Be human.
We have helped countless organizations navigate various crises over the years and we've seen firsthand how tough it is for executives to be compassionate. It is always easier to speak about the problem and all the hassle it creates than it is to acknowledge that people are suffering. Showing everyone dignity and empathy in times of crisis goes a long way, and it's one of the best ways to endear your company to employees and customers.
Speak with one voice.
This is the end result of encouraging the free exchange of information as defined above. In the midst of a disaster and in the immediate aftermath, every person associated with the organization should be considered a spokesperson. They may not present information formally on behalf of the organization or step to the podium at an industry conference, however, each and every employee has a different sphere of influence. In the age of social media and instant messaging, you cannot underestimate how quickly information will travel. Ensure all employees have accurate facts and outfit them with Q&As to guide them in conversations. After all, nearly every person with whom they interact is a social media consumer and can amplify your messages.
Any effective crisis communication plan will include each of these 5 fundamentals. In addition to serving as a framework for building your communication strategy, they will help you make decisions as you plan your next steps. Transparency is key in disaster conversations; data should not be fudged, manipulated, or hidden. In order to cultivate trust among internal and external stakeholders, you must demonstrate that facts matter and everyone's opinion and contribution counts.
Step 6: Understand audience worries.
High-intensity crises have an effect on society at a couple of levels, especially in an era of globalized work and migration. In addition to worries of residents and families, employees and consumers expect to be factored in to any decisions that may affect them. Crisis communication must communicate to their needs, in addition to the needs of the most vulnerable populations like seniors and children.
Step 7: Media engagement.
For key announcements or even for ordinary conversation, organizations should have a way to interact with the media. This can vary from physical/digital press meetings to conversations on the green.
Help lines can be an effective strategy but they will need to be well staffed to give accurate and well timed responses to the media and to the general public, and online help desks must be informative, easy to use, and accurate. Websites and social media channels should cultivate a trustworthy, authoritative persona to calm any anxieties.
Step 8: Communicating with the wider ecosystem.
Crisis conversation inputs and outreach need to include key sectors of the broader economy, such as the health, retail, hospitality, and tourism sectors. Civil society and religious groups should be engaged in helping reinforce key messages. Celebrities or social media influencers can also be harnessed for purchasing buy-in from fanatics and broader communities.
Effective messaging should create a sense of security, compassion, and kindness. Always avoid laying blame, and instead focus on "why it's just plain wrong" that your customers should have to deal with this issue and what can be done about it.
Step 9: Tell a compelling story.
Crisis communication requires calmness, seriousness, and empathy. In addition to statistical analysis and scientific research, use the key elements of storytelling to craft a narrative that is both informative and compelling. Using real life testimonials can help demonstrate compliance, innovation, and excellence (in addition to illustrating how not to do something).
Testimonials and "hero" statements evoke empathy, respect, and resonance. Humor can be really powerful at easing peoples' fears, however, careful attention to context will help you avoid offending anyone. Errors and mistakes should be eliminated through thorough verification, proof-reading, and testing, in order to avoid harm to your organization's recognition and credibility.
Step 10: Leverage digital transformation as a risk mitigation strategy.
One of the larger impacts of the current Coronavirus crisis on businesses is the acceleration of digital transformation. With so many employees now working from home using telecommute solutions like GoToMeeting, organizations are now exposed to a whole host of new vulnerabilities that we'll cover in a later section. This rapid shift towards digital technology could potentially disrupt the entire organizational culture from collaboration to interactions with investors. The best prepared organizations continually assess the security, accuracy, and accessibility of their customer data, marketing collateral, income channels, logistics systems and any critical information systems that may affect their ability to conduct business.
Humanity (and indeed, all species) has confronted and weathered a range of crises throughout history. In addition to operational impact, the larger socioeconomic fallout will take time to fully come into to focus. We should plan for the worst and hope for the best case scenarios.
Payroll, legal troubles, and compliance are other issues that organizations need to tackle head-on, in conjunction with policy and regulatory requirements from local, state and federal government. Many business owners struggle to fully grasp the myriad risks they face. That's why we've built a framework that teaches businesses how to anticipate and navigate risk before it becomes a crisis. Despite the immediate hardships, a crisis is a messenger, and should be framed as an opportunity for introspection and reinvention. Be sure to download a free copy of Chesley Brown's "The Business Continuity and Crisis Management Handbook" for our complete list of tips and strategies for preparing for and navigating a crisis effectively.
Nick Freeman
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How the Events of September 11th Have Impacted National Security
By Chesley Brown	| September 2, 2021
How Has National Security Evolved Since September 11th, 2001? Written by: Dell Spry As I sit and write this paper, Afghanistan is collapsing. It is not my intention to point the finger at anyone and…
A Nation Under Attack: The Colonial Pipeline Attack
By Chesley Brown	| May 20, 2021
Written by: Dell Spry Introduction: Before the Colonial Pipeline Attack In earlier centuries wars were fought between nation states to acquire water and fertile land. Then came the quest for natural resources; gold, silver, oil.…
Reasons to Hire an Outside Security Risk Firm
By Chesley Brown	| February 2, 2021
The security services industry is one of the fastest growing industries in America today. With so many disruptive technologies, emerging threats, and the growing frequency of natural disasters, its easy to understand why — It…
The Ultimate Guide for Small Businesses to Combat Economic Espionage
By Chesley Brown	| January 26, 2021
For decades, the US has been a victim of economic espionage — a foe that is now costing American companies billions of dollars in lost revenue. From hacking government websites to infiltrating research institutions with…
Preventing Civil Unrest
We've all recoiled in disbelief and horror as we watched scenes of violence stream across our TVs. The violent insurrection we saw in the U.S. capitol was unprecedented. That is to say nothing of the …
The Business Continuity and Crisis Management Playbook
It only takes one bad day to destroy your business and livelihood. Why leave it to chance? Download your FREE copy of the Business Continuity Toolkit now and transform the way you think about your most valuable assets. Where should we send it?
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Today Britain's most celebrated amateur boxer, Nicola Adams OBE, announced her decision to turn professional with Hall of Fame Promoter Frank Warren.
The news was revealed at a London press conference today hosted by Mr Warren and his new broadcast partner BT Sport, who will televise Ms. Adams' professional bouts in the UK.
The double Olympic Gold Medallist and flag bearer for women's boxing, will appear in the professional code for the first time on Saturday 8thApril at the Manchester Arena on an event headlined by Terry Flanagan's WBO World Lightweight Championship defence against Petr Petrov.
Ms Adams will then fight in her home City of Leeds on Saturday May 13th at the First Direct Arena, on a bill headlined by local boxing hero and WBC Silver Featherweight Champion Josh Warrington. Both events will be simulcast live on BT Sport and BoxNation in the UK.
The hugely-popular Ms Adams will campaign at Flyweight and is determined to match her unprecedented amateur achievements in the paid ranks and rise to become a professional World Champion.
Ms Adams became a cross-over star during London 2012, where her incredible performances in the ring and beaming smile became some of the most iconic images of an Olympic Games which saw unprecedented success for Team GB's athletes. Ms Adams went on to win Great Britain's first ever Gold Medal for Female Boxing at the Games, famously defeating three-time World Champion Ren Cancan of China in the Olympic final.
During what can be considered one of the successful ever amateur careers for any female or male boxer, Adams secured a clean sweep of amateur titles winning every available accolade in the unpaid code, capturing the only remaining major title to elude her when she defeated Thailand's Peamwilai Laopeam to be crowned the World Amateur Flyweight Champion in May 2016.
After Ms Adams' World Championship victory, she embarked on her second Olympic Games in Rio as the reigning Olympic, World, Commonwealth and European Games Champion and became the first British boxer to successfully defend their Olympic title for 92 years defeating France's Sarah Ourahmoune in the final.
In recognition of her services to boxing, Ms Adams was appointed Member Order of the British Empire (MBE) in the 2013 New Year's Honours list. The British sporting icon was then awarded an OBE in the 2016 New Year's Honours list for her outstanding achievements and efforts to raise the profile of women's boxing.
After a breath-taking amateur career, Ms Adams, managed by Grant Michaels and Ed Griffiths of ROAR Global, is thrilled to be embarking on the next chapter of her remarkable story under the guidance of Hall of Fame Promoter Frank Warren. Boxing fans will be able to follow her career in the professional ranks exclusively on BT Sport and BoxNation.
Hall of Fame Promoter Frank Warren said: "Of all of all the signings I have made in my 35 years in the sport of boxing, this is among the most I have been excited about.
"Nicola is a national icon already and is without doubt Great Britain's most successful amateur boxer of all time. She is a tremendous fighter and has a wonderful personality that lit up both Olympic games where she captured two consecutive Gold Medals.


