To be sure, there's also not anything especially wrong with the textual configurations that most attorneys default to. No judge, clerk, or court official will give a second thought to them, meaning they're safe — but they also miss an opportunity to make a distinct, positive impression.
I realize I am far from the first person in the legal profession to write about this subject. Some courts have even taken it upon themselves to provide members of their bars with detailed guidance on typography. All practitioners would also do well to pick up Matthew Butterick's Typography For Lawyers, a wonderfully practical and comprehensive resource for these issues.
Of course, surveying the various commentary out there reveals that different sources have different opinions on different typography methods. That shouldn't be too surprising, but some attorneys may characterize such divergence of opinion as proof that typography is, at its core, simply a matter of personal taste and therefore not worth spending time on. This misses the point — the usefulness of typography is not in creating a set of unanimously endorsed rules, but, rather, getting writers to simply think about what they're doing.
For whatever reason, lawyers who devote countless hours exploring and developing meticulously reasoned legal arguments for their briefings don't care to make similarly reasoned decisions about what they want their briefings to actually look like. But just how any minimally competent practitioner would craft a brief's content, crafting a brief's presentation is a bit of a process: surveying all the available options, considering the benefits of drawbacks of each, experimenting with various alternatives, and arriving at a deliberate preference. Doing so, you might actually find that, although you've been blindly following the same typographical habits imposed on you by the partner in your first job out of law school, you actually favor something else. More importantly, so would your readers.
Before this post gets into the details of various typographical topics, some ground rules: First, our canvas for today is a blank 8½-by-11-inch sheet of paper — for now, no messing with those tiny U.S. Supreme Court briefing booklets. Second, I am assuming that, by virtue of your reading this on a blog, you have a computer and use that computer to draft your briefs, so don't expect any tips on optimally setting the carriage of your 1933 Remington Rand. Third, the suggestions herein are not meant to be evaluated individually; rather, to keep a proportionate and balanced look, an adjustment to one typographical element may require a corresponding, complementary adjustment to another. Trying out new typographical ideas requires a holistic approach.
With that, let's start with some basics.
To correctly apply appellate rules on type size, it's first necessary to understand the difference between uniformly and proportionally spaced type. Uniformly spaced, or "monospaced," type gives every character the same width — a lower-case "i" (including additional surrounding white space) takes up the same horizontal room on the page as a capital "W" (along with its proportionally smaller cushion of white space). Obviously, those letters do not need the same footprint in your document, so where did this wacky practice originate?
From the source of 95 percent of all typographical evils: the dreaded typewriter. A typewriter's keys were all the same size, so the characters on the keys were all the same size as well. Monospaced fonts were thus created to accommodate a mechanical limitation, not because they offered any aesthetic benefits. As discussed below, there is no good reason to ever use them this day in age, so, although both the Maryland and federal rules set separate size parameters for uniformly spaced type, these can be ignored.
Proportionally spaced fonts assign each character the amount of horizontal area it actually needs. Because these are the fonts you should be using, you have to abide by the corresponding appellate dictates about type size — at least 13-point in the state appellate courts, see Md. Rule 8-112, and at least 14-point in the federal circuit courts, see Fed. R. App. P. ("FRAP") 32(a).
Now comes the big decision: Which font? There are an overwhelming number of typefaces at your disposal — if you're using Microsoft Word, there may be more than 150 built right into your software. To make things a bit more manageable, the Maryland Court of Appeals has set forth a number of "approved" fonts, but even these are "suggested, not mandatory." Notice that Times New Roman is but one of these 16 suggestions.
Despite some perception out there to the contrary, the Federal Rules of Appellate Procedure don't require Times New Roman (or some variant of it) in briefings either. When FRAP 32(a)(6) says, "A brief must be set in plain, roman style," it's simply saying that the text can't be entirely bold, italicized, capitalized, narrow, or condensed — not mandating typeface mediocrity. Indeed, the only hard-and-fast rule under the Federal Rules is that a proportionally spaced font must have "serifs," id. at (5), which are small little lines that accent the tips of character strokes. "Sans-serif" fonts, which don't have the lines, don't fly at the federal appellate level in proportionally spaced fonts (except in headings or captions, but for now we'll bypass the question of using multiple fonts).
There is an endless smorgasbord of fonts laid upon the typography table for connoisseurs to, like an pretentious oenophile at a wine tasting, sample, judge, and offer preposterously nuanced opinions on. But exploring the universe of non-system fonts out there can take considerable time and effort, with decreasing returns. For the busy attorney who just wants to bump the quality of his or her briefs up a notch, it's fine to stick to the plethora of options available right there from the pull-down menu in your word processor. As such, for the sake of brevity — as well as appeasing lawyers' crippling fear of doing something too unorthodox — I'll artificially restrict today's font discussions to those on the Court of Appeals' "approved" list that can be found in Microsoft Word.
That said, Times New Roman isn't a disastrous choice by any means; its biggest sin is just being boring and overused. You could do a lot worse than Times New Roman… and, in fact, the Maryland Court of Appeals explicitly allows you to. For these following fonts, although the venerable court has ostensibly given you permission to use them, resist the invitation.
Arial: Arial was developed as Microsoft's blander, more generic, no-licensing-fee alternative to Helvetica, a font so successful that it became a sort of touchstone for the global, multicultural, modernist era. (Those with an hour-and-a-half to spare are encouraged to check out this entertaining documentary about the Helvetica's history and omnipresence.) Arial is a perfectly comfortable, easygoing, versatile font, much in the way your favorite T-shirt, pair of jeans, and set of loafers makes for a comfortable, easygoing, versatile outfit. Question: Would you wear your favorite T-shirt, jeans, and loafers to an appellate oral argument? Of course not. Don't use Arial for the same reason. (As a sans-serif font, Arial isn't available to you at the federal appellate level anyway.) For a full take-down of Arial, check out this particularly disparaging commentary.
Bookman Old Style: The Court of Appeals has also authorized the use of Bookman Old Style, a curvy, serif font that resurfaced in the '60s and '70s as a way to convey a sense of relaxed sophistication. It's a mellow, sunny font perfect for a little light reading as you let a couple buttons loose on your designer jumpsuit and stretch out on your luxury earth-toned shag carpet. In other words, the Bookman font family is outdated and kind of cheesy. If you're going to use a Bookman font, consider figuring out a way to have your brief softly play a little James Taylor for the judges as they flip through it.
Century Gothic: Yes, it's been well-documented that Century Gothic can save you money on ink (though not in the federal circuit courts, because it's sans-serif). But let me ask you this: Do you want your brief to the highest court in the state to use the same font as Weezer? There's only one answer to this question.
Courier New: Courier New is a monospaced font that was made to replicate the output of typewriters. Courier New's acceptable uses in today's world are therefore pretty much limited to props in Mad Men, yet a baffling number of attorneys still call upon it for their appellate briefs. In what other context would it be desirable to willingly bind oneself to the mechanical shortcomings of a prior era? If you had a Super Bowl party, would you lock your 60-inch ultra-high-definition television in the closet and force everyone to crowd around as you dialed up the game on your dusty postwar vacuum-tube model? If you were buying a brand-new Porsche, would you ask the dealer, "Hey, this is great, but any chance you could make this sucker run just like a 1972 Ford Maverick?" Sometimes things die for a reason. Courier fonts are such things.
Book Antiqua: Disclaimer: Much how Arial lazily mimics Helvetica, Book Antiqua is a shameless knock-off of another successful font, Palantino. Palantino was designed by a professional calligrapher, so Book Antiqua retains the subtle, fluid grace of ink-written materials, making it a frequent go-to font in all kinds of literature. I don't know about you, but I — and other appellate commentators as well — would rather use Book Antiqua and have my brief look more like a formal published text than use Times New Roman and have my brief look like there might be a Sudoku somewhere near the back.
