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Strengthening our Community Tree Canopy Through Education Module #10: Hazard Tree Evaluation Laura Sanagorski, Environmental Horticulture Extension Faculty
Strengthening Our Community Tree Canopy 2012 Urban Forestry Series This series is a part of a project titled Strengthening Our Community Tree Canopy Through Education. This project is made possible by a grant through the 2011 National Urban and Community Forestry Grant Program and the Florida Department of Agriculture and Consumer Services, and the Florida Forest Service.
Hazard Tree Evaluation
Hazard Tree Assessment: The process of evaluating the likelihood that part or all of a tree will fail (fall) and cause damage and/or injury. 4
Arborists perform tree risk assessments to: Enhance public safety Protect workers on the job site Promote tree longevity by predicting and preventing tree structural failure 5
Risk Assessment Basics 6 Failure potential: The likelihood that an entire tree, or part of a tree, will break and fall within a given period of time. Targets: People or property that could be damaged, injured or killed if the tree fails.
Tree stability may be affected by: Construction, grade changes and trenching Removal of adjacent trees that previously served as a wind buffer Replacement of sidewalks and root loss Failure of nearby trees Change in wind dynamics Storms cause most tree failures Either during the storm, Or after the storm as a direct result of damage that occurred because of the storm. 7
Tree Inspection A Visual Tree Assessment (VTA) involves a thorough, systematic inspection, walking completely around the tree. 8 Is there dieback, gaps or discoloration in the trees crown? Is the tree leaning? Are individual branches extending beyond the rest of the crown? Does the tree have good tree taper? Are there abnormalities in the trunk, root collar or root zone? Photo: UF George Fitzpatrick Photo: UF George Fitzpatrick Visual Tree Assessment
9 Trunk Taper Diameter should decrease as you measure higher up the trunk
10 Co-dominant leaders Included bark; little or no connective wood. Photos: UF George Fitzpatrick
11 Photo: UF George Fitzpatrick
12 Photo: Bill DuMond
Common Defects Associated with Tree Failure Codominant stems Included bark Cracks and splits Multiple attachments Cross branching Girdling roots Wounds Decay Cavities Fungal fruiting bodies Bleeding Loose or cracked bark Nesting holes Deadwood Borers, termites ants Leaning tree Overpruned palms Previous failure 13
Tree Decay 14 Fungal fruiting bodies, mushroom like structures, can indicate significant decay inside the tree. Basal rot can lead to tree failure at the base. Potential Indicators of Decay Cracks Seams Bulges Wounds Photos: UF George Fitzpatrick
Useful tools for tree inspections Air excavation devices Binoculars Listening devices Increment borers Electric drills Resistograph 15
Risk Assessment and Management Requires knowledge of both tree biology and structure-function relations in trees All observations, measurements and recommendations should be documented Numeric formulas are available (i.e., Matheny and Clark). 16
Two-page form recommended by the ISA for Tree Hazard Evaluation 17
Hazard Rating FAILURE POTENTIAL (1-4) + SIZE OF DEFECTIVE PART (1-4) + TARGET RATING (0-4) ___________________________________________________________________________
Hazard Rating – Failure Potential 1 = low 2 = medium 3 = high 4 = severe
Hazard Rating – Size of Defective Part 1 = less than 6 inches 2 = 6 to 18 inches 3 = 18 to 30 inches 4 = greater than 30 inches
Hazard Rating – Target Rating 0 = No target 1 = occasional use 2 = intermittent use 3 = frequent use 4 = constant use
Hazard Tree Case Studies 23
The tree in question is a Bael Fruit(Aegle marmelos), that is located in a botanical garden adjacent to a walkway at the main entrance, where a steady flow of pedestrian traffic occurs. The tree possesses numerous codominant trunks with bark inclusion and also has visual signs of decay and cracks at the areas of bark inclusion. Additionally, pruning cuts were made in the past that have not compartmentalized. Beneath one of the codominant trunks is a bench that could be considered a potential target. Furthermore, a hanging basket with a staghorn fern is situated on this particular codominant trunk. Additionally, visitors to the garden should also be considered targets. This tree could potentially experience branch failure from any one of the codominant trunks, especially the one with the excess weight of the fern. Upon inspection, it appeared that this particular 10-12 branch was most likely to fail As such, the trees hazard rating would be: Failure potential:3 Size of defective part:2 Target rating:3 Hazard rating: 8 Case Study 1 24
Failure potential: 3 Size of defective part: 2 Target rating: 3 Hazard rating: 8 Case Study 1 25
This large Live Oak (Quercus virginiana), is approximately 38-40 years old and is 51.5 DBH (diameter at breast height). It is located adjacent to a walkway that leads from a parking lot to the main entrance of a building and receives a fair amount of regular pedestrian traffic. The tree has numerous codominant trunks that are roughly the same diameter and originate low on the tree. There is also bark inclusion present and cracks are forming where the bark is trapped between the trunks. The narrow branch angles on the tree appear to be retaining moisture and could possibly be harboring decay. A number of branches have excessive end weight and there is evidence that a number of pruning cuts and wounds have failed to seal over. Given these defects, this tree has potential of failure from one of the large codominant trunks. Upon inspection, it was determined that a branch with a diameter of approximately 22-24 posed the greatest threat for failure, though, while highly possible, it is not necessarily imminent. As such, the trees hazard rating would be: Failure potential:3 Size of defective part:3 Target rating:3* Hazard rating: 9 * - Picnic tables formally underneath the oak were removed to reduce the target rating, bringing it from a 4 to a 3. Case Study 2 26
Failure potential: 3 Size of defective part: 3 Target rating: 3 Hazard rating: 9 Case Study 2 27
Mitigation Options 28
Mitigation: The process of reducing risk potential. May include: Removing the target Fencing off the area Removal of dead and broken branches Installation of bracing or cabling Lightning protection systems 29
Liability and Negligence Arborists have a duty of care and are held to higher standards because of their higher level of knowledge and experience. 30 Negligence is the failure to exercise due care. Causation in fact means that the damage, injury or death can be traced back to a persons action (or inaction). Act of God is an occurrence due to natural causes that could not have been prevented by ordinary skill and foresight. All landscape trees pose a risk and some trees pose more risk than others. The property owner must decide what level of risk is acceptable. The arborist can assess risk and make recommendations.
Strengthening our Community Tree Canopy Through Education Module #10: Hazard Tree Evaluation Laura Sanagorski, Environmental Horticulture Extension Faculty
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COPENHAGEN STREET STYLE!February 6, 2013
Those Copenhagen cats are at it again, why are they so cool and trendy? It must be something in the water, because we got some awesome snaps of this stylish bunch whilst they shopped around UO during Copenhagen fashion week.
Photographer and UO lady Bettina picked up her camera during the week long fashion parade, unable to dismiss any opportunity to grab some shots of pretty people in handsome clothes. The best shots made their way onto the UO Copenhagen street style wall…
We managed to grab a few fleeting words with Bettina…
What were you looking for during the fashion week?
There were three types of people I took photos of, the street fashion nerds, those who asked to get their picture taken, and the smiling ones! Personally I enjoyed the last category the most. I was very surprised by the response from those that I asked, they all said yes before I even had the chance to explain to them WHY I wanted to take some pictures. It was fun and highly appreciated from both sides of the camera.
And what about the fashion?
A trend that i love love love this season is the 90s look of wollen hats, a East 17-style mixed with Fresh Prince patterns for both girls and boys. That can't go wrong.
We couldn't agree more! Here's some of favourite fashion people, you can check out the rest over on the UO Pinterest board.
What's your favourite Copenhagen style? Let us know in the comments below…


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Lazy rule: can't reach it, Don't need it
If anyone can have it I don't have it
If monday had a face
im not lazy
I tried being normal once
Didn't loose my sanity
bad decisions make great stories
I can't hear you
Where's the food
Hashtag Blessed
hungover or monday
I need a six month vacation twice a year
I'm not afraid of heights have you seen my shoes?
So Quiche
I enjoy long romantic walks to the fridge
Everything I like is either expensive, fattening...
I can't concentrate in flats!
Do not read the next sentence
do not read the next sentence


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Posted by ethan on 2/11/2017, 10:26 am, in reply to "is ur a fag?"107.167.116.x
ha ha #### u u lik the dick in ur mouth dont u, fag ah deeuuuurrrrgggghhhhhhh!
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The CNA Executive Council
The Crestview Neighborhood Association has 5 named officer positions, along with 4 advisory positions. All positions are voluntary and are drawn from residents of the Crestview neighborhood. Elections are held in May – if you would like to serve send a message {email address} To serve as President, one must have already served at least 1 year as an officer/advisor on the Executive Council.
President (term ends 5/2017)
Dave Considine
Vice President (next elected 5/2018)
Mike Lavigne
Secretary (term ends 5/2018)
Nancy Mohn Barnard
Treasurer (term ends 5/2017)
Ali Thompson
Newsletter Editor (term ends 5/2018)
Chip Harris
Advisors (terms ends 5/2017)
Place 1: Elaine Stegant
Place 2: Anne-Charlotte Patterson
Place 3: Julie Reichart
Place 4: Roland Rodriguez