Several accounts have been produced on the seaweeds of the west coast including the Saldanha and Langebaan System, but none has looked at how human activities may have affected the seaweed diversity of Saldanha Bay. The major human activities in the Bay include shipping and mariculture. The major human impacts resulting from these activities are species introductions which often lead to a change in species composition in an area. Six sites were sampled in various visits in autumn and winter 2012. Two of the sites were natural wave exposed rocky shore sites while the rest were sheltered and man-made. Detailed collections of seaweeds were made. The seaweeds were identified to species level and preserved either as herbarium specimen or in formalin. Specimens that it was not possible to identify using morphological features were preserved in silica gel for DNA analysis. Three samples of the red algal genus Gruteloupia, collected from aquaculture systems, which could not be identified using morphological features, were sent to Ghent University where the rbcL gene region was sequenced. Collected species were compared with species lists from previous studies in the same area. The results from this study showed that most of the species collected were west coast species, with a few species with south coast distributions. The numbers of species in the Bay since the study of Day showed an increase from 23 by Day to 58 by Simons' 1960 collection. His 1976 collection reduced to 47. The most species so far recorded are by Schils' 1997 collection with 72. From this study, 69 species were recorded of which 23 are potentially new records. Of these records, two were south coast species Gelidium reptans and Laurencia peninsularis. They were recorded for the first time in the system and they are range extensions. Furthermore, the results provide molecular evidence that two species of Grateloupia have been introduced into Saldanha Bay. Bayesian analysis showed that one of the species was Grateloupia turuturu, a Japanese species which has spread to a number of world regions which culture the Pacific oyster. The other was distinct from other Grateloupias previously recorded in South Africa, and did not match any species in Genbank. The potential vector of introduction for Grateloupia turuturu, importation of the Pacific oyster Crassostreo gigas, is discussed.
Siyanga-Tembo, F. 2012. Seaweed diversity in Saldanha Bay and the effects of human activities. University of Cape Town.


Kolomenskoye used to be Great Princes' mansion in the XIV-XVII centures.
Enjoying a beautiful location within ancient woodland, it is a unique art, historical, architectural and nature-landscape museum-reserve, situated in the South region of Moscow.
Nowadays it is a remarkable place to enjoy Russian folklore performance such as wedding-game, skomorokhs-buffoons and songs.


Parents are required to attend Evaluations for the first 30-45 minutes. Parents will be briefed by AYB Board Members (typically the President and the Level Coordinator) about general AYB policies as well as issues specific to their division.
In an effort to make teams as even as possible, AYB conducts evaluations so that the coaches can assess the players before the teams are picked. It is extremely important that your son/daughter attend evaluations.
Players new to AYB will not be placed on a team until and unless they have been evaluated.
The evaluation schedule is listed below. All evaluations are at the Clearfield Recreation Center. Please have your child there 10 minutes early to sign in. For Boys in grades 3-8 and Girls in grades 3-8, you must have registered prior to evaluations.
If you are unable to make the evaluation listed below, please contact the Level Coordinator (see General Info -> Board of Directors).
Monday, October 14 & Friday, October, 18.
Players will be assigned to teams after player evaluations have been held (see dates and times above). Typically, all teams are scheduled to play for one hour twice a week. Some of these sessions will be practices and some will be games. Each team typically plays 14-16 games during the regular season except for the 3rd and 4th grade levels where more practices are scheduled. All teams make the playoffs.
Your coach will call you late at night on Monday, Oct 22 with details of your first practice.
First practice for all teams will be on Saturday, Oct 27.
Every effort is made to schedule every team to play once during the week (usually in the 5:30-10:00pm range) and once on Saturday (anywhere in 8:00a-8:00p). During the week, it is typical for days and times to rotate so that all divisions share in those times viewed as desirable and those viewed as less than desirable. On Saturdays, times will change from week to week depending on schedules and availability of courts, among other reasons.
Final Date of Play (Championship Saturday): March 2.
Basketfest will be held on Saturday, November 9.
Basketfest is best described as "organized chaos."
This is our kick-off event.
Every team will play 3 mini-games during the day. To be specific, each game will be the first half of a game (using rules for the division that apply to the first half of a game).
Clocks will run, scores will be kept, and for the only time all year, we ask parents/coaches to officiate the games.
Saturday, October 19 & Sunday, October 20: You will get coupons via e-mail that entitle you to 20% off most purchases at the Transit Road location all day on both days.
Girls 8th-12th Grade Division: This division will play only on Saturday afternoons at the Clearfield Recreation Center.
Play begins on Saturday, Oct 26 at 3:00p at Clearfield. Please register prior to this day and show up ready to play!
Starting with the second week, and depending on enrollment, teams may play at either 3:00p or 4:00p.
The Level Coordintor will keep you up-to-date on the time of play.
Final date of play (championships) is Saturday, March 8.
Boys 9th-12th Grade Division: This division will play only on Sunday evenings at the Clearfield Recreation Center. Players are encouraged to play with their friends and/or make their own teams. Games, games, games. No practices in this division. All events are Sunday evening. The Level Coordinator will keep everyone appraised of the schedule.


Starving Trancer feat MAYUMI MORINAGA ( songs) mp3 download or listen to tracks, mp3 albums online word track for free, without registration and sms #1. Ending 11 Glitter - Starving Trancer Remix feat. Mayumi Morinaga by Fairy Tail Addict: Fairy Tunes, Pop music from Fiore, PH on ReverbNation. Second album from Xceon. Download Xceon (Starving Trancer) – Fuyu Tsubaki [ Album] Mp3. Tracklist: 冬椿 ft. Kanae Asaba (Album Long.
Your download has started. Having troubles downloading? Try again. This is the 11th ending theme from Fairy Tail and it was composed by "Another Infinity. Hope you enjoy, and watch Fairy Tail if you haven't already!
MuseScore Search. Browse Community. Choose where you want to share: Link to this page Embed on your site. The score can be downloaded in the format of your preference: Try again TimeLordAaron worked hard on this score. Want to give something back? Give a small token of appreciation! Try again More of this, please? Follow to get notified when TimeLordAaron has uploaded new scores.
To print starving trancer mp3, please upgrade to a Pro account first. Starving trancer mp3 Pro. Select part. Download PDF Print. Why am I seeing this? Uploaded on Aug 11, Log in with Facebook Log in with Google. Email starving trancer mp3 username. Password Forgot your password? Create one. Email address.
Confirm password. Password reset instructions will be sent to your registered email address. Username or email address. The feature is in development now and will be added soon. We will let you know when it's ready. Attribution, non-commercial.


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package gokhanaliccii.hurriyetmobile.db;

import android.arch.persistence.room.Database;
import android.arch.persistence.room.RoomDatabase;

import gokhanaliccii.hurriyetmobile.db.dao.WriterDao;
import gokhanaliccii.hurriyetmobile.db.entity.WriterEntity;

/**
 * Created by gokhan on 08/10/17.
 */

@Database(entities = {WriterEntity.class}, version = 1)
public abstract class AppDatabase extends RoomDatabase {

    public abstract WriterDao writerDao();
}

redpajama_set_name: RedPajamaGithub


Defining your castability, and a pinpoint on the horizon!
One of the first questions I ask an actor is, "What do you think your castability is?" I'm always surprised when they aren't sure. That's like a door to door salesman arriving at your house and not knowing what he or she is selling. In my 28 years of coaching, one thing I know that is paramount in an actor's career is knowing their castability. So often actors say that they'll play anything… whatever comes their way. Knowing what you are selling is of the utmost importance.
Being specific and having a niche is how this business works. Are you a college student, an FBI agent type? Are you a congress woman or an urban mom? Are you a hipster or a doctor type?
What are the roles that you are most likely going to be cast in? Are you more suited for night-time drama or sit-coms? Commercially are you the soccer mom or the lawyer? If you are aware of what you're selling, then how you approach the business will be more effective. Working on your craft from the standpoint of something specific is really helpful. Actors say they want to 'play it all'. That's like saying I want to be a professional athlete in every sport.
Now I would never want to put an actor in a box, that's for sure. But starting from where your greatest castability lays is just good business. If you are more suited to night-time drama… then you also know who the casting directors to target that cast that genre of show. Casting directors go from show to show but they normally stay within the same style of show from year to year.
Also knowing were you are headed is essential. I have had the good fortune of working with many successful actors in my career as a coach. Most have one thing in common. They know where they are headed. Having a specific pin-point on the horizon gives you a pathway to get there. So many actors wait around for their careers to fall into place but are not actively crafting their life's work. For example, if you aspire to star in dramatic studio feature films, then perhaps getting some guest starring or series regular credit in nighttime television might be a good stepping-stone. Or if you aspire to work on a sitcom, perhaps cultivating stand up might be a good way to head out in that direction. Without a defined goal ahead of you, most actors have a tendency to wait around for the business to define them.


Corporate spies are everywhere these days, even hiding in our radio station break room!
It looks like your favorite Townsquare Media Yakima on-air personalities have been caught spilling their guts to the microwave ... which apparently really does have a camera in it.
I'm sure H-R will want to review this video. I guess it teaches us that nothing is sacred these days, not even a microwave oven.


Organisations can no longer ignore the competitive advantage SEO provides when selling or promoting services and products on the internet.
Search Engine Optimisation is the process of attracting organic traffic to websites via search engines such as Google and Bing.
In a world of acronyms SEO is the meaning for "search engine optimisation", "to optimise in SEO means we look to improve incremental variables of the service to deliver better results".
To obtain a more prominent ranking SEO is the ability to influence organically search engine results.
It's likely you will encounter further acronyms like SERP, "search engine results page" this is the preview list you see by typing in a key word.
Many internet customers use PPC "Pay for Click" or "Sponsored Links", this is Googles service for advertisers with a budget who pay to direct traffic to their site. PPC can be expensive as every time someone clicks on your site it diminishes your budget.
How do you find what you're looking for and how do search engines keep everything relevant and up to date?
Google uses a Googlebot a web crawling tool, also called a spider, it trawls the internet using previous listed URL pages as reference points. The Googlebot then locates new links and adds them, any dead or broken links are also referenced and updated in the software's index.
Another task is to index all the page words, a huge task with millions of internets pages on the internet.
How Do you Find What your Searching for?
When you type in any query into the search bar, the search engine looks for indexed matching pages it believes are relevant to the user.