Century Schoolbook: First, Century Schoolbook is not related to that half-baked sans-serif standard-bearer of modernist mediocrity discussed above, Century Gothic — Schoolbook was created in 1919 and is commonly used in textbooks, while Gothic was thrown together in 1991 and was commonly used by Starfleet on Star Trek: The Next Generation. Avoid the latter in your briefs (yes, even you hopeless sci-fi nerds out there), but turn to Schoolbook for an enduring, eminently readable typeface with the simple, elegant professionalism of academic works. If you're nervous about making the switch, take some comfort from the fact that you know at least nine other well-respected attorneys who use a version of Century Schoolbook… the U.S. Supreme Court.
My general philosophy is that a double-spaced, one-inch-margins page of text leaves the right amount of the paper blank — just in the wrong place. The trick then becomes to shift the white space to where it is most effective for your presentation without greatly disturbing the size or per-page word counts of your content. Luckily, this isn't that hard.
First, a question: Why do you double-space text? Chances are, you haven't actually sampled all the various spacing options and decided that double-spacing gives the ideal amount of room between lines — you've just never done anything else. Like all typographical choices, this is not a valid reason for staying the course… and, like a lot of other typographical choices, the reason for the status quo is obsolete typewriter limitations. In this case, the carriage return of a typewriter only moved in increments of one line, so, if you wanted space between lines, you had to leave a full line blank. Again, no one ever decided this was the optimal choice for readers; it was just all the typewriter could do.
Computers obviously don't have such restrictions — you can set your line spacing to whatever you want. Which is good, because full-line gaps actually set the type too far apart, forcing the text into trenches with equal swaths of no-man's-land in between. By cutting back on the spacing, you can bring lines closer together for a more cohesive, consolidated appearance. In my experience, however, lawyers react to the suggestion to ditch double-spacing like you've recommended they write their briefs entirely in Pig Latin.
This response is likely at least somewhat attributable to the federal circuit-court requirement that documents use double-spacing. See FRAP 32(a)(4). (Combined with the mandated minimum of 14-point type, this makes briefings begin to look a little like children books — but, hey, if that's all the judges' poor eyes can take, that's certainly their prerogative.) Maryland appellate courts, on the other hand, only set a minimum of 1.5-spacing, Md. Rule 8-112(a)(2), which is a splendid distance between lines: enough to keep the text from crashing together, but not so much as to cause your type to look like lane markers in a swimming pool of white.
Go ahead: Drop the spacing to 1.5 and take a gander. Did you notice that your 54-page brief that you just couldn't make any shorter now fits within the page limit? Don't put down your red pen quite yet, however — like a cosmetic fat transfer, we've only sucked extraneous white space out of one part of the page to inject it back in somewhere more attractive.
Indulge me in a quick experiment. Take a page of something you've typed up with one-inch margins and hold it up to something that has been professionally framed. Proportionally, which has wider borders? Alternatively, the next time you're at a fancy restaurant, compare your page to one of the courses — chances are, it's being served on a plate that's considerably larger than the food itself. What's up with all this nothingness around the something that you're supposed to be looking at?
A pretty basic tenet of visual design, actually. Blank space (or "negative space") sets the featured content (or "positive space") apart from its surroundings, drawing and focusing the viewer's attention to it. And you just can't get the most out of this effect with one-inch margins.
The Maryland Rules allow no less than one inch of space in the top and bottom margins; to accommodate the possibility of extra margin space for binding purposes, the Rules don't dictate the size of side margins but set a maximum of six-and-a-half inches of text across, the same amount you get with one-inch margins. (The Federal Rules of Appellate Procedure set a one-inch minimum on all four margins, FRAP 32(a)(4), but, given how much extra space will already be in your document courtesy of those Rules, I don't recommend inserting any more.) It's a standard length that you've likely come quite accustomed to, but it's also a length that begins to test readers' patience.
See, people don't actually read character by character, or even word by word: We read groupings of words at a time, taking an ever-so-slight mental pause after each to connect them together. After three or four such pauses on a line, our brains need to briefly hit the reset button before continuing, or else we quickly tire of the process. Accordingly, it's been shown that a person's focus diminishes as he or she continues reading along an unbroken line of text.
In general, six-and-a-half inches of horizontal print starts to push it. Increasing the margins, therefore, can serve two purposes: one, widening the negative space bordering your page to better focus the reader's attention on your text, and, two, bringing your line lengths into a shorter range that won't threaten to impact the reading experience. Exactly how much larger to set your margins is a bit of a judgment call, but, as stated above, I think the total amount of white space on a double-spaced, one-inch-margin page is about right, so I try to keep it roughly equivalent. Typically, one-and-a-half-inch margins — combined with 1.5-spacing — gets more or less the same amount of type and space as the default line spacing and margins, but looks a lot better and is easier to digest.
Even making just these simple alterations to font, line spacing, and margins can immediately give your briefs greater visual appeal and readability, all in total compliance with Maryland's appellate guidelines and rules. But these aren't the only ways to accomplish these goals; if my suggestions don't do it for you, there are countless other typographical elements that, with a little research, you can learn to tweak for a better brief format. However you do it, be guided by modern, digital-age practices that give your legal work the best presentation — not long-outdated practices adopted to make typewriters tolerable.
Wonderful article. I have a perpetual battle with those in my firm, the majority of whom insist on using Arial for everything. I slowly converted a few to Times New Roman, and am now trying to convert them further to Book Antiqua. It is a slow and painful process, but one which I am committed to implement.


text:
Anti-Defamation League joins bigots in opposing Manhattan mosque - Ikhwanweb
Anti-Defamation League joins bigots in opposing Manhattan mosque
The Anti-Defamation League has announced its opposition to the building of an Islamic community center (or mosque, as CNN and others put it) in Manhattan, near ground zero.
Monday, August 2,2010 13:07
sabbah.biz
The Anti-Defamation League has announced its opposition to the building of an Islamic community center (or mosque, as CNN and others put it) in Manhattan, near ground zero. It accepts that the builders have every right to do so, but believes that they should not because its presence there will cause offense and pain.
Proponents of the Islamic Center may have every right to build at this site, and may even have chosen the site to send a positive message about Islam. The bigotry some have expressed in attacking them is unfair, and wrong. But ultimately this is not a question of rights, but a question of what is right. In our judgment, building an Islamic Center in the shadow of the World Trade Center will cause some victims more pain –unnecessarily — and that is not right.
Perhaps the Anti-Defamation League could produce some helpful maps to delineate the areas in our cities where Muslims may live, work and pray without causing more pain.
The original statement was linked to here by others, but it's not currently available. Via CNN.
Source: boingboing
tags: Mosque / Islamophobia / American Muslims / Anti-Islam / Muslim Americans / Anti-Defamation League / Islamic community center / Muslims in the West
Posted in Islamophobia
Wake up Americans
Connecting the Zionist dots: roots of Zionist domination of Britain and the United States
Stephen M. Walt: The Critic of My Friend is My Enemy
Fox in the henhouse: can we trust Israeli security companies?
Delusions and illusions of press freedom
In the Name Of Zionism
'U.S. Administration is Ignoring a Host of Palestinian Provocations'
United States-Israel Showdown?
The Israel Lobby's War on Al Manar TV
Was Homeland Security Created to Protect Those Who Deceived the US?
War Criminals Are Becoming The Arbiters Of Law
"Enough is Enough: A Rebuttal"
The Three Branches of the AIPAC: The Executive, the Legislative and the Judicial
How Islamophobes spread fear, bigotry and misinformation
The Sarah Palin Christian Theocracy
American Jews and the Palestinians
Democracy, Presidential Election and the Lobby
The senator, his pastor and the Israel lobby
The Power of the Israel Lobby

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Every year, we attend a large gathering of reenactors, medieval period, in western pa. in 2010, my children and i were walking around the campground late at night and observed 4-6 flying objects far off, playing with each other, flying around each other too fast for planes, helicopters. sometimes circling, almost seeming to play tag. bizarre. possible link—our group gets buzzed frequently by military planes in the area. they use our campground to practice counting populations in our tent city. our event is called pennsic, if that helps you look us up. 10-12,000 people every year.


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For the nine in 10 Americans not traveling Memorial Day weekend, an air-conditioned movie might be the best bargain around.
Hollywood came of age during the Great Depression, when movies cost as little as a dime. And even today, at $10 a ticket, the industry is beating the recession with a series of hits.