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Design a Logo For Luxury Fashion Website
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Project Budget$30 - $250 USD
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Need a new logo designed for Luxury Fashion Website. Competition is sites like stylelend, rentezvous.
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Advertising Policy
The following guidelines have been established by Sojern to govern various aspects of Advertising. For these purposes, "Advertising and Advertisements" include banner, button, and contextual Advertising, Sponsorship Messages, and Promotions. These regulations govern issues such as acceptance of Advertisements and Promotions by Sojern, the manner of display of Advertisements and Promotions on the Sojern Site(s), and the removal of Advertisements and Promotions from the Sojern Site(s). Sojern has sole and absolute discretion with respect to interpretation and enforcement of this policy and all other issues associated with Advertising and Promotion on the Sojern Site(s). Sojern may change this policy at any time in its sole discretion by posting a revised policy to the Sojern Site(s).
Sojern has sole discretion for determining the types of Advertising and Promotion that will be accepted and displayed on the Sojern Site(s), and under no circumstances shall Sojern's acceptance of any Advertisement be considered an endorsement of the product(s) and/or service(s) advertised or for the company that manufactures, distributes, or promotes such product(s) or service(s).
Sojern will not accept Advertising that, in Sojern's opinion, is not factually accurate and in good taste. Sojern will not permit at any time the placement of any Advertising for illegal or objectionable products. Advertising must not contain fraudulent, deceptive, or offensive material, including material that misrepresents, ridicules, or attacks an individual or group on the basis of age, color, national origin, race, religion, sex, sexual orientation, handicap and political or religious affiliation.
Advertising cannot resemble Airline website, or solicit or collect, or appear to solicit or collect, any personally identifiable information from visitors without their knowledge.
Advertising must not be related to any of the following: alcohol, firearms, ammunition, fireworks, gambling, pornography, tobacco, airlines, political or religion. Advertisements cannot mimic news headlines in design, tone, third person sentence structure or topic.
Advertisers must only offer products or services which are readily available for purchase at the advertised price (with noted tax and delivery fees). Advertiser must fulfill the advertised offer as stated in the advertisement, including without limitation, not altering any offer periods, product offers or prices that are stated in the advertisement unless Advertiser has obtained prior approval. The Advertising must also clearly identify the advertiser. Any Advertising that could be misconstrued as editorial content will be clearly labeled as Advertising.
Sojern recognizes and maintains a distinct separation between Advertising content and editorial and decision making content. All Advertising content on the Sojern Site(s) shall be clearly and unambiguously identified as such, and Sojern will not run any Advertising on the Sojern Site(s) which is not so identified. Additionally, Sojern retains the right to dictate the form and substance of all editorial content appearing on the Sojern Site(s).
Sojern reserves the right to reject, cancel, or remove at any time any Advertising from the Sojern Site(s) for any reason, where the Advertisement Materials or the site to which the Ad is linked. Sojern will provide prompt notice to the advertiser upon rejection, cancellation, or removal of any Advertising, together with an explanation following the rejection, cancellation, or removal. Sojern also reserves the right to determine the appropriate placement of the Advertising on the Sojern Site(s).
It is the responsibility of the Advertiser to comply with all applicable domestic and foreign laws, including applicable laws and regulations of regulatory bodies. Sojern will not monitor compliance with applicable laws and regulations. Sojern reserves the right to review all Advertising for compliance with applicable laws and regulations and, if Sojern becomes aware of any breach or potential breach of any applicable law or regulation or of these guidelines, Sojern may remove the Advertising.
No Advertising shall be permitted which may injure the good name or reputation of Sojern, Sojern Site(s), partners or affiliates.


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Bars have always seemed safe, happy, warm places to me, probably because several beloved family members have worked in them over the years*. I know many women feel uncomfortable in a pub, especially alone, but it's the first place I go to regroup when I alight from the bus.
Consequently, I have run across a strange and motley array of aspiring pick-up artists and other social vultures. You sit, alone with your book and your whiskey, enjoying the pleasant hum of noise around you, perhaps making small talk with your bartender, then BAM. He's at your elbow, asking what you're reading in that slightly-too-aggressive tone, or wondering why a pretty girl couldn't find anyone to go out with her.
The "neg," the back-handed compliment designed to disorient and weaken self-confidence, seems the favored opener for men who operate in a predatory way. Its popularity may result from its romanticization on the page and silver screen, or from its resemblance to the genuinely pleasant banter that many people enjoy.
Most people, though, defuse the potentially aggressive connotations of banter either by limiting it to well-established friendships and flirtations, or by keeping it gentle and light at the beginning. A well-intentioned charmer may open by banter, but he won't touch on any personal subjects, he'll shy away from backhanded compliments, he'll look for reciprocity, and he'll accompany his remarks with lots of personal space and a big smile.
In other words, the charmer is openly teasing, and trying to signal non-aggression in the process. The neg masquerades as compliment, but in such a way that you'll be knocked off balance, doubting your social perception or wavering in self-confidence.
The neg also works as a verbal breach of propriety, a violation of verbal boundaries to lay the groundwork for the violation of physical or spatial ones. Most well-socialized people, for instance, do not criticize the personal appearances or clothing choices of complete strangers. This is weird and intrusive. A pick-up artist might, though, and by framing it as a compliment or positive attention, tries to trick you into accepting it.
The most important step to dealing with these pests is going with your gut, because your gut is precisely what the PUA is trying to alienate you from. If you feel something is creepy or sketchy or just….something, it probably is. If you are getting an adrenaline rush, and you're not aroused, your body probably thinks you're in danger. On the other hand, if you're feeling happy, relaxed, and open, if you feel in control of the situation, if you want to keep talking to this guy, go with it until something changes.
That second point is as important as the first, and less touted. If your response to all male attention is dictated by anxiety, you're going to have no frame of reference with which to compare genuinely alarm-triggering interactions. Rules-based safety advice (don't go to bars, if you must go to bars don't drink too much, don't flirt, avoid men of this race or this class) harms women by shifting their attention from the validity and importance of their own instincts and desires to an abstract behavioral checklist.
So, the guy's neg worked, and now you're feeling disoriented and uncomfortable. Say so.
"Wow, what a personal remark."
"Do you think being rude is going to help make my acquaintance?"
"I really don't know how to reply to that."
"That made me feel uncomfortable."
Bland bluntness is good. Do you remember A Wrinkle in Time, how Meg resists It by reciting the periodic table? Or in the Silver Chair, how Puddleglum thrusts his foot in the fire to break the spell of the witch's voice? The principle is the same here.
The pick-up artist wants to draw you into a little world of his own creating, just for the two of you, where he gets to determine all the boundaries and all the norms. Maybe he's trying to cast you in a role–if you're bookish or noticeably religious, you're the shy ingenue, he's the sophisticate who's going to seduce and liberate you in spite of yourself.
By reacting with mild, disdainful surprise, you are drawing the curtain on Oz the Great and Powerful. You are resisting his attempts to immerse you both in a smoke-world where it's sexy and exciting to be put down by strange men. You belong to the ordinary world, where people are polite to strangers, his behavior is bizarre, and his pathetic scripts and roles have nothing to with you and your life. You are Puddleglum, and you remember the sun.
If someone violates your personal space, same unapologetic vocalization of your desires: "Can you please give me a little more space? I feel uncomfortable with your hand on my back."
Of course sometimes we want to be touched, because we're enjoying the flirtation and want it to progress. Humans are pretty good about signalling their receptiveness to mild levels of contact; the honest flirt will be paying attention to your signals and trying to figure out if you would appreciate a light touch on the arm. The pick-up artist is trying to move the flirtation along unilaterally–he wants to make you accept his touch, thereby convincing you that you wanted it, rather than the other way around. There's no clear-cut way to differentiate the two in the moment, so again, pay attention to your desires and listen to your gut.
If he backs off or apologizes when you call attention to a crossed line, then he's probably more socially inept than predatory. Manipulators will try to punish you for enforcing basic social boundaries; they might call you stuck up, touchy, a princess; they might make a great performance of offense and hurt; they might try a sad sack I-should-have-known-you'd-never-talk-to-a-guy-like-me. They might argue with you about the legitimacy of your reaction, or try to re-eroticize the conversation by calling you "feisty."
Engaging their arguments or outrage won't work, and moreover, why should you? You are not Fanny Price, whose every free moment belongs to the nearest asshole in her proximity. You are at your leisure, entirely the mistress of your own time. When you walk into that bar, you're Emma Woodhouse, and you owe no one anything**.
So when he dials up the manipulation, you don't have to prove your right to end the conversation. Just assert. Repeat yourself, because you were perfectly understandable the first time, and why waste brainpower on this fool?
"I did not like what you said to me. It was intrusive."
"I'm not enjoying this, and this conversation is over now, thanks."
"I'm going back to my book/drink/people-watching now. Have a good night."
Declarative, polite rejection is death to PUAs, because their schtick depends on smoke, mirrors, and the creation of insecurity. At this point, most will leave with an eyeroll, huff, or muttered "bitch." Some, however, will stay to wheedle, verbally abuse, or physically intimidate. Now all civility is at an end. Say loudly, calmly, and firmly that you want him to leave, that you have asked him to leave, that he is making you feel unsafe. If people hear you, so much the better. Arguing with him, reasoning with him, trying to win him over will all be totally fruitless. The only thing you can do is reject his White Queen logic, and make him look like an asshole in the normal world to which you have asserted your allegiance.
Don't be afraid to get the bartenders involved. Most of them, especially in quiet local Irish pubs, have absolutely no interest in anyone harassing their female patrons***. All you have to do is catch his eye, and quietly tell him that you've unsuccessfully asked this this gentleman to leave you alone.
If you've got the righteous wrath in you for something like this, go ahead. They may escalate more quickly to verbal abuse; but I've reacted this way once, having a drink after twelve hours waiting tables, where accommodating entitled asshats is part of the job. I think it was the quiet, near-unhinged fury in my voice that made him reconsider.
Eventually, if you can avoid ensnarement in his illusory world, the urge to dominate won't be worth the social cost and negative attention for him. He won't leave gracefully, but he will leave, at which point I suggest you reward yourself for your dragon slaying with a quick trip to the jukebox.
*Get me to tell you about the time my dad had Chase Utley as a barback.
**Except the bartender. You owe him a decent tip.
***And if you see someone being harassed, support them. Move physically close to them and make it clear you're listening. Ask the harassee if she wants you to get security, if he's bothering her, if she wants to come sit by you. It doesn't matter if you're big or strong. The main point is signalling that a) you are watching closely, and b) you do not think this behavior is ok.
Captain Awkward is the queen of boundaries.