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Winterwatch 2019 is going wild for the Cairngorms
Posted on December 21, 2018 December 21, 2018 by Georgina Mayhew
In a time when our lives are organised with more predictability than ever, nature still offers an immersive experience of wonder and surprise. Many of us travel to far flung corners of the world to find nature-rich wildernesses, forgetting that Europe still offers many remote places ripe for exploration.
Uath Lochans and Inshriach Forest in the Cairngorms National Park, Scotland.
James Shooter/scotlandbigpicture.com
One of the wildest landscapes here in the UK can be found in the Cairngorms National Park in the Highlands of Scotland, the location of the Endangered Landscapes Programme (ELP)'s Cairngorms Connect project. Much to the excitement of the ELP, it was recently announced as the chosen location for the next BBC Winterwatch series in January 2019. As Chris Packham, one of Winterwatch's presenters, put it: "[Scotland is the] land of the brave, home of the wild and hope for the UK's wildlife".
Not everyone has the opportunity to visit such wild and rugged landscapes, but with increasingly sophisticated and immersive cinematography we all have an opportunity to get an insight into the natural world. Michaela Strachan, who also presents Winterwatch alongside Gillian Burke, has expressed her excitement at visiting the "dramatic" Cairngorms and the abundance of wildlife it has to offer, "from golden eagles to mountain hares, wildcats to black grouse, ptarmigan, pine marten, red squirrel, water vole, otter…[The Highlands are] one of those places in the UK where you can really connect with the natural environment."
Reconnecting people with nature through inspiring examples of restoration is one of the core objectives of the Endangered Landscapes Programme. Experiences of nature have been shown to benefit mental and physical health and wellbeing. Although there is no substitute for getting out and surrounding yourself in nature, the real-time images brought to us via Winterwatch are a reminder of the wonders and excitement of the natural world and provide inspiration to visit such places.
Cairngorms Connect forests are home to half of Scotland's lekking Capercaillie population.
Peter Cairns/scotlandbigpicture.com
The 60,000 hectare Cairngorms Connect project is one of eight landscapes in Europe where ELP projects are restoring biodiversity and natural ecosystem processes. Although the project area encompasses the largest surviving remnant of ancient Caledonian pinewood forest and is a stronghold for stunning wildlife such as the capercaillie (a huge woodland grouse), it is a mere shadow of its former glory. Over centuries, it has been impacted by the changing demands of farming, commercial forestry and field sports. An exceptionally forward-looking project, Cairngorms Connect (a partnership between the RSPB, Forest Enterprise Scotland, Scottish Forestry and the Cairngorms National Park Authority) has a 200-year vision to reinstate Caledonian forest to its natural limit, creating a landscape rich in wildlife that can support sustainable local economies.
Programmes such as Winterwatch remind us why we must make every effort to protect and restore landscapes, in the UK and elsewhere in Europe. As we reflect on the progress of the ELP this year, from the selection of our eight funded projects to the Programme's official launch in October, we look forward to sharing their achievements over the coming year.
You can catch Winterwatch on BBC Two in January 2019 (air date to be confirmed).

redpajama_set_name: RedPajamaCommonCrawl


text:
Hitchin Town FC > News > All News > VICTORY BRINGS FA CUP DRAW TO TOP FIELD
VICTORY BRINGS FA CUP DRAW TO TOP FIELD
Posted by SCurtis on Friday 19th October 2018
Hitchin Town now have an even bigger incentive to win their FA Cup Fourth Round Qualifying game against Leatherhead this Saturday. We have learned that the winners of the game will host the draw for the First Round Proper of the Emirates FA Cup on Monday evening.
This will go out live on BBC2 and BT Sport at 7:00pm, being introduced by Mark Chapman, the host of 'Match of the Day 2', with former Premier League striker Dion Dublin among the guests.
The Emirates FA Cup trophy will also be on display in front of a specially invited audience of supporters and officials, and representatives of other clubs in the draw.
The draw is also due to be shown live around the world, bringing the chance of international recognition for the club.
Please get behind the boys this Saturday. Discounted online tickets are available here until 8pm on Friday evening, with cash turnstiles then on available on the day.
ROY IZZARD


text:
The End of Melancholy
Andrew Petroff
St.-Petersburg
"The End of Melancholy" is female fronted alternative | gothic metal band, founded in 2014 by Olly Lex and Andrew Petroff in Saint-Petersburg, Russia. The band have a new point of view on a modern gothic scene, combine in music powerful metal riffs with lyrical ballads. Fans from the US call the band's style as "New European Metal". Since 2015 "The End of Melancholy" had performed with such bands as Theatres des Vampires and Hanzel und Gretyl, Motionless in White, released three music videos, EP, singles, and in the beginning of 2019 released a new album "Paradox Intention". The band supported this release in two European tours in April-October 2019 and visited France, Belgium, Switzerland, Czech Republic, Hungary, Poland, Lithuania and Estonia. Now "The End of Melancholy" preparing to release new material and plans a new European tour in October 2020. Discography: Playing with Shadows (2016) - EP The Demon (2017) - Single Peaceful Warrior (2019) - Single Paradox Intention (2019) - LP Monsters Feeling (2019) - Single Videos: Dolphins (2016) The Demon (2017) Peaceful Warrior (2019) Monsters Feeling (2019) The End of Melancholy are: Olly Lex : vocals Andrew Petroff : guitars, production Roman Laine : bass Eugene Psychedelic : drums
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ACC=American College of Cardiology; AHA=American Heart Association; HFSA=Heart Failure Society of America; ACEi=angiotensin-converting enzyme inhibitor; RRR=relative risk reduction; ARR=absolute risk reduction.
See the Class I recommendation for ENTRESTO.
GFR=glomerular filtration rate; NT-proBNP=N-terminal pro-B-type natriuretic peptide; ARB=angiotensin receptor blocker; ICD=implantable cardioverter defibrillator; CRT-D=cardiac resynchronization therapy defibrillator.
References: 1. ENTRESTO [prescribing information]. East Hanover, NJ: Novartis Pharmaceuticals Corp; November 2018. 2. McMurray JJV, Packer M, Desai AS, et al. Baseline characteristics and treatment of patients in Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure trial (PARADIGM-HF). Eur J Heart Fail. 2014;16(7):817-825. 3. Yancy CW, Jessup M, Bozkurt B, et al. 2017 ACC/AHA/HFSA focused update of the 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America. Circulation. 2017;136(6):e137-e161. 4. McMurray JJV, Packer M, Desai AS, et al. Angiotensin–neprilysin inhibition versus enalapril in heart failure. N Engl J Med. 2014;371(11):993-1004. 5. Cook RJ, Sackett DL. The number needed to treat: a clinically useful measure of treatment effect. BMJ. 1995;310:452-454.


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With Their Debut Album Slated For Release In Early 2019, It's Time To Get To Know GOON Check Out Photos From Their Gig At The Teragram Ballroom 10/15/18
LOS ANGELES, CA- Back in October, I saw the U.K. band Shame rock The Teragram. As I'm organizing the photos I took this year, I noticed that I neglected to post my thoughts and photos of one of the bands that opened for them: Goon. Shame on me.
A quartet of Kenny Beck, Drew Eccleston, Christian Koons and Caleb Wicker, Goon is based out of L.A. and their music is a dreamy, lo-fi wash of alt rock sonics. I had read somewhere that their frontman Kenny is an accomplished painter and after I checked out some of his paintings, I really got a sense of who he was and where the music comes from. For the most part, his paintings are abstract yet based in reality, which mirrors the musicality of Goon's music. Bits of brightness filled out with shades of the sludgy, Goon's music uses a splash of everything that's on the sonic palette.
It was my first time catching the band live, and I dug the vibe that their music imbued on the crowd. It almost felt like I was back in the 90's at a club gig and Pavement were on stage. That's the vibe I got. Layered, fuzzy and loud guitars filled the room as Kenny's nonchalant vocals subtly pierced through the buzz. Though I'm not fully educated on GOON's entire repertoire, I have the sense that a majority of their setlist came from their heavier, grungier, 2016 EP "Dusk Of Punk".
With two EP's under their belt, next year looks to be a significant step in this band's evolution as their debut full length is scheduled to be released in early 2019 by Partisan Records. The two tracks they release in 2018 ("Enter Bethel Admissions" and "Choke Throat") seem to follow the generally lighter vibe of their 2017 EP Happy Omen, so I'm interested to see how much of their new album has got that loud, layered sound that hypnotized me at the Teragram.
Follow Goon on Facebook, Instagram and Twitter.
Goon @ Teragram Ballroom 10/15/18. Photo by Derrick K. Lee, Esq. (@Methodman13) for www.BlurredCulture.com.
#Caleb Wicker	#Christian Koons	#Concert Photography	#DKL PHOTOS	#Drew Eccleston	#Goon	#Indie Rock	#Kenny Becker	#Teragram Ballroom
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Power 106 Tops Itself At Powerhouse 2018 With N.E.R.D., Logic, Nipsey Hussle & More! REVIEW+PHOTOS: POWERHOUSE 2018
Culture, Music
Listen to Rich Chigga's New Song "Seventeen" "Dms blowing up I always say no bitch this dick ain't free."
Swimm and Warpaint Shut Down The Echoplex For The Conclusion of Love You Down III With LYDF III in the Books, We Already Can't Wait Until Next Year
Adam Friedman: Troubadour, Mountain Climber & Bridge Maker [INTERVIEW+PHOTOS] BC ARTIST PROFILE: ADAM FRIEDMAN
Fall Out Boy's Fiery (Literally) Performance Rocked KAABOO REVIEW+PHOTOS: Fall Out Boy @ KAABOO 9/16/16
Ooklah The Moc Brings One Love Some Island Vibes From Hawaii THOUGHTS+PHOTOS: OOKLAH THE MOC @ ONE LOVE CALI REGGAE FEST 2/11/18