CBS News correspondent Anthony Mason reports that with blockbusters like "Star Trek," the movies are boldly going where they've never gone before.
"We're on a record breaking pace," says Hollywood.com president Paul Dergarabedian. "We're gonna hit $10 billion in revenue for the very first time ever."
U.S. box office sales are up almost 16 percent, with nearly half a billion tickets already sold this year. Attendance is up 13 percent, after it actually fell in 2008.
And the summer blockbuster season, which accounts for more than 40 percent of yearly box office, is just beginning.
The recession is good for business, Dergarabedian says, because "human beings need an escape." He calls it recession therapy.
"And at $10 a ticket, that's pretty inexpensive therapy to boot," he adds.
Even the more expensive IMAX films are drawing bigger crowds.
"Well, $20 for a ticket is a little steep. But you make exceptions," says moviegoer Chris Marino.
So popcorn has become the comfort food of this recession. But Americans aren't just going out to the movies more, they're watching more movies at home.
The rental company Netflix has seen its revenue jump 21 percent this year, after adding 920,000 new subscribers.
Netflix's Steve Swasey explains, "A rising tide lifts all boats."
Comedies, adventures, even horror movies -- they're all drawing a crowd.
"I suppose the only movie that wouldn't play well right now would be, like 'Mortgage: The Movie'," Dergarabedian quips.
If this recession were a film, the movie industry might not want it to end.


Hi, excuse my bad english, I am foreigner. I buy parts for my new PC, with AMD Ryzen 2400g and Radeon Vega 11. But drivers for my Windows 8.1 missing!!! I try Windows 10, but this system still load my disc. Windows 10 is very dissapointed for me. I have a classic mechanic disc, and his constantly noise making me crazy. Please, I must have drivers for Windows 8.1. I not want use Windows 10.
Windows 8.1 Driver support is ended (and the right thing to do).
Windows 10 & 7 Only drivers from now on..clean install windows 10 with usb.
I agree with locodicegr, try a clean install of windows 10 via usb drive...windows 10 is not perfect but not as "buggy" as windows 8.
Again, excuse my bad english.
Drivers for Windows 7 NOT WORKING for Radeon Vega 11.
If I know, what the drivers for windows 8 wil not be available, I would NEVER buy this processor. I feel like scammed (or cheated) customer.
For Linux drivers exist, for Windows 8.1 not? Really? This is bad joke?
I were AMD fanboy, but this is history. My next processor wil not be AMD.
AMD is not the only company to stop supporting Windows 8.1. or Windows 7 as far as hardware or software is concern.
For your information only, Your Ryzen 2400g (Raven Ridge) is NOT COMPATIBLE with Windows 7. The only way to get your CPU working correctly is to upgrade to Windows 10.
To the OP, you learned an important lesson. Don't buy AMD if you want good drivers and good support. Windows 8.1 is still a supported OS from Microsoft for several years to come. I stumbled upon this post because I have a workstation with a Windows 8.1 Pro license. I put a Radeon RX 580 in it, and started searching for drivers. The website only lists Windows 10 or 7. Kinda interesting since Windows 7 is being discontinued much earlier than 8.1. Even though 8.1 may not have a terribly large installed base, it is the foundation for the HUGELY popular Server 2012 R2 OS. I guess AMD never had to worry about server OS's for the past decade because their stuff sucked so bad that nobody in their right mind would put it in a datacenter. Therefore, they didn't even have to worry about drivers for 2012 R2. I guess I kinda answered my own question. Now you know.
Windows 8.1 is the worst OS in the universe and full of problems.


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Fruits of procrastination
Jagdish Chandra Bose and Plant Neurobiology
Posted by- July 24, 2020 Posted inUncategorizedTags:Bose, Jagdish Chandra Bose, neurobiology
The paper that I'll be discussing today is Jagdish Chandra Bose and Plant Neurobiology by Prakash Narain Tandon, a celebrated Indian scientist.
When I was a school student in India, I often came across JC Bose's claims of plants being sentient beings, having nervous systems, etc. However, these things were never part of the official curriculum (i.e. we never had to learn these things for tests). Bose's statements in this matter have always been considered to be something of a not-completely-scientific, "the whole universe is connected by prāna"-type of claim by the larger scientific community. This paper assets that despite initial rejection, most of Bose's claims have been proven to be correct by modern science in recent times.
By the time Bose retired from Presidency College, he was a world renowned physicist who was known to have studied radio waves even before Marconi (although the primacy debate is a complex one, there is evidence to suggest that there were scientists in Europe who had studied radio waves even before Bose). After retiring, Bose started working at Bose Institute (which he founded), and guided by his "Unity of Life" philosophy, started studying the effect of radio waves on inorganic matter. Finding their response to be "similar to animal muscle", he now started studying plant physiology (the nervous system of plants). He would expose plants to various stimuli, and record their response through the use of ingenious instruments that he himself designed. His conclusion was that the nervous impulses of plants were similar to those of animals.
Action Potentials
Bose studied both large plant parts and individual plant cells. He would connect microelectrodes to these cells, and record their response to stimuli. He concluded that plants contain receptors of stimuli, nerve cells that code these stimuli electrically and propagate these messages, and also motor organs that purportedly helped in carrying out a response to the stimuli. In this, he concluded that plants and animals have similar nervous systems. Bose said that the nervous system in plants was responsible for things like photosynthesis, ascent of sap, response to light, etc.
Bose said that the action potential of plant neurons follows the unipolarity of animal neurons. But what is Action Potential? This is an amazing video explanation of what it is. Action Potential is the electric potential via which neurons transmit messages. In resting state, the electric potential difference between the inside and the outside of neurons is -70 mV (the inside is negatively charged). When neurotransmitters activate the neuron (because a message is to be passed), this negative potential difference is destroyed by a stream of positive sodium ions that comes into the neuron from the outside. This causes lots of changes to the neurons, including inducing it to release neurotransmitters to activate the next neuron in line. The electric potential difference becomes positive, and then becomes negative again because the neuron loses a lot of potassium ions to the outside. The sodium-potassium pump on the cell membrane expends energy to exchange sodium and potassium ions to ensure that the neuron returns to its previous state before it was excited. Thus, the neuron enters the resting state again. This is the chemical mechanism by which a neuron conducts a message.
Where can one find the "nerves" of plants? Bose localized the nervous tissue in the phloem, which conducted both efferent and afferent nervous impulses. He also measured the speed of the nervous impulse, which he found to be 400 mm/sec. Although Burdon-Sanderson and Darwin had previously reported on nerve impulses in insectivorous plants, Bose's studies over the next three decades were far wider and deeper. Although ignored after the 1930s, his studies have been found to be correct by modern experiments. The author claims that Baluska et al have not only confirmed Bose's major findings, but have also advanced these further utilizing molecular biology, genomics, etc. Baluska seems to have published this paper in a journal that he himself is the editor of. Hence, these claims perhaps need to be investigated further.
Electrical Studies
Along with Action Potentials (APs) (common to plants and animals), Slow Wave Potentials (SWPs) or Variation Potentials (VPs) (found only in plants) are also used by plants to transmit nerve impulses. These SWPs do not propagate electrically, but by hydraulic pressure exerted by tissues found in the xylem. Some plants like Dionaea flytraps were found to possess unidirectional APs similar to those found in cardiac myocytes (cadiac muscle cells). This prompted Bose to poetically state that plants possess hearts that beat as long as they live.
At the molecular level, plants possess voltage gate channels (membranous proteins that are activated by change in electric potential and allow the exchange of ions), a vesicular trafficking apparatus (for the transport of proteins and other molecules within the cell plasma) etc, all of which are also found in animal cells. Trewavas also observed that water soluble ions were responsible for intra-cell communication, and also inducing changes in plants as a response to environmental conditions. We now know that there exist many such water-soluble (these are called cystolic) messengers in plants, as they do in animals.
Plant Roots
Darwin had pointed out that the tip of the radicle (found in the roots) is endowed with sensitivity, and also directs the movements of adjoining parts. In this, it is like the brain.