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*Teaser* Chapter 1 Revision
The sign on the Kame Game Shop said close, as Yuugi reached the door. So he thought nothing of the silence as he made his way into the part of the shop that was the house. Yuugi figured that his Yami, Atem, would be out with the gang.
Now, this bears the question, why was Yuugi not with them as well. To claim it was complicated would be an understatement.
It all began after Atem and the other Yamis received their own bodies. At first all was well, but as time went on things began to change. The others began to get wrapped up in each other, school things like preparing to go to university, and just their own lives in general. This being said some of them went back to old habits without really realizing it, and others developed new habits, and not necessarily good ones.
*Teaser End*
The rest of the Chapter will hopefully be ready soon. I have about 2 pages worth written and am expecting at least another two before its done.
Sorry everybody, this is mostly an author's note. Although, hopefully it will be a helpful/ reassuring one. ^_^
I am really sorry for falling off the grid like I did; it started with writer's block, which was followed by losing the flash drive that had my most recent chapter(s) that I had not written down, and this was soon followed by getting caught up in school, work and life in general.
I'm looking to not only start writing for this story again but also to revise the previous chapters as well. I've already done a couple things to the first chapter, though I am playing with the idea of taking it down and redoing it (almost) completely.
I've also been looking through past reviews, and I think I should answer some of the questions that were asked.
Yes I plan on Yugi being a ninja, though I believe it would suite him best to be a Medic-nin.
I know that I did not really explain the whole 'friends forget Yugi' thing, but I really didn't think my approach through very well at the time. I was still in the phase of 'if it is obvious to me then it is obvious to everyone else too,' and thus did not want to bore people with a detailed explanation.
I do plan on going back and fixing my spelling errors, such as Kankuro and not Kokoro.
I started this story as someone that was new to writing for someone other than a teacher (really I still am) and I honestly did not really plan my chapters thoroughly like I should have. I am considering a Beta, but I'm still not completely sure how that works.
Last thing, I want to be clear on one thing though, stemming from the problem I had over a Flame. I am not against well thought out, constructive criticism. But that does not entail insulting me as not only a writer (especially after I said myself that the story was probably going to suck), but as a person as well. Those types of reviews do not help, it doesn't matter who they are from. If I feel that the review is more of a Flame the person's comments will be dismissed and they will be blocked.
Thank You all for your support and patience! I hope that I can remake this story to be better and that you guys enjoy it!
Oh and before I forget! I am also working on a completely new story, it is a Harry Potter/Ouran crossover, and it will also be a Mori/Harry pairing as well. The first chapter is almost finished, it just needs to be typed. Hope you all will enjoy it!
Yours truly,
Shiroiro tora