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The Sol 2017 is one of the ION harnesses aimed at the more freestyle orientated women rider as it has a slim outline-cut for maximum flexibility. The Neo Belt creates a smooth transition from the belt to the main body of the harness. In addition, this year ION has been able to further enhance freedom of movement, as well as delivering a perfect fit with even more support. Stylish, sophisticated and specialised, the SOL is your ultimate accessory.
Neo Belt: Improved flex, twist and comfort NEW!
The Sol ION 2017 the lightweight option which is the perfect companion for the freestyle orientated women.


In a large bowl combine olives, tomatoes, peppers, onion, garbanzo beans and capers.
In a medium bowl, whisk together vinegar, olive oil and garlic.
Add to olive mixture with basil and oregano; mix well.
Season to taste with salt and pepper. Gently stir in feta cheese.


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Hope through the valleys
Although Debora's journey to get to House of Joy was one filled with valley after valley, she never lost hope and her friend Anita never stopped praying.
Debora* was married to Bashkim* and together they had five children. In 1997, after eight years of marriage, he secretly divorced her and moved to Germany with their children. As is tradition, when Bashkim left Debora remained living with his family. She took care of his parents, and served in their family's household–doing so with love and respect even though she was not treated well.
However, in 2017, after 20 years without her husband, working for his parent's household, Debora was suddenly told she had to leave: Bashkim was coming back to Kosovo with his new wife.
Desperately, Debora called her friend Anita, a fellow believer at the local church. Anita works with OM in Kosovo at the House of Joy project: a holistic programme that reintegrates survivors of abuse into society. The two women had met in 2010 at church, but whenever Anita had tried to talk to Debora about her situation, Debora could not talk openly because her husband's family was always hovering nearby to stop her from sharing what was happening in her life. Ever since they first met, Anita had been praying and asking God how she could help rescue Debora from the house that had enslaved her.
When the day of Debora's expulsion from her home came, Anita and two other OMers picked Debora up and took her to the police station to make a report. From there, they took her to a government shelter. After staying for the legal minimum of one month, Debora transferred to the House of Joy, where she has truly brought the word 'joy' into the home.
Debora shares the gospel with the other ladies in the house, sings worship songs at the top of her lungs, studies her Bible in the common areas and invites everyone to church on Sundays.
One Saturday evening, one of the other girls asked Debora questions about her faith and made the decision herself to accept Jesus into her life. The next day at church, after speaking to the pastor, she made a true and official profession of faith.
Debora is a light, despite all of her hardships; she salts the earth as she walks and believes in miracles. She loves everyone with the love that Jesus has for them because she has felt it herself. She is finally free to live out her faith and find healing through the building of her relationship with Jesus, and through spending time with the programme's psychologist.
Please pray for Debora to be a good influence and have success in her time at the House of Joy. Please pray, too, that she would find healing through Jesus, and accept help that is offered.
*name changed


Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5' and 3' untranslated regions (UTRs) of mature mRNA. The 3'UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin's 3' UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3'UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin's respective 3'UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin's shorter 3'UTR sequence was inconclusive; however, results illustrated that titin's longer 3'UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin's variant 3'UTR sequences.
SHROUT, Tara A. Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3' Untranslated Regions. Vanderbilt Undergraduate Research Journal, [S.l.], v. 9, aug. 2013. ISSN 1555-788X. Available at: <http://vurj.vanderbilt.edu/index.php/vurj/article/view/3772>. Date accessed: 26 apr. 2019. doi: https://doi.org/10.15695/vurj.v9i0.3772.


Tomorrow begins First Baptist Moreland's 6th season offering Upward Soccer. Last fall was our 6th season of Upward Flag Football & Cheerleading. Upward Sports is the largest Christian Sports Ministry - over 2,600 churches partner with Upward. It began in a basketball gym at First Baptist Spartanburg, South Carolina - set-off by a message from Johnny Hunt to Caz McCazlin about the importance of reaching one more.
1). Sports Ministry requires alot of time. If you're looking for something quick and easy, running a football league isn't it.
2). Sports Ministry can be self-sustaining. Sell concessions, sell pictures (you should receive 20% of all sales), and ask for sponsors. People will give for little Johnny to play soccer and learn about Jesus.
3). People like to register and pay all online. The days of evaluations, try-outs, and waiting in a registration line are over. We began using online registration with Blue Sombrero our 3rd season and now the majority of our registrations are online. We meet the player at the first practice.
4). You need an updated Sports Ministry Facebook page. Remember, most parents in your league are in the under/around 40 crowd, which means they're all on Facebook. Post game-day pictures, schedules, and promote the players and coaches on the page. The key is to keep it updated. If your page is boring info updated from last summer - then parents are going to believe your league is boring and outdated. Here's our page.
5). The secret to success are the coaches. Bottom line: good coach = fun experience for the child = they'll return next season. Even if you have sub-par facilities, if your coaches are prepared and pour their lives into the kids - it will be a great season. The coach has the most influence - over devotions, sharing the Gospel and inviting players/parents to church. The coach will determine if they play again. I coach because of this reason. There are people now born-again Christians because I took the time to coach. Your league director/minister of recreation should invest a great amount of time into their coaches.