Bose elaborated on this by saying that the radicle is stimulated by friction and the chemical constitution of the surround soil. The cells undergo contraction at appropriate times, causing their liquid contents to go up. This causes the ascent of sap. Baluska et all carried these claims even further, and stated that within the root apex of the maize plant, there is a "command centre" which facilitates the long distance travel of nervous impulses, and instructs the plant to move towards positive stimuli (and away from negative stimuli). Tandon rejects the notion that such a command centre is anywhere near as complex as an animal brain.
Synapses- Neurotransmitters
Bose found the nerve cells in plants to be elongated tubes, and the dividing membrane between them to be the synapse (the gap between animal neurons where messages are transmitted between neurons). This claim has been substantiated by Barlow, who said that plant synapses share many characteristics with animal synapses. Plants also use many of the same neurotransmitters as animals like acetylcholine, glutamate and -aminobutyric acid.
Plant Memory, Learning and Intelligence
Bose claimed that plants are intelligent, have memory, and are capable of learning. Tandon makes the claim that Trewavas describes a large number of protein kinases in plant neural pathways, and hence finds their nervous system to be similar to that of animals. On skimming Trevawas' paper however, I mostly found it to say that although there do exist protein kinases in plants, the neural systems found in plants differ from that of animals in important ways.
In another paper, Trewavas claims that one important difference between plant and animal nervous systems is the timescale of response- plants respond much more slowly to external stimuli. Hence, we need time scale photography to properly study the plant neural response. Also, if intelligence can be thought of as "a capacity for problem solving", then plants show signs of intelligence as they change their architecture, physiology and phenotype in order to compete for resources, forage for food, and protect themselves against harsh elements of the environment.
Barlow substantiates these arguments, claiming that plants rapidly convert external stimuli to electrochemical signals, which cause them to change their physiology. He also claims that plants do have memory, as their decision making would involve recollection of previously stored memories. Barlow also says that roots experience four stimuli at once (touch, gravity, humidity and light), and have to decide how to obtain the optical mix of all. Hence, plants do possess the decision making aspect of intelligence.
With regard to plant intelligence, Baluska et al make the following claim:
'Recent advances in chemical ecology reveal the astonishing communicative complexity of higher plants as exemplified by the battery of volatile substances which they produce and sense in order to share with other organisms information about their physiological state"
Gruntman and Novoplansky from Israel also claim that B. Dactyltoides are able to differentiate between themselves and other plants, and if a plant has multiple roots, each set of roots identities the other as belonging to the same plant (and hence these roots don't compete with each other for resources). But how do plants recognize themselves and others? The authors claim that this is from the internal oscillations of hormones like auxin and cytokines. The frequency of this oscillation is unique to each plant, and can be measured externally by roots.
Bose claimed that
"these trees have a life like ours……they eat and grow…….face poverty, sorrows and sufferings. This poverty may……induce them to steal and rob…….they help each other, develop friendships, sacrifice their lives for their children"
The author finds that this sentiment is not yet fully supported by the scientific data collected by Bose. However, these claims may be further ratified when more experiments are done in this realm.
Bose single-handedly created the field of Plant Neurobiology. Although the establishment of this field has its opponents, even the most vocal of these opponents cannot find fault with any of Bose's scientific claims. The author hopes that plant and animal neuroscientists communicate better with each other in the future, and find the time and resources to study this field more. Hopefully, such studies will ratify even more of Bose's revolutionary ideas and claims.
How Plants Learn
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HomeNaija NewsCorps Members To Undergo COVID-19 Tests... See Why
Lifestylegist November 03, 2020
For safe re-opening of the National Youth Service Corps orientation camps across the country, the Nigeria Centre for Disease Control has said all corps members will be tested using the new antigen-based test.
The centre disclosed that the process of procurement of the antigen-based test was almost complete.
Speaking at the Presidential Task Force on COVID-19 briefing in Abuja on Tuesday, the NCDC Director-General, Dr Chikwe Ihekweazu, said, "We will test everyone (corps members) and that will enable us to keep our camps safe and also help us understand what is going on in the rest of the country
Ihekweazu also revealed that the government had launched Infection Prevention and Control call vanguard in all the states, noting that young people interested in IPC were being trained for self-management and self-regulation in the camps as they opened.
Ihekweazu said, "We know that corps members are normally young mobile population. Again, we are counting on not only the young people going to camps but parents; so we need the parents to support young people to do what they have to do.
"We have been working extremely hard over the last two months with NYSC to reopen youth service camps. We have made a lot of progress to ensure that we can do that safely.
Education Naija News


In 1747 Spanish privateers attacked and ransacked the town of Beaufort. Three were captured and two hanged. Come join in the fun as the event is reenacted this summer.
Where are you guys out of? You sure get around!!
The Crewe of the Archangel has members in WI, IL, PA, NJ, NY, VA, FL, TX, MI and Georgia... hope I'm not forgetting any one...ack.
I'll probably be there on Thursday and have to fly out on Sunday night.
I soooo need a vacation!


Toilets in the bathroom and intercoms on the door were the innovations of yesteryear. In future, our houses will be equipped with a level of comfort that still seems like science fiction to many of us today. But even now, household appliances deploy a wealth of sophisticated technology to save energy and make our lives easier. This potential can be further increased with a central control system. Thanks to their cross-system usage possibilities that can be intuitively controlled via an iPhone, iPad or tablet PC, bus systems will in future automate a wide and diverse range of functions.
Even in the bathroom consumers are using more technical products – from MP3 players over flatscreens all the way to computers and online services. The technologies herald an improvement in day-to-day life and stand for a new generation of products that focus firmly on their users' needs. Taking a bath has now become even more comfortable with the new Kaldewei Comfort Select. Therefore Kaldewei offers an electronic fitting with digital, user-friendly operating panel to regulate water inlet and outflow, water temperature, and shower or bath function.
Especially in times of constantly rising electricity costs, it is important to adopt an efficient approach to energy consumption. It makes sense, for instance, to charge the batteries of electric cars or start the washing machine automatically at night, when electricity is at its cheapest. Unused energy from batteries can then be used to run the refrigerator during the day.
In addition, when leaving the house, the occupant can use integrated control technology to switch off everything that doesn't necessarily need a constant supply of electricity – on a per-room basis too, if he likes. This also helps minimise the amount of electronic smog in the bedroom, for instance. The heating can be conveniently switched on at the push of a button, even when there's nobody home. And when they're on holiday, homeowners can control the blinds, lighting, hi-fi or television automatically to make it seem as if the house is occupied. In the bathroom, the tub can be filled with water at just the right temperature so you can slip straight in as soon as you get home from a tiring day at work.
Berlin real estate company Thamm & Partner goes one step further with its vision of the future. When a client buys an apartment, he is given an iPad along with the deeds – and thus also convenient online access to a concierge service. If he wants, he can then have his laundry picked up, the apartment cleaned by reliable staff, the shopping done, concert tickets booked or a table reserved at his favourite restaurant. When he's away, the concierge service takes care of the apartment, letterbox and plants. As a designated representative, the concierge is always reachable. This type of service is becoming increasingly important – especially for the elderly – and the more people become accustomed to making use of such possibilities, the lower their inhibition threshold when it comes to taking advantage of such services. As a result, people can spend more years of their lives living safely and comfortably in their own four walls. Components such as a reminder function, visual bell signals or emergency call services are easy to integrate into Bus systems. In addition, the higher technical performance level means care and service capacities can be efficiently planned and the quality of the support provided improved.
The web concierge is intended to be the helping hand in the household at any time of day or night. The service was developed as part of the intelligent living concept offered in the Berlin real estate company's construction projects. The web concierge service is for instance included in the price per square metre for a new build project in Potsdam by the name of "LEIBL22". When a client buys an apartment, he is given an iPad that he can use to contact the web concierge online at the push of a button – from anywhere in the world. The aim of the intelligent living concept is to redefine the way people live in their homes and thus set new standards in the housing industry.


I heard one of the most thoughtful PGA Tour stars refer to his peers as being "precious" during a US Open interview. That is probably accurate. Golf as a solitary sport shapes many of the stars to be more insular and reserved. Over the years there are multiple examples of champions who do not fit this generalization.