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Role Of Polymer Architecture On Polycation Biology Essay
Published: Last Edited:
This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
Polymeric cations are widely used as nonviral transfection agents. A fundamental understanding of the structure-internalization relationship that underlie the cytotoxicity of these molecules, however, is still lacking to date and mostly based on empirical observations. We have assessed the cytotoxicity profile of a polymer library synthesized from (built from?) L-lysine monomer units containing 17 members in order to systematically investigate the influence of all the structural parameters on cellular toxicity. The library contained linear, hyperbranched and dendritic L-lysine analogues in a broad range of molecular weights 400-140 000 g/mol. Hyperbranched poly-L-lysine, which is newly designed alternative to polyethylenimine in gene delivery, was compared to linear and dendritic poly-L-lysine for its toxicity. We investigated the effect of the molecular weight (Mn), degree of branching and polydispersity on the mechanism of short and long term cell toxicity in vitro in two different cell lines. The mechanisms underlying cell death at various stages of cell exposure to polycation were identified. Acute cell death was triggered via lethal membrane injury and increased osmotic pressure. In contrast delayed cell death was related to the intracellular accumulation of the polycation resulting eventually to mitochondrial collapse. The onset and extent of these specific modes of cell death were shown to be dependent on the molecular weight and degree of branching of the poly-L-lysine analogues. The number of cells displaying signs of early apoptosis was related to the degree of branching and molecular weight in similar fashion as for the acute cell death. Most importantly the novel L-lysine analogue, hyperbranched poly-L-lysine, elicits the same stress induced response in cells as its structural analogue dendritic poly-L-lysine of comparable molecular weight and polyethyleneimine.
Keywords: linear poly-L-lysine, L-lysine dendrimer, hyperbranched poly-L-lysine, degree of branching, apoptosis, toxicity, acute cell death.
Increasing number of studies (investigations) focuses on the application of newly designed polycations for delivery of nucleic acids for transient gene expression. Polyethylenimine (PEI), linear Poly-L-lysine (LPL) or polyamidoamine (PAMAM) represent examples of widely used polycations in biotechnological or medicinal research for the gene delivery to mammalian cells in vitro and in vivo.1 The native, unmodified form of these polymers is directly investigated for this purpose, however structural derivatization of PAMAM, PEI and LPL lead in many cases to polycations with improved properties like increased degradability2, enhanced transfection efficiency3 or targetability4. These results provide motivation to understand the effect of the polymer architecture on the basic biological properties and to advance the rational design and synthesis of more efficient carriers. The most important criteria in focus for gene delivery are delivery efficiency5, internalization properties6-8, proper intracellular trafficking9, 10 and toxicity/degradability11, 12. These properties are directly determined by structural characteristics of the polymer and can be fine-tuned for example by modulating the polymer sequence, its final molecular weight or by optimizing the (co)monomer(s) type13, 14.
The polycation induces in mammalian cells two types of stress related responses which are separated in time - immediate and delayed cell death. These two mechanisms induce distinct morphological and molecular changes in the cells that can be differentiated.
The relationship between immediate toxicity and molecular weight of a polycation was documented in the past and it was demonstrated that the extent of the induced acute cell death is clearly concentration and molecular weight dependent for polycations.15 The positive charge of the polycations is effective in disrupting plasma membranes in living cells or model membranes16-18. Membrane lysis induced by electrostatic interactions thus has been proposed as the main cause of immediate toxicity induced by polycations. This observation correlates with the extensive hemolysis of red blood cells or lactate dehydrogenase release in vitro caused by polycations19, 11. Suppression of the lytic activity of polycations can be achieved by end group modification aimed to reduce the overall charge density. In this perspective PEGylation as well as esterification with acetic acid or long chain fatty acids lead to 10 fold increase in cell viability (biocompatibility). 20 21, 22
Long term impact of the polycation exposure to the cells has been recently analyzed in vitro 23-26. These studies demonstrated that at delayed phases after the polycation treatment the delayed cell death program in the cells is lanced via a cascade of predefined events. It has been proposed that once internalized into the cells polycations may elicit the apoptotic response by distinct yet simultaneous mechanisms27 and that, based on their structure polymers have variation in their mechanism of cell death24. In general polycations have been shown to induce apoptosis through alteration of mitochondrial activity, where depolarization of mitochondrial membrane potential and Cyt C release occurs with activation of caspase-9 and caspase-3 27, 28. However, most of the newly designed and synthesized polycations are only very rarely tested for their pro-apoptotic activity; consequently their long term cell compatibility is generally unknown. Similarly systematic studies on the apoptosis as a function of polymer architecture are missing. Apoptosis, as a long term mode of cell death, has been investigated to this time for poly-L-lysine 24 PAMAM dendrimer 29 and linear or branched PEI 23, 27, however these studies did not directly focus on the concrete role of the architecture of the given polycation.
In this manuscript we present a detailed investigation focusing on the cytotoxicity profile of a polymer library based on a single trifunctional monomer unit L-lyisine. Our library consists of linear- (LPL), dendritic- (DPL) and hyperbranched poly-L-lysine (HBPL) polymers of varying size, and contains altogether 17 members. LPL and DPL are monodisperse and well characterized polymers and widely studied as vehicles for drug and gene delivery applications .HBPL, a recently developed polycation, is a branched analogue of linear poly-L-lysine, that can be cost effective synthesized on a large scale via a one step polycondensation process30, 31. HBPL is a promising candidate for applications in gene delivery (ZK manuscript in preparation). Due to its efficiency in gene delivery, low production costs, and improved biodegradability, HBPL may be especially suitable for in-vivo applications and large scale production of recombinant proteins via transient gene expression. Thus the additional aim of this study was to characterize the cytotoxic properties of HBPL in comparison to LPL and DPL. For this purpose we used industrially relevant cell lines CHO DG4432. Our systematic investigation of the influence of degree of branching, molecular weight and architecture of polycation on the execution and extent of apoptotis may be of help to further optimize the design of the polycation vectors used for large scale gene delivery purposes.
Experimental Section
Supporting Information
See the Supporting Information for Techniques section
Methods-Synthetic procedures
See the Supporting Information for Methods-Synthetic procedures section
Cell culture
CHO DG44. Suspension cultures of CHO DG44 cells were routinely maintained in square-shaped glass flasks (250mL) in serum free ProCHO5 medium (Lonza AG, Verviers, Belgium) supplemented with 13.6 mg/l hypoxanthine, 3.84 mg/l thymidine, and 4 mM glutamine (SAFC Biosciences, St. Louis, MO) as described by Muller et al 33. Cultures were maintained according to standard protocols at 37 °C in a 95% humidified incubator with 5% CO2.
Fluorescence Microscopy. Zeiss Axiovert 200M inverted microscope was used for fluorescence light microscopy, equipped with Zeiss AxioCam MRm camera. Images were analyzed with Axiovision software.
Flow cytometry. Flow cytometric analysis was performed using a Cyan ADP (Beckman Coulter, Fullerton, CA) equipped with three lasers (405nm / 488nm / 635nm) using Summit 4.2 software (BeckmanCoulter, Fullerton, CA). Data were further processed with Flow Jo Software.
Osmolarity measurement. The values of osmolarity were measured with a freezing point osmometer Multi Osmette Osmometer (Precision SystemsInc, Natick, MA) calibrated with a range of standards 100, 500, 1000, 1500 and 2000 mOsm/kg. Each sample was measured in three separate experiments with three separate solution preparation.
In vitro assessment of acute cytotoxicity. In vitro assay applying Viacount Reagent (Guava Technologies) was performed as following. For typical cell sample at a concentration of 2*106 CHO cells per mL 20uL of cell suspension was mixed with 180 uL Viacount Reagent in a 5mL round bottom polypropylene tube. Cells were resuspended and incubated for 5 minutes. The total volume was diluted with 1mL PBS. Samples were filtered prior the flow cytometry analysis. Experiment was carried out in three independent replicate. All polymer samples were tested in the concentration range of 10-2 M to 10-10M,
Membrane permeabilization assessment. Cells were treated with the polycation with defined range of concentration for 1hour. Cells were afterwards washed with PBS and centrifuged at low speed. Dextran-rhodamine solution in PBS was added to the cells and incubated for 15 min at 4°C with shaking. Cells with permeabilized cell membrane presented diffusive cytoplasmic staining of cytosol. Experiments were performed in triplicate. Standard errors were within 15 % of the mean values.
Mitochondrial Membrane Potential Measurement. The change in the mitochondrial membrane potential (ψm) was analyzed by fluorescence wide field microscopy and flow cytometry using the ψm-sensitive dye 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Invitrogen AG, Basel, Switzerland). Briefly, cells were treated with polycation for up to 4 hours. Cell aliquots harvested at defined time point (0.5h, 1h, 2h, and 4h), were extensively washed in PBS and resuspended in culture medium containing 1 mM JC-1. Followed incubation at 37 °C for 20min, cells were washed with PBS and analyzed by wide field microscopy and flow cytometry. The emission maxima of JC-1 monomers and aggregates are 527 and 590 nm respectively. . Camptothecin at a concentration of 10 μM was used as a positive control for this experiment.
Annexin V-FITC/PI assay for apoptosis. Apoptotic cells and dead cells were detected by Annexin-V binding and nuclear PI incorporation, respectively. Approximately 2*105 cells were sampled and washed in cold PBS. Binding buffer was prepared beforehand (Hepes buffer: 10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2). FITC-Annexin V was diluted at a concentration of 1 mg/mL in binding buffer and cells were resuspended in this solution. 0.1 mL of PI solution was added (final concentration,3 μM) and incubated in the dark at room temperature. Samples were diluted with PBS to the final volume of 2 mL and quantification of the apoptotic / dead cell population was carried out with flow cytometry.
Immunocytochemistry - Detection of caspase 3, caspasae 9, cytochrome C. To evaluate the presence of caspase 3, caspase 9, and cytochrome C within the cells immunocytochemistry analysis were performed on PC12 cells. Morphological features were examined by growing them on 35 mm round coverlips. Before treatment, cells were seeded into 6-weel plates with coverslips inside, at density of 1.5X105/well, left overnight to attach to the substratum and to acquire the normal flattened morphology, and finally challenged with veichles (they were treated for 12h with different polymers). After the treatment period cells were washed twice with 1X PBS and then fixed with 4% paraformaldehyde for 30 min on ice. Fixed cells were washed again with 1X PBS and permeabilized with 1X PBS containing 0.1% Tween, for 15 minutes at room temperature. Non-specific binding was blocked by 30 minutes incubation at 37°C with PBS containing 5% secondary antibody serum (goat for caspases 3 and 9, rabbit for cytochrome C). For proteins visualization coverslips were stained with specific primary antibody (from Sigma-Aldrich, Milan, Italy) 1:200 in 1X PBS containing 1% secondary serum overnight at 4°. Then they were washed three times with 1X PBS and subsequently incubated with secondary antibody FITC conjugated (from Sigma-Aldrich, Milan, Italy) 1:500 in 1X PBS, for 45 minutes in the dark at room temperature. Cells were washed three times with 1X PBS and subsequently were incubated for 20 minutes in the dark at room temperature with 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma), which selectively stains DNA and allows for the evaluation of nuclear morphology. Slides were mounted with DABCO glycerol. Cells were examined under a fluorescent microscope equipped with the appropriate filters (Nikon Italy).
Results and Discussion
Generation of a polymer library base on L-Lysine monomers