Congratulations to the IBDP Class of 2017!
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Congratulations to our British Program Students!
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text:
The authors used this protocol in:
Similar Protocols
沉积物中溶解性有机质的提取
培菌白蚁肠道簇虫分离与分子鉴定的方法
Reproducibility Feedback
Materials and Reagents
En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos
Sébastien Darras
sebastien.darras@obs-banyuls.fr
Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins (BIOM), Banyuls-sur-Mer, France
Go to author page
DOI: 10.21769/BioProtoc.4160
Published: Vol 11, Iss 18, September 20, 2021
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Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Darras, S. (2021). En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos. Bio-protocol 11(18): e4160. DOI: 10.21769/BioProtoc.4160.
Coulcher, J. F., Roure, A., Chowdhury, R., Robert, M., Lescat, L., Bouin, A., Carvajal Cadavid, J., Nishida, H. and Darras, S. (2020). Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos. Elife 9: e59157.
Ascidian embryos are powerful models for functional genomics, in particular, due to the ease of generating a large number of transgenic embryos by electroporation. In addition, the small size of their genome makes them an attractive model for studying cis-regulatory elements that control gene expression during embryonic development. Here, I describe the adaptation of the seminal method developed 25 years ago in Ciona robusta for en masse DNA electroporation for in vivo transcription to additional species belonging to three genera. It is likely that similar optimizations would make electroporation successful in other ascidian species, where in vitro fertilization can be performed on a large number of eggs.
Keywords: Ascidians, Tunicates, Electroporation, Transgenesis, Cis-regulatory DNA, Phallusia, Ascidia, Molgula
Ascidians are marine invertebrates that are the closest vertebrate relatives. Their fast, stereotyped, and external embryonic development gives rise to tadpole-like larvae closely resembling other chordates (vertebrates and amphioxus). Their compact genome, together with the availability of simple and efficient methods to manipulate gene expression and function, have made ascidians interesting model organisms for functional genomics (Lemaire, 2011; Satoh, 2014). In particular, the introduction of plasmid DNA into a fertilized egg by electric shock (electroporation) allows the generation of hundreds to thousands of transient transgenic embryos, which promoted Ciona robusta as the reference ascidian species almost 25 years ago (Corbo et al., 1997). DNA electroporation is a widely used method for in vivo transcriptional assays (for characterization of the activity of candidate cis-regulatory elements using reporter genes), over-expression/knockdown, and live imaging.
Ascidians are a diverse group of animals containing around 3,000 species that have been proven as interesting systems to study the evolution of developmental mechanisms since their genomes have been extensively rearranged, but their embryonic development is strongly conserved (Dardaillon et al., 2020). In particular, DNA electroporation has been applied to various species (Roure et al., 2014; Stolfi et al., 2014; Colgan et al., 2019; Coulcher et al., 2020). Here, I provide a detailed protocol to perform electroporation in four species: Phallusia mammillata, Phallusia fumigata, Ascidia mentula, and Molgula appendiculata (Figure 1), which cover almost 400 million years of evolution.
Disposable scalpels
300- and 120-µm homemade sieves
Note: Cut a 50-ml plastic tube at ~4-5 cm from the opening. Cover a heating plate with aluminum foil. Fuse a piece of nylon mesh (Sefar Nitex, catalog numbers: 03-300/51 [300-µm opening], 03-120/49 [120-µm opening]) to the cut tube using the heated plate.
15-ml glass centrifuge tubes (Dutscher, catalog number: 092305)
15-ml plastic conical tubes (e.g., Sarstedt, catalog number: 62.554.502)
60-mm plastic Petri dishes (e.g., Sarstedt, catalog number: 82.1194.500)
60-mm and 92-mm agarose-coated Petri dishes. Alternatively, gelatin-coated dishes (GF) can be used as in Sardet et al. (2011)
Note: Melt the agarose in sea water (1 g per 100 ml sea water). Fill a 92-mm dish with the hot solution. Make a thin agarose layer by pouring the agarose solution into the next dish; repeat until you have enough dishes (you will need two dishes per electroporation, plus a dozen for dechorionation, fertilization, and washing).
6-well plates
Note: Eggs tend to stick to new plastic material. To avoid this, reuse the same plates; simply wash with tap water and air dry.
Gloves (Phallusia blood is a powerful stain!)
Glass Pasteur pipets
Note: Soak them in tap water to avoid sticking of the embryos. Smooth the opening using a lighter. Prepare some with a larger opening (2-3 mm) using a diamond for egg collection in Phallusia.
100 ml glass beaker
Horizontal rotating shaker (e.g., VWR, catalog number: 444-2900)
1.5 ml microtubes
Low-binding 1.5 ml (e.g., VWR, catalog number: 525-0230) and 2 ml (e.g., VWR, catalog number: 525-0232) microtubes
4 mm electroporation cuvettes (e.g., Dutscher, catalog number: 38191)
Depression slides: 4 mm (Dutscher, catalog number: 065230) and 1.5 mm (Dutscher, catalog number: 65227) of thickness
Tungsten needle (Roboz Surgical Instrument Co., catalog number: RS-6065)
Adult ascidians are collected from the ocean by diving, using a trawl or a dredge and are provided by the French node of the European research infrastructure EMBRC (Station biologique de Roscoff: Phallusia mammillata and Ascidia mentula; Station marine de Banyuls-sur-mer: Phallusia mammillata, Phallusia fumigata, Ascidia mentula, and Molgula appendiculata)
Supercoiled circular plasmid DNA (20-100 µg) of the reporter construct
NaOH, 2.5 M
NaOH, 1 M
1 M Tris, pH 9.5
0.96 M D-mannitol solution (VWR, catalog number: 25311.297)
TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
25% glutaraldehyde solution (Sigma-Aldrich, catalog number: G6257)
Sodium thioglycolate (Sigma-Aldrich, catalog number: T0632)
Pronase (Sigma-Aldrich, catalog number: P5147)
0.2 µm filtered natural sea water or artificial sea water (BASWH: see Recipes)
Phallusia and Ascidia dechorionation solution (see Recipes)
Molgula dechorionation solution (see Recipes)
PBTw (see Recipes)
10× PBS (see Recipes)
X-gal staining solution (see Recipes)
X-gal stock solution (see Recipes)
Scissors and tweezers
Temperature-controlled room set at 18°C
Dissecting scope for live embryo manipulation (e.g., Discovery V8 from Zeiss)
Square wave electroporator (Harvard Apparatus, BTX ECM830, catalog number: 45-0052)
Dissecting scope equipped with a digital camera for staining analysis (e.g., Discovery V20+AxioCam Erc 5s from Zeiss)
The in vivo transcriptional assay proceeds according to these successive steps: 1) gamete collection, 2) egg dechorionation, 3) fertilization, 4) DNA electroporation, 5) embryo fixation and staining, and 6) data collection and analysis. The first 4 steps differ between species. Here, I present two versions: one for P. mammillata, P. fumigate, and A. mentula (with minor changes among species); and one for M. appendiculata.
Figure 1. Adult ascidian species. (A) Phallusia mammillata (5-20 cm long). (B) Phallusia fumigata (5-20 cm long). (C) Ascidia mentula (4-10 cm long). (D) Molgula appendiculata (3-6 cm long).
Gamete collection
Ascidians are hermaphrodites. The aim is to collect both eggs and gametes, separately, from each individual.
Phallusia and Ascidia
P. mammillata, P. fumigate, and A. mentula belong to the same family, the Phlebobranchia. Gametes are collected from the gonoducts by dissection. To avoid unwanted self-fertilization, eggs are collected first. All species have transparent eggs.
Open the animals with a scalpel by cutting between the two siphons (Figure 2A). Phallusia tunic is thick and hard.
Incise the tunic toward the base of the animal on both sides. Do not cut too deep to avoid damaging the animal.
Open the tunic by pulling apart with your fingers.
Transfer the animal to a dish on its left side (you should see the heart beating at the base of the animal, opposite to the siphons). If the animal is on the right side, you should see a large, yellowish (Phallusia, Figure 2C) or reddish (Ascidia, Figure 2B) oviduct full of eggs.
Make a small cut in the oviduct and collect the eggs with a Pasteur pipet (Figure 2D).
Note: The eggs in the oviduct are very compact and embedded in a gelatinous substance. Use a wide-bore Pasteur pipet for Phallusia; otherwise, their aspiration through the small opening of a pipet may cause extreme deformation and egg death. Dead eggs are easily spotted since they turn opaque.
Figure 2. Gamete collection in Phallusia and Ascidia. (A) Schematic diagram illustrating the successive incisions (numbered red dotted lines) to open the tunic (left: lateral view with the two siphons pointing to the left; right: side view with the two siphons pointing toward the experimenter). (B) Dissected A. mentula individual where the gonoducts are exposed. (C) Dissected P. mammillata individual where the oviduct is clearly visible. (D) Egg collection in P. mammillata using a wide-bore Pasteur pipet. (E) P. mammillata egg. (F) A. mentula egg. Scale bar: 100 µm in D and E.
Transfer the eggs to a 6-well plate filled with sea water. Phallusia individuals normally have a high number of eggs (0.5-2 ml eggs). Ascidia usually have fewer eggs, but the amount can reach 0.5-1 ml.
Check the egg quality (transparent and uniform egg cytoplasm, Figures 2E and 2F) and transfer them to a glass tube for several washes with sea water. Eggs can be kept overnight at 14°C, spread on a Petri dish, with no major decrease in quality.
You should see the spermiduct (sharp white duct) underneath the oviduct once you have removed the eggs. Make a small cut and collect the sperm into a 1.5-ml tube using a Pasteur pipet for Phallusia or a micropipet with a yellow tip. Avoid collecting sea water, debris, and blood. Dry sperm remains active for several days at 4°C.
Molgula
Contrary to Phallusia and Ascidia, very few eggs are present in the tiny oviduct (Figure 3D). Gametes are therefore collected by gonadal dissection. The oocytes collected from the gonad represent all stages of oogenesis. The procedure aims at collecting mainly fully grown oocytes that are not yet mature, as evidenced by the presence of a large germinal vesicle (GV). Fortunately, these oocytes mature spontaneously in sea water, and germinal vesicle breakdown (GVBD) occurs within 30-60 min.
Open the animals with scissors, starting from one siphon around the animal all the way to the other siphon (Figure 3A) (M. appendiculata are covered with debris, shells, and stones; use large scissors and make your way around these obstacles).
Pull open the tunic and drag the animal still attached by the siphons (Figure 3B).
Cut the body wall from siphon to siphon with fine scissors and pull open the animal (Figure 3C).
Under the dissecting scope, locate the gonads (one on each side of the animal, Figure 3C) under the pharyngeal basket with tweezers. You should see the short gonoducts pointing toward the siphons and the large ovary covered on one side by the testis (Figure 3D).
Cut out each gonad with scissors and place them in the same well of a 6-well plate filled with sea water (Figure 3E).
Figure 3. Gamete collection in Molgula appendiculata. (A) Schematic diagram illustrating the incision (red dotted line) to perform to open the tunic (left: lateral view with the two siphons pointing to the top; right: side view). (B) An individual (right side) after tunic (left side) removal. (C) Cut open the animal with a gonad visible at the center on each side. (D) Each gonad is composed of an ovary surrounded by a testis. The inlet shows a close-up view of the tiny spermiduct (top) and the oviduct (bottom), both openings pointing to the right. (E) A 6-well plate where pairs of gonads from 4 individuals have been collected. (F) A fertilizable egg is obtained after spontaneous maturation of a fully grown oocyte (scale bar: 50 µm).
Under the dissecting scope, release the oocytes from the ovary using tweezers, and transfer the testis to a Petri dish filled with sea water (60-mm diameter). You should see a full range of oocytes (from tiny transparent oocytes to large, opaque, fully grown oocytes with GV).
Transfer all the oocytes from one individual (2 ovaries) through a 300 µm sieve placed in a 100 ml glass beaker (gonad debris will not go through).
Transfer the contents of the beaker to a 120 µm sieve and wash extensively with sea water to remove sperm and small oocytes.
Transfer the oocytes to a 60 mm agarose-coated dish.
Wait 30-60 min until GVBD occurs and produces fertilizable oocytes.
Release the sperm from the testis of all individuals that have been collected in the same Petri dish by dissociating each testis with tweezers (discard the testes afterwards). Collect the concentrated sperm solution into a 15-ml plastic tube and keep at 4°C until use.
Egg dechorionation
The chorion that protects the egg is made of an inner vitelline membrane consisting of extracellular material and an outer cellular layer of follicle cells. To perform electroporation, it is necessary to remove it; this is achieved using a mixture of sodium thioglycolate and pronase. The concentration of each compound and the duration of the dechorionation varies between species.
Note: Dechorionation is usually performed before fertilization, and the naked eggs can still be fertilized. For Molgula, since the procedure is rather quick, this can be performed after fertilization.
Collect the eggs in a glass tube and allow them to settle to the bottom.
Discard the excess sea water.
Activate the dechorionation solution by raising the pH (add 3 drops (around 100 µl) (Phallusia/Ascidia) or 6 drops (around 200 µl) (Molgula) 2.5 M NaOH to 7 ml dechorionation solution using a Pasteur pipet, and mix).
Proceed to dechorionation
In an agarose-coated dish for Phallusia/Ascidia, by adding the eggs to the dechorionation solution (~7 ml for a 60-mm dish and ~14 ml for a 92-mm dish). Mix well and place on a horizontal shaker at ~70 rpm.
In a glass tube for Molgula, by adding 3-4 ml dechorionation solution. Mix well by pipetting up and down.
Regularly check the dechorionation status under the scope. Dechorionation takes 30-45 min (Phallusia/Ascidia) and 7-15 min (Molgula) depending on the egg density.