Gary Sheppard, one of PerryGolf's Golf Travel Specialists plays to a one handicap, but beyond that he has famous golf connections. His maternal grandfather is the highly respected caddie Willie Aitchison, who saw Lee Trevino home for back to back Open Championship victories in 1971 & 1972 at Royal Birkdale Golf Club and Muirfield respectively.


The need for children to be active is a core principle of the way we work at Shere. Human beings were born to be movers and living a sedentary life is a real disruption to our nature. Research shows that exercise and physical activity increases the level of serotonin, norepinephrine and dopamine, which are crucial neurotransmitters trafficking our thoughts and emotions through our body. Essentially, exercise has a profound impact on cognitive ability and mental health. Brain growth and development relies on physical movement.
At Shere we will ensure that children have access to high quality sports provision through directed lessons twice a week. In addition we will ensure that physical activity is part of each and every day, through active and explorative learning, Forest School and active break and lunchtimes. Physical education at Shere will not be limited to the gym but will be a prime element of a child's daily entitlement.


Looking to jump-start your faith life?
Spiritual Life & Campus Ministry is offering a discipleship process called The Jesus Experiment that will help you answer the question, "How would Jesus live my life if he were living it?" Pastor Brian Beckstrom will host an introductory meeting Wednesday, Jan. 18, 7 p.m., in the Chapel Commons. Everyone is welcome. Click here to find out more about The Jesus Experiment.
Consider submitting a proposal for a project to be completed domestically or internationally this summer that promotes peace and addresses the root causes of conflict among parties. Projects should be creative, engaging, meaningful, and measurable. Project drafts are due Friday, Jan. 27, to Kristin Teig Torres in Student Life. The Davis Projects for Peace committee will meet the week of Jan. 30 to rank the proposals. The top two proposals will be forwarded to Wartburg College administration by Feb. 6. Final proposals will be submitted to Davis by Monday, Feb. 13. Direct questions to Teig Torres.
Communion bread, sadly, doesn't bake itself. If you are interested in learning how to bake the bread for Sanctuary on Wednesday nights or for Sunday services, email morgan.riedesel@wartburg.edu.


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Buy now, pay later plans help to reduce online cart abandonment
By Alexi Horowitz-Ghazi
If you have shopped online recently, you may have noticed a new kind of payment option available. It's called buy now, pay later. And the basic model, offered by companies like Afterpay and Klarna, allows customers to buy what they want immediately and pay for it later in four interest-free installments. Alexi Horowitz-Ghazi of our Planet Money podcast explains the potential pros and cons.
ALEXI HOROWITZ-GHAZI, BYLINE: When you use buy now, pay later, you are essentially taking out a little loan. It's kind of like paying with a credit card.
TERRI BRADFORD: The difference is you aren't paying interest. It almost is, like, too good to be true.
HOROWITZ-GHAZI: Terri Bradford is a payments specialist at the Kansas City Fed. And she says buy now, pay later feels too good to be true because as long as you're able to clear a pretty low bar, like a soft credit check that won't show up on your credit score, these companies will front you the money for whatever you're buying. Then they'll collect in four installments from whatever bank account or debit or credit card you want. And it's all interest-free, which raises a pretty big question.
BRADFORD: Interest-free loans. Where's the value proposition in that? So where was the money coming from, right?
HOROWITZ-GHAZI: Right. Lending money is usually profitable because of some combination of interest and fees. But Terri and her research partner, Julian Alcazar, found that the fees in this model generally aren't high either.
JULIAN ALCAZAR: I was always expecting this gotcha moment. You know, when you watch a scary movie and you can hear the suspenseful music and then there's Michael Myers behind the door, that's what I was expecting.
HOROWITZ-GHAZI: It turns out instead of gouging their customers with hidden fees, buy now, pay later companies are taking their cut from the other side of the transaction by charging the businesses actually selling the goods. It costs them between 4 and 9 1/2% per sale. Credit card companies usually charge less, from 2 to 4%, which raises another question - why are more and more retailers opting for buy now, pay later?
BRADFORD: Yeah, it doesn't quite seem to jibe, does it?
HOROWITZ-GHAZI: What makes it jive (ph) is that buy now, pay later is helping businesses sell more stuff because it helps them reach people who don't usually buy on credit, people with bad credit or younger customers. On top of that, Julian says, buy now, pay later helps retailers with a problem they call cart abandonment, when people fill their shopping carts, but then when they head to check out...
ALCAZAR: They'll be like, I don't need to spend $200. What buy now, pay later does - it actually reduces cart abandonment because it makes large purchases seem smaller to the consumer.
HOROWITZ-GHAZI: Which can be dangerous. Amelia Schmarzo experienced this a couple of years ago when she started buying clothes online using buy now, pay later.
AMELIA SCHMARZO: It literally made the price, like - I kid you not - like, $8. So in that mindset, I was like, oh, my gosh, I can afford the world.
HOROWITZ-GHAZI: Amelia fell into a kind of shopping spiral. Before, her credit card statement had only ever been a few hundred dollars at the very most. But when she got the bill that month...
SCHMARZO: I saw that it was $2,000, which was my limit. And then I saw my debit card went from $700 to, like, $20. And that's when I was like, I am going to throw up.
HOROWITZ-GHAZI: Stories like Amelia's are part of the reason that regulators, like the Consumer Financial Protection Bureau, are now looking into how these services fit into existing credit regulations and mapping out the risks they may pose to the people who use them. Alexi Horowitz-Ghazi, NPR News.
(SOUNDBITE OF LOVE ABOVE'S "MOKHOV") Transcript provided by NPR, Copyright NPR.
Alexi Horowitz-Ghazi is a host and reporter for Planet Money, telling stories that creatively explore and explain the workings of the global economy. He's a sucker for a good supply chain mystery — from toilet paper to foster puppies to specialty pastas. He's drawn to tales of unintended consequences, like the time a well-intentioned chemistry professor unwittingly helped unleash a global market for synthetic drugs, or what happened when the U.S. Patent Office started granting patents on human genes. And he's always on the lookout for economic principles at work in unexpected places, like the tactics comedians use to protect their intellectual property (a.k.a. jokes).
See stories by Alexi Horowitz-Ghazi


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Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells
Jafar Vatandoost 1 , * , Behnaz Dolatabadi 2
1 Department of Biology, Hakim Sabzevari University, Sabzevar, Iran
2 Department of Biotechnology, Sabzevar Branch, Islamic Azad University, Sabzevar, Iran
* Corresponding author: Jafar Vatandoost, Department of Biology, Hakim Sabzevari University, Sabzevar, Iran. Tel: +98-5144013329, Fax: +98-5144013365, E-mail: j.vatan@hsu.ac.ir
Published Online: April 30, 2017
Accepted: March 30, 2017
DOI : 10.5812/gct.13096
Background: Selecting a host system for the expression of recombinant proteins should be carefully evaluated prior to the initiation of any bio therapeutic development programs. Since different hosts express proteins with various efficiencies and with different posttranslational modifications, changing hosts may impact the expected activity of the protein. The main expression systems have members of the mammalian cell family, however, there are remarkable differences between the species. The most generally used mammalian hosts for the production of recombinant proteins are CHO and HEK293 cells.
Objectives: In order to compare the differences between HEK versus CHO cells, the human coagulation factor IX in a transient and stable expression was examined.
Methods: After transfection of CHO and HEK cells with an hFIX-expressing mammalian plasmid, pcDNA-hFIX, transient expression was analyzed on the culture supernatant during 72 hours. The stable clones were also recovered after 10 weeks of geneticin selection. The expression and activity of the hFIX was evaluated by performing enzyme-linked immunosorbent assay (ELISA) as well as a coagulation test, respectively. The γ-carboxylation of the hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate.
Results: Although the stable CHO clones secreted hFIX protein 30% higher than HEK cells, the transient HEK cell line proved to be superior in the production of total hFIX protein (%42 increased) and functional hFIX (%29 higher) relative to the CHO cell line. Moreover, specific activity and the fully γ-carboxylated hFIX are almost constant in transient and stable expression, indicating that γ-carboxylation efficiency in both CHO and HEK cell lines is almost equal.