In order to determine how the architecture of polymers may contribute to their potential cytotoxycity we have synthesized a polymer library based on L-lysine. Our library consisted of three structural types of polymers, i.e. linear, branched, and dendrimeric (Fig. 1), and contained samples with varying molecular weight, degree of branching and polydispersity. The type of poly-Lysine counterion, was homologous for all the samples, i.e. HCl, in order to limit the crosstalk between additional parameter 34, other than the Mn, DB and PDI. The final aim is evaluation of the role of the polymer architecture on cell cytotoxicity we initially characterized all the synthesized polymers for their degree of branching, molecular weight, polydispersity, number of accessible amino groups and buffering capacity.
Insert figure 1 here
Synthesis of linear Poly-L-lysine. Linear poly-L-lysine was synthesized via polymerization of N-carboxy anhydride monomer. This strategy allowed us to successfully synthesize linear poly-L-lysine molecules with a molecular weight of up to 20 000 g/mol (Table 1). For the different analogues, the theoretical molecular weight, determined by the ratio of initiator to monomer was in agreement with the values obtained by NMR analysis. On the other hand GPC measurements (Figure S4) lead to a certain overestimation of the molecular weight. GPC analysis also revealed a relatively low polydispersity for all analyzed samples.
Insert table 1 here
Synthesis of dendrimer of L-lysine. Dendrimers were synthesized up to the 6th generation via standard peptide coupling strategy, employing HOBT and HBTU as coupling agents (Table 2). The structure of dendrimers was verified by 1H NMR (Figure 1), integrals of the structural unit correspond with theoretical values and the deviation does not exceed 8 % (table S1). Dendrimer displayed relatively low polydispersity as documented by GPC chromatograms (Figure S3). However, further MS (ES+) analysis revealed a certain heterogeneity especially for those dendrimers of generation higher than the forth. The divergent strategy used here for the construction of the dendrimers is prone to induce structural errors, mainly due to incomplete coupling occurring during the sequential steps of polymerization. Similar types of irregularities were previously reported for the synthesis of poly (propyleneimine) and PAMAM dendrimers35.
Insert table 2 here.
Synthesis of hyperbranched poly-L-lysine. Polycondensation of the AB2 monomer L-lysine, are known to lead to inherently heterogenous structures with relatively high polydispersity. In addition, the thermal degradation of the monomer units or of the growing polyamide chain can generate additional structural imperfections 36. We attempted to limit these side reactions and thus reduce heterogeneity, by carrying out the polymerization reaction under a continuous stream of nitrogen. When determined experimentally by ???,compounds obtained that way, exhibited a slightly lower number of free amino groups than the theoretically predicted value. In order to distinguish and to understand the influence of the structural irregularities of HBPL on the toxicity, all the in vitro studies were carried out simultaneously with structurally faultless analogues LPL and DPL.
As evident from the table 3 the polycondensation and subsequent fractionation, leads to multibranched peptidic structure of very high molecular weight. Contrary to the conventional peptide coupling strategy or NCA polymerization used for LPL and D respectively, polycondensation is easily scalable and cost effective synthesis method. Furthermore, the reaction conditions are free of any potentially toxic catalyst or reagent making the purification procedure extremely easy.
Insert table 3 here
Acute toxicity has been traditionally defined as the ability of a substance to cause severe biological harm, or death, upon short time or low doses exposure (Hagan, 1959). In our studies we determined acute toxicity induced by the different polymers belonging to our library, by quantifying cell viability after cell exposure to polymers for a time period of 1 hour. In order to determine the half maximal effective concentration (EC50) values, relative to the different polymers, we tested increasing concentrations of the single compounds, ranging from 10e-8 M up to 10e-1 M. Our data show a direct correlation between EC50 values and the size and structure of the different polymers (Fig. 2). In particular we observed a direct correlation between enhanced cytotoxycity and increasing molecular weight, independently from the architecture of the compounds. Moreover at similar molecular weights the EC50 values for the branched poly-L-lysine forms (i.e. Dendrimer and HBPL) were up to xy fold lower than those measured for the linear compounds, suggesting a toxic effect induced by the free xy groups present in the branched polymers. . The EC50 values measured for the L-lysine dendrimers ranged from 0.009+/- XY to 7x10e-5 +/- XY M, for molecular weights of 900 to 20000 g/mol respectively. These data are in agreement with previous studies, where the MTT assay was used to determine dendrimer cytotoxicity on various cell types.7, 37, 38. Likewise the observed evolution of the toxicity of L-lysine dendrimer with increasing generation type was similar to the one reported for the dendritic PAMAM polymer 8. At equivalent sizes dendritic and hyperbranched poly-L-lysine induced acute in vitro toxicity at similar levels, especially for polymer sizes ranging from 3000 up to 20000 g/mol. (25 kDa) PEI is one of the most effective vectors for gene delivery, consequently it is extensively used in both biotechnology and gene therapy research (add ref). Interestingly despite both HBPL and dendrimer polymer types, were overall showing the highest cell toxicity, their EC50 values were only slightly lower than the one measured for PEI when size analogous were tested. It should also be noticed that whereas the PEI sample was expected to have a Mn of 25'000 g/mol as suggested by the supplier, its molecular weight assessed by GPC (?), was only of 10'000 g/mol.
. The slope of the function f(Mn HBPL)= EC50 decreases for high molecular weight samples and the gradual change of EC 50 with Mn is slower than for PL and dendrimer. This observation indicates either of the role of polydispersity and structural imperfections of hyperbranched poly-L-lysine or of aggregation of high Mn samples of HBPL due to lower solubility.
The impact of high polydispersity on EC 50 value was confirmed by measuring the EC 50 value of crude (yet dialyzed) HBPL 7. This sample of nonfractionated HBPL contains contaminants of very high molecular weight. These "impurities" are extremely effective in triggering acute cell death at low doses. Indeed, the EC 50 value of HBPL 7 is approximately equal to the EC 50 value of high moleculer weight HBPL 3 or the EC 50 of the 6th generation dendrimer. The variability of the EC 50 value of this sample was notably high, although the testing conditions were identical as for all the previous samples. We observed that linear poly-L-lysine displayed the lowest toxicity of all the tested analogues.
Insert Figure 2 here
We further investigated the potential causes of the immediate cell death observed upon polymer exposure. Osmolarity is one of the most important variables affecting the cell viability, indeed increased osmolarity of the extracellular fluid has been proposed among the possible causes of immediate loss of cell viability 39-41. In their protonated form, polycations can contribute to the osmotic stress by every single charged amino group and as well by the corresponding chloride counterion and thus exerting a cytotoxic effect by increasing the osmolarity of the cell culture medium.., Optimal osmotic values of cell culturing media lie in the range of 280-320 mOsm/kg11, and changes in morphology and cell viability can be already observed at an osmotic pressure of 450 mOsm/kg for suspension cell culture39. In order to investigate cytotoxic effect due to changes in osmolarity, the osmotic pressure of cell culturing medium supplemented with the tested polycations at concentrations corresponding to their EC50 values was measured (Table S2). Interestingly the low molecular weight polycations LPL 1 and HBPL 1 showed the highest osmotic values of 630 and 570 mOsm/kg, respectively, whereas In a model experiment aimed to test whether these osmotic values are indeed detrimental to cells, cell populations were incubated in a NaCl solution having comparable osmolarity (Table S2). This abrupt change of the osmotic pressure, induced necrosis of cells as evidenced by trypan blue inclusion in to the cytoplasm of the cells (Figure S5-3B). These data suggests that the cytotoxicity observed for low weight polycations may directly result from changes in osmolarity of the polymer solution compared to cell culture medium. On the other hand, when high Mn polycations were added to cell cultures at their EC50 concentrations, only a limited increase of the osmotic pressure was observed (Table S2). Furthermore such levels of osmolarity were not detrimental to cells as verified by equi-osmotic NaCl solutions (Figure S5-2A and 2B). We can thus suggest that for high molecular weight polycations osmolarity may not be the predominant factor inducing cell death.
Apart from the hypertonicity, an additional event that may contribute to early cell death upon exposure to polycations, is the potential ability of the polycation itself to promote cell membrane pore formation42-44. This process has been evidenced and analyzed experimentally in both, model biomembranes and in living cells (add ref). The ability of the polycation to disrupt cell membranes is commonly followed by the spectrophotometrical quantification of lactate dehydrogenase release (add ref). Alternatively, plasma membrane permeability can be evaluated by the cellular uptake of tracer molecules that are normally excluded from the cell, like for example high molecular weight dextran-fluorescent conjugates 45.. This approach is more relevant to the application of the polycations as gene delivery compounds (explain way?). On the basis of the previously published data showing enhanced LDH release upon cell incubation with polymers of higher Mw (REF?) we hypothesize that the high molecular weight polycations trigger the cell death by compromising the cell membrane integrity, and the extent of this incident is charge density dependent. In an attempt to investigate such hypothesis and determine cell membrane disruption induced by polycation exposure, cells were incubated with the various polycations and analyzed for membrane permeability to 70 kDa rhodamine-dextran entry. For each polymer three different concentrations, below their respective EC50 values were tested. Following polymer exposure, cells were washed and incubated in media containing rhodamine-dextran. Finally, dextran uptake into the cytoplasm of damaged cells was visualized by confocal scanning microscopy (Figure S6) and the percentage of cells presenting rhodamin-dextran in their cytoplasm cells was quantified by flow cytometry (Figure 3). From. the heat map in Figure 3, representing the percentage of rhodamin-dextran positive obtained by flow cytometry, we can observe that cell membrane permeability increases with increasing polymer molecular weight and DB. These data suggest a direct dependency of the degree cell membrane permeabilization upon the polymer charge density. We can therefore hypotize that the e cell membrane damaging is a key factor during induction of acute cell death especially for the branched poly-L-Lysine types.
Insert figure 3 here
Activation of pro-apoptotic proteins (ref), direct mitochondrial permeabilization (ref) and perturbation of the membranes of intracellular organelles with the subsequent release of apoptosis inducing factors29, are among the proposed mechanisms by which polycations may trigger the apoptotic response.
The pioneering work of Moghimi et col23 defined the important events in late stage cytotoxicity of polycations. Their study focused on PEI and poly-L-lysine and identified them as apoptotic inducers. Authors pointed out the importance of the molecular weight of linear poly-L-lysine24 and PEI and its role on the mechanism of apoptosis. On the basis of their results the authors hypothesized that various polycationic macromolecules may initiate the apoptotic signaling pathway differently and with different efficiency. In relation to this hypothesis we investigate the extent of apoptosis induction as a function of polycation architecture. Distinct features characteristic for early apoptotic progression were monitored following cell exposure to polycations. These events involve translocation of phosphatidylserine from the inner to the outer side of the cell membrane, mitochonodrial depolarization and Cytochrome C (Cyt C) release. Further on we followed induction of the apoptotic pathway to its later stages via detection of caspase 3 and caspase 9 activation. Our final aim was to understand the temporal pattern of intracellular changes in response to polycation exposure, and how such events may correlate to the architectural structure of the different polycations.