Stop dechorionation when most eggs are clearly devoid of the chorion. Phallusia/Ascidia: gather the eggs at the center by swirling the dish and transfer them to a 15 ml glass tube. Add sea water to the top and gently mix by pipetting up and down. Molgula: discard as much dechorionation solution as possible. Add sea water to the top and gently mix by pipetting up and down.
Note: Dechorionation is more difficult to follow in Molgula: while follicle cells are quickly lost, the vitelline membrane is more difficult to see (look closely, change the scope mirror orientation; the presence of test cells on the egg's surface is a good indication that the eggs are not fully dechorionated).
Wash extensively 2-4 times by allowing the eggs to settle down, replacing the sea water, and gently mixing with a Pasteur pipet. Debris (chorion and dead eggs) will float, while naked eggs rapidly sink to the bottom of the tube. Naked eggs are fragile and explode easily; be gentle and avoid air bubbles.
You can proceed to the next step using the same tube or collect the eggs in an agarose-coated dish for later use. Dechorionated, unfertilized eggs can be stored overnight at 4-14°C with no major decrease in quality.
(Phallusia/Ascidia only) Add 5 µl dry sperm to a 1.5-ml microtube containing 1 ml sea water.
(Optional) Fertilization is usually efficient with straight sperm solution; however, to achieve a complete fertilization rate and high synchrony, it is better to activate the sperm solution by raising the pH (in the ocean, sperm are activated by the slightly basic pH of the sea water). Add either 50 µl 1 M Tris pH 9.5 (Phallusia), 4 µl 1 M NaOH (Ascidia), or 8 µl 1 M NaOH (Molgula) to the sperm solution.
Note: Phallusia sperm can also be activated by incubating the 1 ml mixture for 15 min with 20 µl chorionated eggs as in Sardet et al. (2011).
Check the sperm motility by diluting an aliquot 50-100× in sea water and looking under the dissecting scope at high magnification. Sperm should be swimming frantically.
Add ~200 µl (60-mm dish) or 400-500 µl (92-mm dish or 15-ml glass tube) sperm solution.
Mix well, making the eggs float in the medium. You should see the sperm swimming intensively.
Check for egg deformation that occurs following fertilization and that should be visible within the first few minutes.
At 10-15 min post-fertilization, proceed to the washes to remove the sperm, since this protocol uses enormous quantities of sperm. (Fertilization in a Petri dish) Gather the eggs at the center by swirling the dish and transfer them to a 15-ml glass tube using a Pasteur pipet. Allow the eggs to settle to the bottom of the tube, discard most of the sea water using a Pasteur pipet, add clean sea water up to the top by gently pouring down the side of the tilted tube; resuspend the eggs thoroughly by flushing sea water with the pipet. Repeat this wash 1-3 ×.
Note: Embryos without their protective chorion are fragile and explode as soon that they come into contact with the water/air interface. Be cautious; gently manipulate them, avoiding air bubbles.
DNA electroporation
Prepare the DNA (20-100 µg) in 50 µl TE.
Add 200 µl 0.96 M D-mannitol and mix well.
Collect the fertilized eggs at the bottom of a glass tube.
Mark with a pen, the 100 µl level on the low-binding 1.5 ml tubes.
Transfer the fertilized eggs into the 1.5-ml low-binding tubes. Try to add the same number of eggs to each tube (one per construct to be tested).
Adjust the volume to the 100 µl mark with sea water.
To each tube, add 250 µl DNA/mannitol solution.
Note: Mannitol is used to reduce the salt concentration (and thus avoid electric arching during electroporation) while keeping the osmolarity at a sufficient level.
Transfer the total mixture to an electroporation cuvette (use a different Pasteur pipet for each tube to avoid mixing DNA).
Place the cuvette into the electroporator cuvette holder.
Apply a single electrical pulse of 37 V (Phallusia/Ascidia) or 20 V (Molgula) for 32 ms. The pulse should be done toward the end of the first cell cycle (but before cleavage): at 50-60 min post-fertilization.
Note: Electroporation can be performed any time after fertilization, and the timing does not affect the electroporation efficiency; however, the fertilized eggs are more robust to electroporation (but also to microinjection) during the last third of the first cell cycle.
Proceed to the electroporation of the next construct.
Once all electroporations are done, add some sea water to the cuvette and transfer the eggs to 92 mm agarose-coated Petri dishes (usually at least two dishes for one electroporation).
Note: A total of 4-6 electroporations are routinely done per fertilization round. With experience, 10-12 electroporations can be performed.
Check the dish under the dissecting scope. You should see intact embryos (since they are toward the end of the first cell cycle, you should see the myoplasm and/or deformations corresponding to the preparation of the first cleavage) and debris of exploded eggs.
Note: For species with transparent eggs (Phallusia/Ascidia), dead eggs are easily spotted since they become opaque. In most cases, there is a small proportion of eggs that do not survive the electroporation; these correspond to fragile or unfertilized eggs. In cases where you find no surviving embryos, there are two main explanations: the fertilization failed (unfertilized eggs systematically explode after the electrical pulse) or the voltage was too high. Try to make two experimental controls: non-dechorionated and non-electroporated embryos, and dechorionated and non-electroporated embryos.
Transfer the dishes to an incubator set at the desired temperature.
Note: 14-19°C is the common range of temperature for all species. They can also develop well at lower temperatures with a significant decrease in speed. Phallusia/Ascidia also develop well up to 22°C.
Spread the embryos by flushing sea water with a Pasteur pipet (they have a very high tendency to stick to each other if they are too close). Avoid moving the dishes before the 8-cell stage since blastomeres are very loosely attached to each other.
Embryo fixation and staining
Depending on the reporter gene, the course of action may differ. Here, I describe the use of LacZ as a reporter gene (detecting β-galactosidase activity using the chromogenic substrate X-gal).
Allow the embryos to develop until the desired stage.
Swirl the dish to gather the embryos at the center.
Collect the embryos using a glass Pasteur pipet and transfer them to a 2-ml low-binding microtube.
Allow the embryos to settle and discard the excess sea water.
Fill the tube with fixative (0.2% glutaraldehyde in sea water) and rotate the tube in your fingers for 30 s.
Fix for exactly 30 min at room temperature (over-fixing will abolish β-galactosidase enzymatic activity, too light a fixation may lead to embryo disintegration).
Remove half of the fixative and add 1 ml PBTw. Mix by inverting 2-3 ×.
Once the embryos have settled, replace the solution with 2 ml PBTw, mix by inversion, and wait 10 min. Repeat this wash 1 ×.
Rinse for 5 min in X-gal staining solution.
Replace with 250 µl staining solution containing 0.4 mg/ml X-gal (dilute the X-gal stock solution 100×).
Incubate at 37°C (an incubator is preferable to a water bath since no evaporation takes place in the tube).
Check the blue staining from time to time (it starts within a couple of hours depending on the strength of the tested regulatory region).
Once the staining is adequate (this may take several days, but this is a staining procedure that never produces any background), wash 2 × 5 min in PBTw. Typical examples of staining with a version of β-galactosidase targeted to the nucleus may be found in Roure et al. (2014) and Coulcher et al. (2020).
Post-fix for 1 h at room temperature or O/N at 4°C in PBTw containing 3.7% formaldehyde.
Samples can be stored long-term.
Discard the fixative and wash 2 × 10 min in PBTw.
Transfer all the embryos to a thick depression slide.
Transfer part of the embryos to a thin depression slide.
Using a dissecting scope and a tungsten needle, score the number of stained embryos in the target tissue until you reach 100-200 scored embryos or until all the embryos in the tube have been scored (score only those embryos that have developed normally) (Roure et al., 2014; Coulcher et al., 2020).
Embryos can be manipulated easily using a tungsten needle, a cat whisker, or an eyelash.
Depending on the aim, the approach can be refined by scoring not only the number of stained embryos, but also the number of stained cells per embryo; however, this may be time consuming.
Do not forget that this transgenesis method yields mosaic transgene expression (each cell has randomly inherited a variable amount of plasmid DNA) and that each embryo is an independent event; hence, the activity of a given region is determined by compiling the expression in all the embryos that are analyzed.
Take images of representative embryos.
Evaluation of cis-regulatory activity and comparisons
The assay provides qualitative data (i.e., the spatial domain of activity of a given piece of genomic DNA), but the percentage of stained embryos also reflects the strength of the regulatory region.
The score obtained may vary from experiment to experiment according to the ascidian batch, plasmid DNA preparation, and embryo concentration during electroporation; thus, we usually perform at least 3 biological replicates (different ascidian, different day of experimentation) and calculate an average score and standard deviation.
Note: Long (4-10 kb) genomic regions are usually strongly active (staining is visible within 1 h of incubation at 37°C, with over 90% of the embryos stained) and do not display much variation between experiments.
Often, the goal of this assay is to locate essential cis-regulatory elements; thus, one needs to compare the activity of various constructs. Obviously, the plasmid backbone should be the same. We also favor comparing the results of constructs that have been electroporated during the same experiment.
Banyuls-sur-mer Artificial Sea Water HEPES (BASWH)
BASWH is a 'Mediterranean type' sea water with the following features: salinity of 37.9 g/L, osmolality of 1219 mOsmole, Na+/K+ ratio of 47, and a pH of ~8.0.
For 1 L, mix:
30.34 g NaCl
0.83 g KCl
1.47 g CaCl2·2H2O
4.98 g MgCl2·6H2O
6.29 g MgSO4·7H2O in MilliQ water
Adjust to 1 L
At this step, the solution can be stored at room temperature for months.
Before use, add 0.18 g NaHCO3 and 5 ml HEPES 1 M pH 8.0, and filtrate (0.2 µm). Can be stored for 1-2 months at 4°C.
Phallusia and Ascidia dechorionation solution
1% sodium thioglycolate
0.05% pronase in sea water
Mix thioglycolate in sea water by shaking, add pronase on top – no mixing – and keep at 4°C for a few hours before use.
Can be used for 1-2 weeks when kept at 4°C.
Bring aliquot to room temperature before use.
Molgula dechorionation solution
0.5% sodium thioglycolate
0.1% pronase in sea water
PBTw
PBS 1× containing 0.1% Tween-20
PBS 10×
For 1 L, add:
80.0 g NaCl
26.8 g Na2HPO4·7H2O
2.0 g KCl
2.4 g KH2PO4
Adjust pH to 7.4 with NaOH
Bring to 1 L, and autoclave
X-gal staining solution
1 mM MgCl2
3 mM K4Fe(CN)6
3 mM K3Fe(CN)6 in PBTw
X-gal stock solution
40 mg/ml X-gal in dimethylformamide
This protocol was used in the study 'Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos' by Coulcher et al. published in eLife in 2020 (doi: 10.7554/eLife.59157). This work was made possible by provision of adult ascidians thanks to the knowledge and generosity of G. Diaz (professional fisherman in Port-Vendres, France) and staff (divers and boat crew) at the marine stations of Banyuls-sur-mer and Roscoff (French node of the European research infrastructure EMBRC). I wish to thank members of my group. This work was supported by CNRS and Sorbonne Université, and by specific grants from the ANR (ANR-11-JSV2-007 and ANR-17-CE13-0027), the CNRS (DBM2020 from INSB), and the European project Assemble Plus (H2020-INFRAIA-1-2016–2017; Grant No. 730984).
There are no conflicts of interest or competing interests.
Colgan, W., Leanza, A., Hwang, A., DeBiasse, M. B., Llosa, I., Rodrigues, D., Adhikari, H., Barreto Corona, G., Bock, S., Carillo-Perez, A., Currie, M., Darkoa-Larbi, S., Dellal, D., Gutow, H., Hokama, P., Kibby, E., Linhart, N., Moody, S., Naganuma, A., Nguyen, D., Stanton, R., Stark, S., Tumey, C., Velleca, A., Ryan, J. F. and Davidson, B. (2019). Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements. Evodevo 10: 24.
Corbo, J. C., Levine, M. and Zeller, R. W. (1997). Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124(3): 589-602.
Dardaillon, J., Dauga, D., Simion, P., Faure, E., Onuma, T. A., DeBiasse, M. B., Louis, A., Nitta, K. R., Naville, M., Besnardeau, L., Reeves, W., Wang, K., Fagotto, M., Gueroult-Bellone, M., Fujiwara, S., Dumollard, R., Veeman, M., Volff, J. N., Roest Crollius, H., Douzery, E., Ryan, J. F., Davidson, B., Nishida, H., Dantec, C. and Lemaire, P. (2020). ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates. Nucleic Acids Res 48(D1): D668-D675.
Lemaire, P. (2011). Evolutionary crossroads in developmental biology: the tunicates. Development 138(11): 2143-2152.
Roure, A., Lemaire, P. and Darras, S. (2014). An otx/nodal regulatory signature for posterior neural development in ascidians. PLoS Genet 10(8): e1004548.
Sardet, C., McDougall, A., Yasuo, H., Chenevert, J., Pruliere, G., Dumollard, R., Hudson, C., Hebras, C., Le Nguyen, N. and Paix, A. (2011). Embryological methods in ascidians: the Villefranche-sur-Mer protocols. Methods Mol Biol 770: 365-400.
Satoh, N. (2014). Developmental genomics of ascidians. Hoboken, New Jersey, John Wiley & Sons, Inc.
Stolfi, A., Lowe, E. K., Racioppi, C., Ristoratore, F., Brown, C. T., Swalla, B. J. and Christiaen, L. (2014). Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. Elife 3: e03728.
Copyright Darras. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Developmental Biology > Morphogenesis
Molecular Biology > DNA > Transformation
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Highly upgraded loft at Andersonville's Map Factory. Duplex 1 bed + den timber loft. Enormous chef's kitchen w/ tons of cabinetry, granite island, high end appliances. Exposed brick, hardwood floors throughout, 14' ceilings, fireplace, large living space, dining area, in-unit W/D. Renovated bathroom w/ soaking tub. Professionally organized walk-in closets. Common rooftop deck. Walk to Andersonville strip, Metra, CTA.