Conclusions: In conclusion, transient transfection studies with HEK cells provide a simple and strong method for a high production of recombinant proteins in weeks, a process which is more time consuming in the CHO cell line.
HEK Cells CHO Cells Transient and Stable Expression Coagulation Factor IX
The production of proteins in adequate quantity and quality is a necessary demand of the present time. Before we begin any bio therapeutic development program, we must pay close attention in choosing a suitable host system for the expression of recombinant proteins. Since a gene can be expressed in multiple systems and different hosts, and it can express proteins with different efficiencies as well as variant posttranslational modifications, determining the best system for expression of recombinant proteins is one of the most important issues in biotechnology. A recombinant protein expression system must be able to produce the biological material with the highest biological activity and safety while it has the lowest cost. Scientists must also consider the expression system's productivity, consistency, and the current regulatory environment (1).
There are numerous available expression systems for large scale production of recombinant proteins including Escherichia coli (E. coli), yeast, insect, and several mammalian based systems. Each of these has its own relevant advantages depending on cost, simplicity, and their post-translational modification profiles (2). Now, some famous companies are using eukaryotic systems to produce recombinant proteins. Among them, mammalian expression systems are the main expression systems for the production of recombinant human proteins. Mammalian cell expression systems have several advantages over other eukaryotic expression systems (3). Mammalian expression systems are able to fulfill the appropriate post-translational modifications, which are important for protein folding, product assemblies, and biological activity. However, it should be noted that there are differences between species. A large number of mammalian cells including myeloma (NS0 and SP2/0) cells, Chinese hamster ovary (CHO) cells, human retina-derived cells (PerC6), COS and Vero (both green African monkey kidney), Human cervical cancer (HeLa), human embryonic kidney (HEK) cells, as well as baby hamster kidney (BHK) cells have been successfully used to express recombinant proteins (2).
Although CHO cells are the first candidate cell lines for vitro cultivation and recombinant protein production, HEK 293 cell line has been extensively used as an expression tool for production of mammalian and non-mammalian recombinant proteins, both by transient transfection and by the formation of stable cell lines (4, 5). So far, the main focus of expression approaches has been on employing members of the CHO cell family, however, little has been published notably on side by-side comparisons of CHO and HEK293 cell. In addition to transfection efficiency comparison of both cells with reagent-FreeStyleTM MAX (6), polyethyleneimine (PEI) reagents (7), or in a suspension culture (8), multiple studies have reported differences in the glycosylation patterns of proteins when produced in CHO versus HEK cells (9, 10). Furthermore, a few single publications report differences in expression levels of recombinant proteins in HEK and CHO cells (11, 12). It has been showed that the expression of bispecific T cell engager (BiTE) antibody in transient HEK cells is higher than CHO cells (11). A recent review by Durocher and Butler (12) report differences in expression levels as well as biological activity. In vitro and in vivo linked to the capacity of individual host cell lines for secondary modifications such as glycosylation patterns based on stable transfected cell lines, however, similar differences may be observed in transient expression of proteins. It has been shown that high glycosylation heterogeneity for HEK293 and mono- to tetrasialylated glycans for CHO-derived material. Furthermore, sialic acid content of N-glycans was 25 % lower for HEK293- than for CHO recombinant factor IX (10).
Since the current treatment for hemophilia B patients consists of replacement therapy with recombinant factor IX (FIX) (13), selecting a host system for the expression of recombinant proteins should be carefully evaluated. On the other hand, the production of cost-effective and high-quality recombinant human FIX would have a remarkable impact on the treatment of hemophilia B patients worldwide. Moreover, to quickly evaluate proteins, it is better to use faster approaches such as transient gene expression technology (TGE) in the production of recombinant proteins. Therefore, the choice of an appropriate host cell system for a production of novel protein target is currently a question of availability of systems and experimental testing. Furthermore, it is unlikely that CHO cell and their derivatives systems will be capable of expressing all target proteins. Therefore usage of other established cell lines with different cellular origin may be extremely beneficial for production of research proteins (4).
Our aim in this work is the comparison of 2 different mammalian cell lines that are used for transfection, which can finally lead to gene expression of human factor IX.
3.1. Materials
All cell culture reagents were provided from the thermo fisher scientific, except for penicillin G as well as streptomycin (Sigma-Aldrich). Geneticin (G418) and protease inhibitors were purchased from Roche.
3.2. Construction of Recombinant Plasmids
The cDNA of hFIX (1.4 kb) was PCR-amplified from a human liver cDNA library with pfu-DNA polymerase, using oligonucleotide pairs, hFIX-KpnI (5GGGGTACCGCCACCATGCAGCGCGTGAAC3) and hFIX-XhoI (5 CCGCTCGAGATCCATCTTTCATTAAGTGAGC3) as forward/reverse primers. Following subcloning into T-vector, the KpnI-XhoI fragments were inserted into the pcDNA3 vector, 3' to the CMV (cytomegalovirus) promoter. The resulting plasmid was designated pcDNA3-hFIX (Figure 1). The identity of the fragments was confirmed using restriction digestion followed by nucleotide sequence analysis.
Figure 1. Plasmid Construction Diagram
The hFIX cDNA was amplified from liver cDNA library and ligated to pTZ57RT. Following KpnI/XhoI digestion of T.V-hFIX, hFIX cDNA was subcloned to pcDNA3 plasmid.
3.3. Transfection and Preparation of Stable Clones
CHO and HEK cells (a kindly gift of Dr. Zomorodipour, NIGEB, Iran) were grown in a 5% CO2 and 37°C, subcultured at a density of 2 × 105 cells in a volume of 2 mL in 6-well plates (13), and transfected with 2 µg pcDNA3-hFIX using the calcium phosphate method. Individual clones were expanded in the presence of 450 µg/mL Geneticin for 2 months. The expression media containing 6 µg/mL vitamin K1 and 10% (v/v) FBS were added to ~70% confluent cells, upon which the individual clones were screened for FIX production during 3 days.
3.4. RNA Preparation and RT-PCR Analysis
Total RNA from 106 cells expressing human FIX was extracted according to the protocol provided by the manufacturer (Tripure kit). First-strand cDNA synthesis was performed using random primers and M-MuLV reverse transcriptase (Sinaclone, Iran). The generated fragments were subsequently used as a template for the PCR-amplification of the double stranded cDNA, corresponding to the hFIX coding sequence.
3.5. Quantitative and Qualitative Analysis of Human Factor IX
Human FIX was quantified in a conditioned media employing an ELISA (Asserachrom IX:Ag, Stago, France) following the procedure provided by the manufacturer. In addition, intracellular accumulated FIX was assessed, for which, the cells were centrifuged at 100 g for 5 minutes, upon which pellet were suspended in 500 µL of ice-cold lysis buffer (100 mM KCl, 2 mM MgCl2, 10 mM, HEPES pH 7.5, 0.5% Triton X100) containing an antiprotease mix (complete Protease Inhibitor, Roche). Subsequently, the lysate was centrifuged at 12,000 g for 10 minutes at 4°C, upon which the supernatant was assessed for FIX. The aPTT assay was also used to examine functional activity of FIX (13). Human plasma immuno-depleted of FIX (100 µL; Stago) was mixed with conditioned media (100 µL) and aPTT reagent (100 µL; BioMerieux). After 3 minutes of incubation at 37°C, 100 µL of a prewarmed CaCl2 solution (25 mM) was added to the mixture, and the clotting time was recorded. Normal human plasma (prepared from the Iranian blood transfusion organization) was used as a reference and 1 unit of FIX activity corresponded to the amount of FIX in 1 mL of normal plasma (~ 5 μg/mL).
3.6. Quantification of γ-carboxylated Factor IX
The barium citrate precipitation of γ-carboxylated human FIX was adapted from previously described methods (14). Briefly, sodium citrate (14 mg) and 1 M barium chloride (95 µL) were added to 2 mL of conditioned media, which were incubated for 1 hour at 4°C with gentle mixing. The mixture was centrifuged at 100 g for 5 minutes and the supernatant was kept for the analysis of unabsorbed FIX lacking γ-carboxyglutamic residues. The pelleted γ-carboxylated FIX was washed with ice-cold 5 mM BaC12, subsequently centrifuged at 100 g for 5 minutes, suspended again in 0.1 M sodium citrate (1 mL), and adjusted to 10% ammonium sulphate to dissolve the adsorbed hFIX. After 30 minutes, at 0°C, and subsequent centrifugation at 100 g for 5 minutes, after which the barium sulphate pellet was discarded, FIX was determined in the supernatant by ELISA as described.