Detection of the phosphatidylserine translocation belongs to the well established method of identifying population of early apoptotic cells (add ref). The plasma membrane of the early apoptotic cells loses its asymmetry and the phosphatidylserine is exposed to the outer leaflet as an external signal of cell demise.46 We have quantified the percentage of the apoptotic cells in CHO DG44 culture exposed to polycations via flow cytometry, employing the phosphatidylserine binding protein, Annexin V-FITC. The concomitant propidium iodide staining was used as indicator of cell necrosis. The histograms presented in Figure 4 represent Annexin V-FITC binding occurring 3 hours after the challenge of the cells with the polycation at a concentration of 20 μg/mL. Since this concentration is largely below the EC50 values previously measured, we assumed that under these experimental conditions, apoptosis was the primary mode of cell death. This was confirmed by the very low number of necrotic cells detected in all samples tested, as assessed by propidium iodide inclusion measurements (Figure 4B). As shown in Figure 4 for equi-size polycations, apoptotic induction detected by phosphatidylserine cell membrane translocation, reveals following tendency LPL< HBPL < DPL.
Insert figure 4 here
The next signaling event in the apoptosis cascade implicates mitochondria which membrane depolarization finally results in the release of Cyt C into the cell cytoplasm (add ref maybe :Susin et al., 1998). Cyt C plays a central role in the regulation of apoptosis by acting as an activator of a series of caspase cascades (add ref maybe Cai et al., 1998; Liu et al., 1996). The JC-1 reagent was used to assess the dissipation of cell mitochondrial potential as response to polycation exposure. JC-1 is a lipophilic probe bearing a delocalized positive charge wich depending on mitochondrial transmembrane potential (Δψm) accumulates either as red emitting aggregates in functional hyperpolarized mitochondria or, following mitochondrial membrane collapse, as a green monomer in the cytoplasm of apoptotic cells. (add ref Cossarizza A et. al Biochem Biophys Res Commun. 1993 Nov 30;197(1):40-5). Cells, per-exposed to polycations, were stained with the JC-1 compound and immediately analyzed either via flow cytometry (Figuer 5) or by fluorescence microscopy (Figure S7). In all the experiments, any cell fixation procedure was omitted in order to preserve mitocondria integrity. In accordance to the previous results on phosphatidylserine translocation, the highest degree of apoptotic induction, determined by the maximal number of the cells presenting green diffusive JC-1 fluorescence, occurred 2-3 hours after addition of the polymers to the cell cultures. Flow cytometric analyses demonstrate that the induction of mitocondria depolarization is directly dependent on the molecular size of the polycation. Indeed only in case of exposure to high weight polycation, cells presenting depolarized mitocondria were detectable upon JC-1 staining (Figure 5). On the other hand cells exposed to low molecular weight polycations did not show signs of dysfunctional mitochondria even after extended periods of incubation with the polymer samples (Figure 5). Since equi-size polymers showed similar result the structure of the polymer itself seems not to have influence on the onset of mitochondria collapse.
Insert Figure 5 here
It is generally accepted that the release of Cyt C from mitochondria is one of the very early events in the development of an apoptotic program that is a prerequisite for subsequent engagement of caspase cascades (add ref). Cyt C belongs to a family of mitochondrial intermembrane proteins47 and its cytoplasmatic release is often defined as a "point of no return" in the course of apoptosis (add ref). According to the work of Mather and Rottenberg 48 dual mechanism is responsible for the release of Cyt C from the mitochondria after the treatment with polycations: in a first putative mechanism Cyt C release might be a direct consequence of the mitochondrial depolarizarion induced during apoptotic cell death, a second hypothetical mechanism rely on the direct destabilization of the outer mitochondrial membrane through the electrostatic interactions of polycations with the anionic phospholipids of the mitochondrial membrane. Nevertheless both of these mechanisms contribute to the apoptotic program. The presence of the Cyt C in the cytoplasm of PC 12 cells was detected by indirect immunocytochemistry 12 h after addition of the polycation to cell cultures. For each polymer sample increasing concentrations were tested ranging from 10-8 to 10-4 M. Confirmatory to the trend observed for the depolarization of the mitochondrial membrane, cytoplasmatic Cyt C was detectable in cells incubated with high molecular weight polymers already when using concentrations as low as 10-7 M. On the other hand for low molecular weight samples, Cyt C release was evident only when high concentrations of polycations were added to the cell cultures (Figure 6). Such a trend was generally consistent with the tree types of polycation analogous, i.e. LPL, HBPL and DPL. Although the branched types of polymers induced a stronger response.
Insert Figure 6 here
Finally, the last stage, the activation of the caspase zymogens was detected and its presence was compared. Interestingly both apoptotic signals - Cyt C release and Caspase 9 activation - are detectable at a concentration range were apoptosis was already detected for PAMAM dendrimers.29 This may suggest …???. Once activated caspases cleave host of cellular substrates, leading to morphological hallmarks of apoptosis including DNA fragmentation and condensation of cellular organelles. The fragmentation of chromatin after 12 hour treatment with polymer was observed for all the samples except LPL samples in CHO cells (Figure S8).
All the check points in the intrinsic apoptosis pathway were observed if the cells (CHO and PC 12) were challenged with low doses of branched structures of high molecular weight. The extrinsic apoptotic pathway (caspase independent) was ruled out following the absence of caspase 8.
Insert Figure 7 here
The polycations which are the subject of this study are the main tools in transient gene expression for delivering the required genetic material for recombinant proteins expression in mammalian cells either for biotechnology purposes or in the view of medicinal therapy. For this reason the exposure of the cell to the polycation must not lead to lethal injury or must not become a triggering event for apoptosis. If a (sub)lethal injury occurs the cell may enter into the commitment step of the death program without any chance to recover. Such a cell is ineffective producer of recombinant protein either in "bioreactor" 49-51 or in vivo23, 52, 53.
All four types of polycations exhibited similar trends in the commitment of the cell death, although branched analogues were more effective in causing stress related response of the cells. The differential cytotoxicity of the polycations results most probably from the different degradability pattern and degradation rate of these analogues. Dendritic and hyperbranched poly-L-lysine contains ε amide bonds, therefore partially resists to the cleavage by proteases. The high molecular weight of hyperbranched poly-L-lysine HBPL 5 and HBPL 6 probably contributes as well to the structural hindrance between a protease (i.e. trypsin) and the bond to be cleaved. These two samples were not well sustained by the cells. The degradation behavious is going to be further studied for our library… (These results will be contained in the next paper on gene delivery)
In summary we have shown that the novel L-lysine analogue, hyperbranched poly-L-lysine, elicits the same response in the cells as PEI and dendritic poly-L-lysine of the same molecular weight. In agreement with previous studies we have identified two separate mechanism of the cell death caused by polymers: acute cell death & apoptotis. The immediate cytotoxicity causing the cell death by the polycations demonstrates the ability of polycations to affect the cell morphology, metabolism and cell membrane permeability. The acute cell death can be caused by lethal cell injury caused via fatal membrane permeabilization or high osmolytic shock. The extent of the acute cell death and apoptosis is directly linked to molecular weight and DB of the polycation. Low molecular weight polycations trigger the acute cell death at high concentration by osmolytic shock. Polycations of low Mn do not initiate apoptosis even at high concentration, irrespectively of their architecture. Our data indicate that the molecular mechanisms of the apoptosis is mediated via mitochondria, we have detected phosphatidyl serine translocation, mitochondrial depolarization, cytochrome C release and caspase 9 activation.
To conclude the objective of this study was to clarify the relationship between architecture and Mn of the polycation with the degree of their in vitro toxicity. The mechanism of the acute cell toxicity and delayed cell death was investigated together with the underlying mechanism. Additionaly, a novel L-lysine analogue, hyperbranched poly-L-lysine, was investigated for its biocompatibility and the data proves that it is a suitable candidate for gene delivery applications based on toxicity criterion.
Figures & Tables
Figure 1. Schematic representation of the architecture of the poly-L-lysine analogues. A) dendritic poly-L-lysine, B) hyperbranched poly-L-lysine, C) linear poly-L-lysine.
Table 1. Ring opening polymerization of N-carboxy-(N-ε-(tertbutoxycarbonyl) L-lysine anhydride.
[I0]/ [M0]a
Mtheorc [g*mol-1]
M1H NMRd [g*mol-1]
Mn e [g*mol-1]
Mwf [g*mol-1]
a Ratio of initiator to monomer; molar concentration in the polymerization feed, b polymerization time c Theoretical Mn determined from initial [M]:[I] ratio and conversion. d Determined by 1H NMR spectroscopy by comparing the initiator-derived resonance to 2H attached to epsilon carbon e, f,g Experimentally determined by GPC.
Table 2. Synthesis of dendrimer of L-lysine
ESI MSb
ESI MSc
Mn d [g*mol-1]
[ g*mol-1]
a theoretical molecular weight, b Experimental value, c m/z theoretical, d,e,f Experimentally determined by GPC, ¯ multiple peaks present in the spectra
Table 3. Synthesis of hyperbranched poly-L-lysine
Mn h [g*mol-1]
Mw i [g*mol-1]
NNH k
DB l
h, i, j Experimentally determined by GPC. k number of amino groups experimentally determined by acid base titration. l Average number of branches were calculated as described in SI, Section Techniques.
Figure 2. Quantification of acute cytotoxicity. CHO cells were incubated for 1h with various polymers as indicated. Acute cell death was then assessed by flow cytometry. EC50 values were extrapolated by assessing cell viability after incubation with increasing polymer concentrations. For each sample at least 10 concentrations were tested ranging from 10-8 to 10-1 M.
Figure 3. The cell membrane integrity was assessed by its permeability to rhodamine-dextran. The diffusion of rhodamine-dextran into the cytosol of CHO cells after 1h incubation with a polymer at a concentration range of 10, 20 and 30 μg/mL. Color scale of the heatmap (a) was obtained by measuring the sample of the cells treated with 0.1% solution of triton (positive control) and PEG 5000 at a concentration of 100ug/mL (negative control). The heatmap represents the percentage of the fluorescent cells after the treatment with HBPL (b), DPL (c), and LPL (d) the percentage of fluorescent cells was determined by flow cytometry. .
Figure 4. Phosphatidyl serine translocation upon polycation treatment. CHO cells displaying phosphatidyl serine were indetified via Annexin V binding 3h after incubation with LPL, DPL or HBPL polymers. Non viable cells were excluded by PI staining.
Figure 5. ΔψM as a function of the incubation time and the Mn of polycation. The percentage of cells displaying depolarized mitochondria after the exposure of the cells to LPLs (a), DPLs (b) or HBPLs (c) was determined. The ΔψM of mitochondria was analyzed at 30min (â- ), 60min (â-), 120min (â-²), 240min (â-¼) post incubation with polycation. Analysis was performed by flow cytometry using the JC-1 stain. Wide field fluorescent microscopy of cells with depolarized mitochondria (green staining of the cytoplasm) and with intact mitochondria (punctual red staining). Cells treated with negative control (PEG 5000) at a time 0 and 4h (d and e respectively). As a positive control, cells were treated with camptothecin and images were acquired at the time 0(f) and 4h (g). Images h and i shows mitochondrial depolarization after the treatment of the cells with HBPL 6 (0h and 4h).
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Figure 6. Detection of Cyt C cytoplasmatic release in PC12 cells after 12h incubation with different polymers. The release of Cyt C into the cytosol is preceded by the decrease in mitochondrial transmembrane potential after the stress inducing factor. Cyt C release is evident in almost all samples, for lower molecular weight samples the release is detectable only when high concentration is applied. The presence of Cyt C is more pronounced for the dendritic and hyperbranched samples and samples of higher molecular weight. The Cyt C was not detected for the low molecular weight HBPL and LPL.
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Figure 7. Caspases 9 activation in PC 12 cells after 12h treatment with polycation. Caspase 9 is a crucial upstream activator of the Cyt C-dependant apoptotic pathway., and its presence (green) was detected mainly with high molecular weight samples -DPL 6, HBPL 6, LPL 5.