Acuity Eyecare is happy to provide care for both your eye health and your visual system. Insurances require that routine or wellness exams be billed to vision plans, and any medical eye care or testing will be billed to your medical/health insurance plan. We are on many medical insurance panels (such as Blue Cross Blue Shield, Medicare, United, etc), as well as vision discount plans (Eyemed, VSP, Superior, etc) to make this comprehensive care possible. We inform our patients about exam and treatment costs at the beginning of your appointment. If you have any other questions about your insurance, please let us know.
Please keep in mind that some insurances go by different names so if you don't see your insurance listed above, please give us a call to see if we are providers for your insurance.
Many employers are offering Flexible Spending Account (FSA) options to employees. These plans are designed to let you save money in an account, pre-tax, to pay for additional medical expenses such as eye exams, glasses, contacts, and often laser vision surgery. Check with the benefits administrator at your work to see if you are eligible for this program. FSAs can typically be used in conjunction with any insurance plan to offset any out-of-pocket costs on your behalf.
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Homes on parade: 23 domains open doors to paying public today | Tri-City Herald
Homes on parade: 23 domains open doors to paying public today
By Loretto J. Hulse, Herald staff writer
Whether you're planning on building new, looking for remodeling ideas or looking to the future, you'll enjoy the 2008 Parade of Homes.
The 23 houses on the tour open at 9 a.m. today.
They're scattered throughout the Tri-Cities and West Richland and run the gamut in size from 1,551 to 5,225 square feet, said Jeff Losey, executive director of the Home Builders Association of Tri-Cities in Kennewick.
"That's the beauty of a Parade of Homes," Losey said. "The houses range from your luxurious dream homes to something more practical, a little closer to the budget of a young family."
The houses are open until 5 p.m. today and from 9 a.m. to 5 p.m. Sunday and Sept. 13-14. They're also open from 1 to 7 p.m. Wednesday.
Cost is $7 per person. Tickets, which include a guidebook giving directions to each house and details on the construction and the contractors, are available at all Tri-City Sun Marts.
Tickets are valid for all five days but for only one visit per home. Tour the houses in any order. Visit all the homes in a single category -- marked on the back of the ticket -- have it validated and you'll be eligible to vote for your favorite. Visit all 23, turn in your validated ticket and you may win $500.
* Loretto J. Hulse: 582-1513; lhulse@tricityherald.com.
Explore these Tri-Cities gardens for a cause
Here are eight vegetables to grow during winter