The ANOVA program for analysis of variance followed by a Tukey post-hoc test was performed for the evaluation; P < 0.05 was considered statistically significant. All statistical analyses were carried out with SPSS 16 (SPSS Inc., Chicago, IL, USA).
4.1. RNA Expression Analyze
Factor IX (FIX) expression by the CHO and HEK cell lines was further confirmed employing RT-PCR, which demonstrated the presence of FIX mRNA in both cell types (Figure 2).
Figure 2. Gel Electrophoresis of RT-PCR Products of hFIX from CHO and HEK Cell Lines
Electrophoresis of PCR products obtained by RT-PCR on 2% agarose gel electrophoresis. Lane 1, 1kb DNA ladder (thermo scientific); lanes 2 and 3, the amplification product of hFIX (1389 bp) in CHO and HEK cell lines, respectivly; lane 4, negative control.
4.2. Selection of Stable Clones Expressing Factor IX
The selection of single cell-derived CHO clones that stably express human FIX was performed in a period of 2 months and resulted in the selection of 12 high-producer clones. Selection of an hFIX-expressing cell line derived from stable transfected HEK cells was performed in the time span of 10 weeks, and the numbers of stable transformed HEK cell lines, which express rhFIX (4 clones), were lower than stable CHO cells. This suggests that efficiency of delivery to the target region of genome, loss of the plasmid, and resistance to antibiotic may contribute to differences in these cellular vehicles.
4.3. Characterization of rhFIX Secreted from Transiently Transfected HEK and CHO Cell Lines
To evaluate the transient expression of rhFIX in the HEK and CHO cells, both conditioned media and cell lysates were examined for FIX antigen levels. While the CHO cell line secreted up to 96 ng/mL/106 cells FIX after 72 hours, FIX expression time in the media by the HEK cell line was increased up to 42% and accumulated to 137 ng/mL/106 cells (Table 1). Consistent with these findings, the HEK cell line expressed up to a 29% higher FIX activity as compared to that of the CHO cells (58 vs. 45 mU/mL/106 cells). However, the CHO-expressed FIX displayed a 20% enhanced specific activity relative to that expressed by the HEK cell line (0.48 vs. 0.4 U/mg). Taken together, these findings show that the transiently transfected HEK cell line produces higher levels of functional rhFIX as compared to the CHO cell line.
Table 1. Factor IX expression by transfected HEK and CHO cells. Factor IX expression was determined in conditioned media after 72 hours of FIX expression. The data are the mean ± SD of 3 similar experiments.
FIX Antigen in median, /mL/106 Cell
FIX Activity, mU/mL/106 Cell
FIX Specific Activity, U/mg
Recovery%
CHO 96 ± 4 45 ± 1 0.46 80
HEK 136 ± 5 58 ± 3 0.40 78
4.4. Expression of rhFIX from Stable HEK and CHO Cell Lines
After a selection of single cell clones that stably express rhFIX, comparison of the rhFIX expression following expansion of these clones in a 25 cm2 flask to 95% confluence revealed that the stable CHO cells are capable of expressing up to 30% more of human FIX proteins as compared to HEK cells (148 vs. 113 ng/mL/106 cells, Table 2). Consistent with the findings obtained on the FIX antigen levels, the activity levels of CHO-expressed FIX were also increased by 40% in comparison with those of the stable expressing HEK cells. Specific activity of rhFIX in both cells was 0.3 U/mg.
Table 2. Factor IX expression by stable HEK and CHO cells. Factor IX expression was determined in conditioned media after 72 hours of FIX expression. The data are the mean ± SD. of 3 similar experiments.
FIX Antigen, ng/mL/106 Cell
FIX Activity, mU/mL 106 Cell
CHO 148 ± 3 47 ± 2 0.3 82
HEK 113 ± 4 33 ± 2 0.3 79
4.5. Recovery
In order to assess the FIX recovery, the general characteristics of vitamin K-dependent (VKD) proteins, which can be absorbed to barium citrate via it's γ-carboxylated, glutamic residue was used to find out whether the recombinant cells were capable of the proper γ-carboxylation of human FIX (15). During this process, neither poorly γ-carboxylated molecules nor non- γ-carboxylated were trapped by the barium action and were remained in the soluble fraction; the barium-bound hFIX can be subsequently eluted from the precipitate (15). Following barium citrate precipitation, the supernatant obtained from the HEK and CHO cell lines was enriched for FIX. Quantification of FIX recovered from the barium citrate precipitation showed that around 80% of the CHO and HEK cell-produced FIX was recovered employing barium citrate adsorption (Table 2), and therefore was most likely fully γ-carboxylated. Moreover, the amount of fully γ-carboxylated rhFIX is similar in CHO cells rather than HEK cells.
4.6. Secretion Efficiency
As defined, the secretion efficiency of a protein is the secreted fraction ratio to its total amount (16). On the basis of the data obtained, the secretion efficiency of FIX in transient HEK and CHO cells were 68% and 81%, respectively. The secretion efficiency of human FIX by both of the stable HEK and CHO cells reached up to 66% and 78%, whereas the intracellular FIX content remained minimal all through the experiment.
Over the past decades, the production of r-proteins has been successfully undertaken in a variety of mammalian cell lines, however, there are still some concerns regarding the expression levels and long periods of clonal selection when moving forward toward a large-scale production for therapeutic purposes. The most commonly used mammalian hosts for the production of r-proteins are CHO and HEK293 cells, although other mammalian cell types have also been used. We generated a clonal, FIX-expressing HEK cell line, which required up to 10 weeks of selection. In contrast, using the CHO cell system we were able to generate a clonal cell line in the short time period of 7 weeks. Moreover, the number of stable clones was small in the case of HEK cells. This was surprising because during the time transfectability in HEK cells was higher comparing to that of CHO cells, the time of stable clones preparation and the number of high producing clones were less. It has been shown that the transfectability of HEK cells is 90% versus 40% of CHO cells, when using the calcium phosphate transfection method (17). Gonçalves has also indicated that the transfection of HEK293T cells in a suspension is more efficient than transfection of CHO cells in terms of the number of transfected cells (8). Liu et al. has also shown that transfectability of HEK293 cells is extremely high and able to tolerate a wider range of cultural conditions during transfection, although transient transfection efficiency with cationic lipid-based transfection reagent-FreeStyleTM MAX is similar in both 293-F and CHO-S cells (6). Suen et al. indicated that although transfection of HEK cells is generally performed using inexpensive transfection reagents such as polyethyleneimine (PEI), the CHO cells are commonly transfected with more expensive lipofection reagents (7). Therefore, although HEK cells are generally more likely to be transfected than CHO cells, the generation of high-producing stable HEK cell clones is more time-consuming than CHO cells. This may be due to these cellular vectors, which are usually derived from different species and tissue source and are likely to differ in their efficiency of delivery to the target region, loss of the plasmid, as well as resistance to antibiotics.