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text: How do you deactivate the bomb in megaton? 1,070,044questions onWikianswers Add New Page Edit Edit source History Talk0 Share This should help. http://fallout.wikia.com/wiki/The_Power_of_the_Atom Retrieved from "http://answers.wikia.com/wiki/How_do_you_deactivate_the_bomb_in_megaton?oldid=4821027" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Fallout series Answered questions Add category Cancel Save


text:
RSM Legal LLP provides legal advice to RSM clients on employment, partnerships, corporate and commercial contracts as well as regulated legal services.
RSM Legal LLP is closely aligned with and complements the whole range of our extensive client service offering and employment solutions, HR services and employer services in particular.
To find out more about RSM Legal LLP, please contact Carolyn Brown, Fiona Bell or your usual RSM adviser.


text:
About us
Hello from team Love Patchwork & Quilting!
Love Patchwork & Quilting is the bestselling modern quilting magazine that shares your passion for fabric! We publish 13 times a year, featuring must-make projects, essential techniques, interviews, news and reviews from the world of modern quilting. Every issue also comes with a FREE gift!*
Jenny Fox-Proverbs, Senior Editor
Jenny has been editor of Love Patchwork & Quilting since we launched in October 2013 (has it really been that long?) and now is the editor of our new sister mag, Today's Quilter, as well as overseeing LP&Q. She previously worked as managing editor on our sister cross stitch titles. Before taking on the world of craft mags, Jenny had an illustrious career commissioning craft books, and has worked with some of the biggest names in the craft world. At home Jenny has two much-loved, if rather elderly, cats and loves nothing more than cuddling up on the sofa with her fur-babies, a good murder mystery and a jar of nutella!
Alice Blackledge, Commissioning Editor
Alice is our Commissioning Editor, having worked on LP&Q since its launch. She's kept super-busy keeping the team on track and seeing that we don't get buried under the mountain of fabric and quilts that arrive each month (Jen and Sarah are both total hoarders!). She's the creative force behind the mag, working with our wonderful contributors to come up with the fun, fresh projects that feature in every issue. She can often be found tracking down the best new quilt designers and planning beautifully-styled photo shoots. She's also the office EPP queen, whipping up gorgeous quilts with impeccably embroidered details! Her guilty pleasure is trashy crime drama and she can eat an entire tub of Haagendaz in a single sitting (impressive, right?)
Sarah Griffiths, Senior Technical Editor
Sarah is our resident tech-expert, as at home whipping up quilt samples as she is tackling complex quilt-math with the aid of her trusty calculator, so she's an essential part of Love Patchwork & Quilting team! She's got a fabric stash that any quilter would envy (and yes, it's a stash – not a hoard!) and an almost indecent number of projects on the go at any one time. She'll often bring finished projects into work and wow the team with her sewing skills. Sarah's our in-house American, originally from Ohio, she loves a good cup of tea (spearmint's her favourite) and comes from a long tradition of family quilters. It's Sarah's hands that you see if our tech feature every month, and when she's not being photographed she has a definite penchant for fancy nail varnish.
Katharine Bennett, Production Editor
Kate is our yoga-practising, happy-making Production Editor. She works on all the features in the mag, so next time you settle down with a cuppa to read about your favourite designer or lust after a gorgeous fabric collection, you can thank lovely Kate! She spends most of her days discovering the best upcoming fabric releases and resisting the urge to buy all of the beautiful things she sources for the news pages. Compared to the rest of the team, Kate is a bit of a quilting newbie, but she's fast catching up with beautifully neat EPP stitching (watch out Alice!) and a project list as long as her arm. When she's not crafting, Kate can be found walking her Doberman Juno on The Downs or wandering around Harvey Nichols, hoping to grow her handbag collection.
Tiffany Jackson, Sales Executive
Tiff is sunshine personified and our favourite person to chat to on a Monday morning! As Brand Sales Executive she works with all of our lovely advertisers to make sure that we always have the very best names in LP&Q. She's no one-trick-pony though – our Tiff is an avid stitcher and is mid-way through a whopping great big double bed spread pieced entirely by hand – only 208 hexies to go! Her Mastermind specialist subject is Harry Potter (no, really) and she's got a bit of a sweet tooth, in fact coining the phrase "Cake always wins!". So if you're interested in advertising in the mag, drop her a line.
You can get in touch with us on Facebook, Twitter , Instagram and Pinterest. We'd love to hear from you!
* Free gift only available with the print edition


text:
Find your new home in Brightwood, VAYou are viewing homes 1 to 1 of 1 home for sale
{"ListingID":"0xDBF438F51997CC76F8E9FEB91A9B07C8","StreetAddress":"2302 Thoroughfare Rd","StreetAddressFull":"2302 Thoroughfare Rd, Brightwood, VA","Price":"$225,000","PriceDisplay":"$225,000","PriceForMap":"$225,000","Bedrooms":"3","Bathrooms":"2","Prop_BedsBaths":"3 beds 2 baths ","HalfBathrooms":"0","PropertyType":"Single Family","Latitude":"38.401204","Longitude":"-78.167191","Distance":5579.1930549383,"Thumbnail":"https:\/\/propertyimage.leadingre.com\/2686\/0xDBF438F51997CC76F8E9FEB91A9B07C8\/1_thumb.jpg","Href":"\/listingdetails\/usa\/va\/brightwood\/2302-thoroughfare-rd-22715_0xdbf438f51997cc76f8e9feb91a9b07c8\/"}
Learn more about homes in Brightwood, VA
Brightwood Home inventory breakdown by property type
Of the 1 homes for sale in Brightwood, the breakdown of property types
is as follows:
100% Single Family.
100% Single Family
Area details, home facts and figures
Homes for sale 1
Average Home Size 0 ft² / 0 m²
Price Range ($225,000 - $225,000)
Information from in and around Brightwood...
You are Viewing 1 to 1 of 1 homes for sale in Brightwood. These homes are comprised of Single Family. The average list price of these property results is 225,000. Prices are shown as US Dollar. For more information on these homes for sale in Brightwood please contact our member brokerages to schedule a showing or view an open house.
The 1 homes for sale in Brightwood, VA are represented by 1 member brokerages serving the Brightwood area. Member brokerages representing the 1 homes include Montague, Miller & Co. Realtors.


text: On Tue, Aug 28, 2012 at 9:47 AM, Nelle Varoquaux <nelle.varoquaux@...>wrote: > Hello, > > I've just checked out the source code from github, and I ran the > tests. Unfortunately, I get loads of errors (more than 1k), most of > them such as the following: > > Looks like you triggered a code-path that most of us haven't encountered. The code in question checks for which version of numpy is installed and uses the faster bincount if it is available and falls back to the older testing code if it isn't available. Unfortunately, the older code seems to have been bastardized in the process. This should defintely be fixed. I might have a moment today to submit a PR for this. Ben Root


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Employee Scheduling & Workforce Management systems for Fire Rescue agencies
Whether your department is strictly volunteer, part-time or full-time staff; scheduling and resource management is made simple with just a few clicks of a button. CrewSense offers a customizable, rules-based solution for your agencies specific, and possibly, daunting – scheduling needs. Designed from the ground up as a powerful, flexible and easy to use employee resource management solution, such daily headaches as complex Fire Department scheduling rotations can be quickly added to the system.
- Connector.
Staffing Rules
Implement complex staffing rules; abide by union contracts or hiring policies. No other system offers the flexibility of CrewSense
- Connector.
Policy / Union Compliance
Establish maximum work hour limits. Establish maximum work hour limits per day, week or month. Ensure Union, FLSA, and Obamacare compliance.
- Connector.
Payroll / Time Tracking
Establish work type time tracking, accrue hours based on pay periods, days, weeks or months. Deduct hours per your Organizational rules.
- Connector.
Fair and equitable Overtime
Fill open shifts quickly, equitably and fairly using our CallBack feature
- Connector.
More affordable
Nearly 10x less the cost of our nearest competitor. Easier to use, and with a lower barrier to entry – makes CrewSense the right choice.
- Connector.
Easy to Use
Easily move employees from one assignment to another with drag and drop flexibility. Reschedule employees, de-schedule lines, move employees to a pending position, and more – all while maintaining complex staffing rules
CrewSense interfaces directly with the best RMS platforms. No more double data entry!
Rules based scheduling means you can get back to leading your people.
Every Fire Department is unique and requires its own set of rules and procedures. The CrewSense system will accommodate any size Department. Implement rules based scheduling using qualifiers and minimum staffing levels, and watch in real time as the system analyzes the daily schedule for any deficiencies. There is nothing else like it on the market.


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Check out some of The Frugal Girls Best Southern BBQ Recipes!
I wanted to share with you some of our very Best Southern BBQ Recipes we've whipped up over the past few years. These include some delicious rubs, sauces, pork, chicken and beef recipes.
If you love barbecue, you'll really love the tasty simplicity of these yummy BBQ Recipes!
Best Southern BBQ Recipes
Best BBQ Rubs ::
Ultimate Smoked BBQ Rub Recipe
This Rub is flavor packed and simple to make! The perfect compliment to your favorite Barbecue Sauce!
Best Ever Chicken Rub Recipe
If you need a versatile Chicken Dry Rub Recipe, you just found it!
Best Homemade Barbecue Sauces ::
Easy Homemade Sweet BBQ Sauce Recipe
Making your own deliciously sweet Barbecue Sauce is a snap!
Easy Homemade Hickory BBQ Sauce Recipe
Looking for a smokin' good Homemade BBQ Sauce Recipe?
Best BBQ Pork Recipes ::
Easy Crockpot BBQ Coca-Cola Ribs
Craving some crazy-good Fall-off-the-Bone BBQ Ribs??
Crock Pot Root Beer BBQ Ribs
Grab the napkins… or better yet, the whole roll of paper towels. Things are about to get messy good!!
Easy Country Style BBQ Pork Ribs
Craving some comfort food? Let me introduce you to these Ribs!
Crockpot BBQ Coca-Cola Pulled Pork Recipe
Get ready for some mouth-watering Pulled Pork!
Best Crock Pot BBQ Chicken ::
Crockpot Root Beer Barbecue Chicken
Grab a sweet dose of nostalgia and childhood fun!
Crockpot Coca-Cola Barbecue Chicken
This chicken is incredibly easy to make, and full of spicy kick! {yum!}
Crockpot BBQ Bacon Ranch Chicken Recipe
This is by far one of my family's favorites… it's DELISH!! Everything is better with bacon! 🙂
Crockpot Dr. Pepper BBQ Chicken
This chicken is just what the doctor ordered!
Crockpot Honey Mustard BBQ Chicken
Get your BBQ Mustard fix with this yummy Chicken recipe!
Crockpot Kansas City BBQ Bacon Chicken Sandwiches
This sandwich is an explosion of crazy yummy flavors!
Crockpot BBQ Whole Chicken
Get summer barbecue flavor in your Crockpot all year round!!
Crockpot BBQ Ranch Chicken Drumsticks Recipe
Grab the napkins, this dinner is going to be finger licking delicious!
Best BBQ Burger Recipe ::
Best Homemade BBQ Burger Recipe
Hands down this is the Best Homemade BBQ Burger Recipe ever! It's simple and out-of-this-world delicious!
Best Southern BBQ Side Recipes ::
Crockpot BBQ Bacon Ranch Beans Recipe
Ultimate Cornbread Recipe
Crockpot Macaroni and Cheese Recipe
Bacon Lover's Red Potato Salad Recipe
Easy Zesty Coleslaw Recipe
Don't miss out! Follow along for more fun Recipes and Tips with The Frugal Girls right here…


text:
Retailer Target examining data breach involving 'millions' of shoppers
Reports have begun to surface that retailing giant Target has been the subject of an enormous data breach. Potentially at risk are "millions" of customer records, including credit and debit card information. The periods at risk are November 29 through December 15, and affects "nearly all" US Target stores, but not the online store.