Today's hottest trend is Copper! Give your kitchen a warm, industrial look with our 'Old Copper' splashback. The weathered effect makes it special. Very beautiful when combined with white and green colours.
Curious about our materials? Let yourself be suprised by the quality!


It's 20 years since Britney's iconic first studio album 'Baby One More Time' took the pop world by storm and now fans can go back in time to celebrate the anniversary of its release, a staggering two decades ago, on January 12, 1999.
Launching across the US today and now the UK, the Pop Princess is being honoured with 'Britney OS '99' which recreates a Britney Spears fan's computer desktop circa 1999 to interact with a virtual version of the iconic star.
Taking fans right back to the nineties, they use a retro messaging app and users can speak with a 'Britney Bot'. They are also able to send GIFs, make calls and chat to create a shareable playlist that reflects their mood.
Users are rewarded with hidden exclusives, free content and a chance to win prizes by interacting with all areas of 'Britney OS '99', such as the web browser or documents folder.
Creative technical agency Modern English, which has just completed projects for Dua Lipa, Mariah Carey, The Beatles and even Elvis in recent months, designed the site for New York based Legacy Recordings, the catalogue division of Sony Music Entertainment.


The following articles are written by Rene Vaz and are published with permission of Fairfax magazines, having been first published in Fish and Game New Zealand magazine. To learn more about Rene Vaz CLICK HERE to read his bio.
Going With the Flow - An article about fishing dry flies down stream to get better drag free drifts.
Fishing the Film - An article about fishing flies in the surface film vs on top of it.
Dealing with Double Handers Part 1 - Rene explains the different types of Spey rods available and the lines and casting techniques used to work them.
Dealing with Double Handers Part 2 -Rene explains more about how to fish double handers going into more specifics about their use in new Zealand.
Fishing for Gold - Rene tells of his experiences of competition fly fishing in New Zealand and discusses it's merits.
Fly Tackle For Stillwaters - Rene discussed the types of tackle that enhance success on stillwaters and their application to modern techniques.
Modern Loch Style Fishing - Rene explains the techniques and tackle involved with drift fishing lakes from a boat.
Fishing New Zealand Lowland Streams - Who said that the best fishing is hiden away in the back country? Rene explains the benefits of exploring spots that are often closer to home.


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Tag: michelle monaghan
Movie Project #25: Kiss Kiss Bang Bang [2005]
June 19, 2012 January 6, 2015 27 Comments
Due to the surprising success of my initial Movies Project, I decided to do a part two for 2012. This time around I put a greater emphasis on directors I am not familiar with, but I also tried to compile a mix of different genres and eras. This will be an ongoing project with the finish date being sometime this year.
Kiss Kiss Bang Bang [2005]
Director: Shane Black
Genre: Action/Comedy/Crime
Starring: Robert Downey Jr., Val Kilmer and Michelle Monaghan
Look up idiot in the dictionary. You know what you'll find?
A picture of me?
No! The definition of the word "idiot"! Which you fucking are!
Now where in the hell did this movie come from? It's rare that a film can combine dark comedy, action and mystery so effortlessly in one package. Kiss Kiss Bang Bang has all of this, with a satirical spin on Film Noir to boot.
The always charismatic Robert Downey Jr. stars as Harry Lockhart, a common thief who acts as the meta narrator for our story. After a botched robbery attempt, Lockhart cleverly evades police by running into an ongoing audition for a detective movie. The part, conveniently enough, is eerily similar to the exact situation Lockhart is going through at that moment. He nails the gig, eludes the cops and gets a part in the movie. Now THAT'S how you run from the law.
To train for his role, Lockhart is teamed up with private investigator Perry van Shrike (Val Kilmer), commonly known as "Gay Perry". While working on a case, the duo unwittingly stumble upon a vast conspiracy involving kidnapping and murder, and they soon become swept into a web of crime. Also caught in the heat of things is Lockhart's high school sweetheart, Harmony Lane (Michelle Monaghan), who he happens to run into at a Hollywood party.
What transpires is an expertly written mystery that is both hysterical and suspenseful. The movie moves at a brisk pace, one that is occasionally hard to keep up with but always entertaining. The laughs are dark and crude, without resorting to slapstick or lazy humor. The story doesn't take long to spiral out of control and it stretches the boundaries of believability (okay, it is hardly believable at all), but that doesn't matter. This is a self-aware movie that revels in its irreverence.
And who better to play the lead narrator/thief/pseudo-actor than Robert Downey Jr.? The man's comedic timing and delivery is impeccable, and he plays the lead with just the right amount of cynicism. His chemistry with the surprisingly buoyant Val Kilmer is off the charts. In fact, there are moments where Kilmer steals the scene. His "Gay Perry" delivers some brilliant wisecracks, a perfect complement to Downey's zaniness. Bonus points go out to Michelle Monaghan who is as stunning as I have seen her, and boy does she know it in this film.
Kiss Kiss Bang Bang is one of the last decade's more underrated films. Somehow this slipped under my radar (and many others, apparently) and I can't believe it took me so long to discover this gem. Sure, the plot can be hard to keep up with, but damn if this isn't an fun and wild ride. I am very glad to have included this in my project.
Movie Review: Source Code [2011, Jones]
August 22, 2011 January 6, 2015 14 Comments
Source Code [2011]
Director: Duncan Jones
Genre: Mystery/Sci-Fi/Thriller
Groundhog Day meets… Speed?
Colter Stevens (Jake Gyllenhall) is having a hell of a day. He wakes up on board a Chicago-bound Metra commuter train, and he is no idea how he got there. Worse yet, he appears to be living in someone else's body. This person, a school teacher named Sean, is sitting across from his good friend Christina (Michelle Monaghan). Everything appears to be business as usual until the train explodes after exactly eight minutes, killing everyone on board. Stevens wakes up in an unfamiliar location and is quickly contacted by Air Force Captain Colleen Goodwin (Vera Farmiga), who briefs him on his situation. Stevens learns that there was a bomb on the train, and that he is being used as part of a secret U.S. Military program to figure out who placed the explosives. He is forced to repeat the same eight minute sequence over and over until he determines who was responsible for the destruction.
Source Code is pretty intense, with several twists and turns amidst the frantic sci-fi thriller pacing. Even though it presents some interesting philosophical and ethical questions, the movie has a mostly benevolent attitude throughout (somewhat similar to The Adjustment Bureau in this regard). There are several moments where one will need to suspend their disbelief, but this is to be expected given the plot premise.
It's easy to get behind the character of Colter Stevens thanks to a great performance from Gyllenhall. He brings a certain human element to his character, even bringing the laughs during dire moments. Michelle Monaghan does well despite having what is essentially a throwaway role. I was most impressed with Vera Farmiga, who excels despite being on a computer screen during many of her scenes. She is quickly becoming one of my favorite actresses. Jeffrey Wright is also another standout here as the leader of the Source Code program, effortlessly showing he is the man in charge.
I enjoyed Source Code about as much as I enjoyed The Adjustment Bureau, which is to say quite a bit. Duncan Jones' title digs deeper and is perhaps "smarter" overall but both are a lot of fun. I would have opted for a different ending in Source Code, but it hardly ruined the experience for me. Fast-paced, intelligent and suspenseful, Source Code is one of the better movies to come out this year.
Movie Review: Somewhere [2010]
June 20, 2011 January 6, 2015 1 Comment
Somewhere [2010]
Director: Sofia Coppola
Language: English/Italian
Somewhere opens with a shot of a black Ferrari aimlessly driving around in circles in an open area. This goes on for a few minutes, after which popular actor Johnny Marco (Stephen Dorff) steps out and gazes into the horizon. End scene. This minimalistic shot sets the stage for the rest of the movie, one that most either seem to love or hate, with very few settling somewhere in between.
Johnny Marco is an apathetic actor who shows little emotion to what surrounds his life. Twin strippers, hotel parties, movie press conferences — he seems disillusioned by it all. As he inches through his life, his 11-year-old daughter Cleo (Elle Fanning) is left to live with him for an undetermined amount of time by his ex-wife. This brings a sense of change to Johnny's life, and now he has no choice but to begin to show some signs of responsibility.
Not a lot happens in Somewhere. The movie focuses on Johnny's life outside of the movies and it shows the strain of his relationship with his daughter, but there are no major events that take place. We are merely taken in on a period of this movie star's life, one who is marred with some sort of depression. Director Sofia Coppola lets the film crawl along, often invoking the use of extended scenes, many of which will leave some people scratching their heads in bewilderment. Occasional quirks are brought up but never resolved. I found these traits to be enjoyable and oftentimes humorous, but it's easy to see how they could bother certain audiences.
The best moments in Somewhere happen when both Johnny and Cleo are on screen together. Their relationship is simplistic, but there is noticeable chemistry between the two. This is helped by strong performances from the two leads. Stephen Dorff is effective at showing minimal emotion, and Elle Fanning's acting feels natural and authentic. Since the movie is strictly about these two characters, there aren't many noteworthy roles played by others. A seemingly random selection of Chris Pontius (of Jackass fame) plays Johnny's friend and roommate, Michelle Monaghan plays an actress that he has worked with before, and Ellie Kemper (The Office) briefly escorts Johnny around in Italy.
While Somewhere's slow pace sometimes works against itself, I found the movie to be a very interesting character study overall. It requires a fair amount of patience and the right mindset, but if you are willing to stick with it you should be able to get something out of it. While not up to the level of Lost In Translation, Somewhere is still an involving experience that can be utterly compelling at times.
OK. Time to watch Dazed and Confused for the 109th time. theringer.com/movies/2018/9/… 2 years ago
RT @simpsonsfilms: Phantom Thread (2017) (dir. Paul Thomas Anderson) https://t.co/ksywOBXZaK 2 years ago
RT @drgonzo123: This photo of Toronto is unreal. Looks like Blade Runner. https://t.co/nD5KzD3xut 2 years ago
Initial reaction to Brawl in Cell Block 99: fuuuuuuuuuuuuuuuuuuck! 2 years ago
RT @GenePark: The @StarWars fandom rn https://t.co/FsXMhijw7U 3 years ago
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