Retail security analyst site Krebs on Security claims that "the type of data stolen -- also known as 'track data' -- allows crooks to create counterfeit cards by encoding the information onto any card with a magnetic stripe. If the thieves also were able to intercept PIN data for debit transactions, they would theoretically be able to reproduce stolen debit cards and use them to withdraw cash from ATMs."

An anti-fraud analyst at a US bank card issuer quoted by Krebs believes that "the breach window is definitely expanding. We can't say for sure that all stores were impacted, but we do see customers all over the US that were victimized."

Target Brands, MasterCard, and Visa have had no comment on the reports. It is unknown who the parties responsible for the breach are, where in the world they are located, or how the data theft was accomplished.
These hackers think they are on top of the world. They should be treated just like any other big time drug dealers.
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text:
Which Discs Of Brake System For BMW 5-Series.
While servicing BMW 5-Series 2.5, look at remaining thickness of disc and pads. The vital thing for driving BMW 5-Series is normal capacity of brakes. When choosing BMW original or analog parts for 3.4 motor, you should be careful. If you need front brake discs for 5-Series with N62B40A coded motor, choose such accessories: Diameter: 348 mm Thickness: 30 mm Height: 72.20 mm Number of holes: 5 Diameter of central bore: 79 mm. Also note that rear discs for brakes in BMW are such: Diameter: 300 mm Thickness: 10 mm Height: 61.50 mm Number of holes: 5 Size of bolt holes: 15 Diameter of central bore: 75 mm PCD: 120 mm. If you possess the vehicle with M50B (206S1) engines made in 1993, you need special brake disc size.
Buying rear brake discs, purchase these: Diameter: 300.0 mm Diameter of central bore: 75.0 mm Height: 95.0 mm Thickness: 10 mm Number of holes: 5. Don't forget your 1990 BMW 5-Series has front discs at sizes: Diameter: 302 mm Thickness: 22 mm Height: 76 mm Number of holes: 5 Size of bolt holes: 14.60 Diameter of central bore: 79 mm PCD: 120 mm. But if your BMW shows bad condition of disc, buy special parts for 2.5 vehicle. You won't need disc replacement in 2004 5-Series often, in M54226S1 number of engine they are quite ok.


text:
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Living Water – Art Exhibit
Loading Map....
Thursday, April 20 - Sunday, May 21
12:00 am
IHM Motherhouse
The "Living Water" exhibit features the work of student artists from Detroit Cristo Rey High School, Marian High School and Monroe-area high schools. Download the flyer.
The exhibit is free and open to the public Monday-Friday from 10:30 a.m.-4:30 p.m., and Saturday by appointment. For more information, contact Sharon Venier at 734-240-9754 or email@example.com.


text:
Speed Reducer, C-Face, 56C, 25:1
BOSTON GEAR| Zoro #: G0419027| Mfr #: F713-25-B5-G
Zoro #: G0419027Mfr. Part #: F713-25-B5-G
$662.00 /ea
This item ships free. DetailsStandard ground shipping
Technical Specifications
|Zoro #: G0419027 Mfr #: F713-25-B5-G
|Shaft Orientation: Right Angle
|Mounting: Any Position
|Gear Material: Hardened and Ground Steel
|Pinion Material: Hardened Steel
|Single/Double Output/Universal: Single Output
|Standards: AGMA
|Finish: Powdered Epoxy Paint
|Nominal Ratio: 25:1
|Base: Optional
|Seal Type: Double Lip
|Solid/Hollow Output Shaft: Solid
|Rotation: Forward/Reversible
|Lubrication: Klubersynth UH1 6-460
|Input RPM: 1750
|Speed Reducer Type: C-Face
|Worm Gear Material: Bronze
|Housing Material: Cast iron
|Item: Speed Reducer
|Bearings: Tapered Roller
|Gear Type: Worm
|Nominal Output RPM: 70
|Number of Stages: 1
|Duty: Standard
|Max. Output Torque: 234.00 in.-lb.
|Reducer Model No.: F713-25-B5-G
|Output Torque @ 1/40 HP: 3600 in.-lb.
|Frame: 56c
|Output Torque @ 1/4 HP: 117 in.-lb.
|Dimension U: 5/8"
|Output Torque @ 1/3 HP: 175 in.-lb.
|Output Torque @ 1/2 HP: 234 in.-lb.
|Output Shaft Height: 1-3/4"
|Output Shaft Dia.: 5/8"
|Overhung Load: 200 lb.
|Worm Center Distance: 1.33"
|Max. Input HP: 0.3
|Bore Dia.: 5/8"
|Country of Origin (subject to change): United States
Product Description
Speed Reducer, C-Face, Right Angle Shaft Orientation, Standard Duty, 1750 Input RPM, 70 Nominal Output RPM, Worm Center Distance 1.33 In., Bore Dia. 5/8 In., 56C Frame, Overhung Load 200 lb., 25:1 Nominal Ratio, Max. Input HP 0.3, Max. Output Torque 234.00 in.-lb., Number of Stages 1, Forward/Reversible Rotation, Optional Base, Any Position Mounting, Tapered Roller Bearings, Hardened Steel Pinion Material, Worm Gear Type, Cast Iron Housing Material, Single Output, Output Shaft Dia. 5/8 In.
Restrictions and Compliance
- *1 : California Proposition 65 : This product contains a chemical known to the State of California to cause birth defects or other reproductive harm.
- *2 : California Proposition 65 : This product contains a chemical known to the State of California to cause cancer.


text:
Great First Time Crystal Experience, ......But Probably Last: Crystal Symphony Cruise Review by mdbgoldfamily
Overall Member Rating
Great First Time Crystal Experience, ......But Probably Last
Destination: Canada & New England
Embarkation: New York (Manhattan)
The staff was phenomenal with a couple exceptions (rude photographer and assistant concierge were each quite full of themselves....arrogant beyond belief). The Internet is overpriced and too slow like on most ships.
We found the violinist to be excellent and lectures at sea to be wonderful. We did not enjoy the otter entertainment options.
Disembarkation was simple and easy. Off ship and through customs in 5 minutes.
The ultimate question is Crystal worth twice the cost of Holland, Princess, Celebrity or Royal? Is the extraordinary service and exceptional food worth twice the cruise fare? We think not. Crystal is not Regent....smaller cabins and more limited food offerings. Some people love to pay to be pampered and for you Crystal is perfect. Candidly, 85 percent of the service is available on Celebrity and Holland for half the price..or close.
Beyond a couple of items mentioned above, there is not much negative to say about Crystal. The staff works its collective butt off. I just don't think the final package was worth the cost. Many others will disagree, based on their own priorities. Less
Published 10/19/11
Cabin review: A
Read Cruise Critic's Crystal Symphony Review >>
Balmoral Blues
Good - Except for the food/Wom...
OK not great: Easy to make it ...
First class crystal
Still up there
Ease at sea
Taking the trip just for the s...
Crystal Does not disappoint
First class crystal
Sponsored Links


text:
Definition of keen–scented
: having a keen sense of smell a keen-scented hound
Love words? You must — there are over 200,000 words in our free online dictionary, but you are looking for one that's only in the Merriam-Webster Unabridged Dictionary.
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text:
The optional software modules for SiPass integrated make it possible to tailor the system to meet almost any security requirement.Highlights
This option makes it possible to add Salto offline components (doors) to a SiPass integrated system. Access control rights can be assigned to both online and offline components simultaneously in the SiPass integrated software.
The photo ID and image verification option enhances the cardholder record keeping function by making it easy to capture photographs and signatures.
The message forwarding option enables the system to automatically send customized text messages to the pagers, mobile telephones or e-mail addresses of key personnel whenever security is breached or other important events occur.
The Apogee interface enables integration between the Apogee Insight building man agement system and SiPass integrated.
The visitor management option enables the use of the same graphical user interface that is used for permanent cardholders to also be used to register visitors. It is possible to capture visitors' facial images, paste in existing image files, record personal details, print access cards and locate a visitor easily.
The graphics option makes it possible to design, import and construct customized graphical maps that are used by security operators to visually handle alarm conditions and continually monitor the status of all points within the system.
Mifare card technology makes it possible to use a single card for a variety of purposes, including unlocking doors and paying for goods and services. Complete Mifare card encoding and profile configuration is a unique feature of SiPass integrated. The system can also supports the encoding of both 1K and 4K Mifare cards, as well as Mifare DESfire cards.
Using this software interface, it is possible to begin recording from any camera with a single click of the mouse button using recording-based shortcuts that can be easily created and placed on graphical maps. All recording events and their statuses appear in real time in the audit trail, and can be played back instantly by simply clicking on the recording event.