We also noted that although the stable CHO clones secreted slightly higher levels of hFIX protein than HEK cells (30% higher), transient transfection of CHO cells leaded to a very poor protein production (42% lower). In contrast, the transient expression of hFIX in HEK cells resulted in a quick production of secreted protein and more important, the use of the same plasmid vector as for the stable CHO cell clone generation. In this regard, Diepenbruck, C. et al. noted that although stable CHO clones secreted slightly higher levels of r-protein than HEK cells (17% higher), transient HEK cells have higher expression r-protein than CHO cells (11). They also showed the CHO cells are not well suited to deliver reasonable amounts of protein following transient transfection (11). Suen et al. has also indicated that the protein expression yield is generally 2 - 5 times higher for PEI-transfected HEK vs. CHO cells (7). Other studies has also indicated that using a CHO-based transient production of proteins was limited by poor transfection efficiencies, viabilities, and production of insufficient quantities of recombinant proteins so that transient production of recombinant scFv-Fc antibodies in HEK293 is efficient and robust (18). The productivity of transient transfections depends on various factors such as the amount of cell population transfected, the gene transcription and translation efficiency in the host cells, innate property of producer cells related to protein folding and the extent of post-translational processing, as well as modification required for optimal protein function (6). It has also been shown that mRNA stability is very important for the transgene expression levels and mRNA stability in transient HEK cells is more than CHO cells (19, 20). Liu et al. showed that the EPO specific mRNA expressed in transient HEK cells remained constant, whereas the EPO mRNA level has been dramatically reduced in CHO cells after 4 days post-transfection (6). On the other hand, although a threefold elevation of EPO mRNA level was expressed in HEK cells compared to CHO cells, the maximal accumulations of EPO were greater in FreeStyleTM CHO, being sevenfold higher than the maximum accumulations obtained from FreeStyleTM HEK cells (6). It could be due to the lower secretion efficiency of HEK cells rather than that of CHO cells. We also indicated that the secretion efficiency in CHO cells was 20% more than HEK cells, which may be due to provision of different intracellular signaling pathways such as protein glycosylation and folding (5).
The hFIX as a vitamin K dependent protein (VKD) undergoes multiple post translational modifications (PTMs), among them, γ-carboxylation has a considerable effect on its biological activity (21). Our findings suggest that the FIX produced in both CHO and HEK cell lines was properly γ-carboxylated, since up to 80% of both cell-produced FIX was recovered employing barium citrate adsorption, indicating that the majority of the FIX produced was γ-carboxylated. It was also interesting that the specific activity and the fully γ-carboxylated FIX were almost constant in both cell lines in transient and stable expression, indicating that the carboxylation efficiency in both CHO and HEK cell lines has been almost equal.
High functional FIX expression levels are another requirement for a successful therapeutic protein production. In our expression systems, the transient HEK cell line proved to be superior in the production of total FIX protein (42% increased) and functional FIX (29% higher) relative to the CHO cell line. It is important to note that due to the increasing demand for the fast and efficient production of proteins in expression systems, many industrial laboratories prefer to use the host cell line in transient gene expression (TGE) system to produce r-proteins for pre-clinical purposes. Although the best method for large-scale production of recombinant proteins in mammalian cells is to establish stable clones, this approach can be laborious, expensive, and time-consuming. As an alternative, the large-scale transient transfection of mammalian cells can produce remarkable amounts of recombinant protein in a very short period of time (5, 22). The ideal mammalian cell line for the transient production of r-proteins is highly susceptible to transfection, naturally inclined to have a high cell specific productivity, and easy to maintain on a large-scale (23). Although, for the stable expression of recombinant proteins, CHO cells are still prior to HEK293, however, the HEK293 is the preferred cell line for its transient expression. This is attributable to the fact that the large-scale transfection of CHO cells has low efficiency and low productivity when cost-effective transfection reagents are used. On the other hand, CHO cells, which are widely used for stable transfection, are not well suited to deliver adequate amounts of protein following transient transfection (11). HEK cells have generated the highest volumetric yields in TGE systems, and are therefore the most widely used (23). For many applications, the use of transient HEK, rather than CHO cells, appears to be superior, since HEK cells are generally more easily transfected (7).
In conclusion, our findings suggest that the large-scale transient HEK cell system has the potential of reducing the cell development time significantly, while maintaining FIX function. More studies and extensive efforts need to be carried out on this expression system in order to maximize protein production in transient expression systems by the optimization of host cell lines, vector systems, and cell culture conditions. Overall, these observations support that the HEK cell system is a respectable candidate for the efficient production of recombinant FIX.
The authors greatly appreciate the gift of HEK and CHO cell line from Dr A. R. Zomorodipour, national institute of genetic engineering and biotechnology, Iran. This study has been performed at the Hakim Sabzevari university in 2016, supported by the center for international scientific studies and collaboration (CISSC) as well as received a grant (number, 200) from Hakim Sabzevari university.
Implication for Health Policy Makers/Practice/Research/Medical Education: Our findings suggest that the large-scale transient HEK cell system has the potential of reducing the cell development time significantly, while maintaining FIX function.
1 Kim KR, Kim YK, Cha HJ. Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture. J Biotechnol. 2008; 133(1): 116-22[DOI][ PubMed ]
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8 Goncalves C, Gross F, Guegan P, Cheradame H, Midou P. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1. Biotechnol J. 2014; 9(11): 1380-8[DOI][ PubMed ]
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10 Bohm E, Dockal M, Graninger M, Hasslacher M, Kaliwoda M, Konetschny C, et al. Expression of recombinant human coagulation factors VII (rFVII) and IX (rFIX) in various cell types, glycosylation analysis, and pharmacokinetic comparison. BMC Proc. 2011; 5 Suppl 8[DOI][ PubMed ]
11 Diepenbruck C, Klinger M, Urbig T, Baeuerle P, Neef R. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells. Mol Biotechnol. 2013; 54(2): 497-503[DOI][ PubMed ]
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13 Vatandoost J, Zomorodipour A, Sadeghizadeh M, Aliyari R, Bos MH, Ataei F. Expression of biologically active human clotting factor IX in Drosophila S2 cells: gamma-carboxylation of a human vitamin K-dependent protein by the insect enzyme. Biotechnol Prog. 2012; 28(1): 45-51[DOI][ PubMed ]
14 Vatandoost J, Bos MH. Efficient expression of functional human coagulation factor IX in stably-transfected Drosophila melanogaster S2 cells; comparison with the mammalian CHO system. Biotechnol Lett. 2016; 38(10): 1691-8[DOI][ PubMed ]
15 Jallat S, Perraud F, Dalemans W, Balland A, Dieterle A, Faure T, et al. Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines. EMBO J. 1990; 9(10): 3295-301[ PubMed ]
16 Kim YK, Shin HS, Tomiya N, Lee YC, Betenbaugh MJ, Cha HJ. Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. Biotechnol Bioeng. 2005; 92(4): 452-61[DOI][ PubMed ]
17 Preuss AK, Connor JA, Vogel H. Transient transfection induces different intracellular calcium signaling in CHO K1 versus HEK 293 cells. Cytotechnology. 2000; 33(1-3): 139-45[DOI][ PubMed ]
18 Jager V, Bussow K, Wagner A, Weber S, Hust M, Frenzel A, et al. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol. 2013; 13: 52[DOI][ PubMed ]
19 Tienen LM. Transient gene expression in mammalian cells for the production of recombinant protein therapeutics. 2012;
20 Sarah W. Transient recombinant protein expression in mammalian cells: The role of mrna level and stability. 2009;
21 Tie JK, Zheng MY, Pope RM, Straight DL, Stafford DW. Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function. Biochemistry. 2006; 45(49): 14755-63[DOI][ PubMed ]
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Jafar Vatandoost: [PubMed] [Scholar]
Behnaz Dolatabadi: [PubMed] [Scholar]


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Sound Magic Amati Violin Captures The Sound Of A Rare Instrument From 1664
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A nationalism that's anti-national
CSSNational Affairs 0
The recent outreach by the Rashtriya Swayamsevak Sangh (RSS) at Vigyan Bhavan in Delhi seems to have succeeded in its principal objective: an image makeover for a niche audience. Thanks to an obsequious media and a commentariat ever willing to suspend disbelief, the event has yielded the soft, liberal gloss the RSS needed and desired. Sadly, the critics limited themselves to questions that the RSS anticipated, indeed wanted: Does the RSS exercise influence on this government? Is the RSS anti-Muslim?
It is time we asked a harder and deeper question: Is the RSS anti-national?
On the face of it, this is an odd question. Nationalism, Indian-ness and Hindutva are very much the calling card of the RSS. This is not put on. I have known the RSS from inside and outside. Having met hundreds of swayamsevaks and many pracharaks, I know that an average RSS volunteer carries this nationalist self-image. I can also attest that just like the communists or old-time socialists, an average RSS worker tends to be more honest and idealist than a run-of-the-mill political leader. I am aware that on more than one occasion, the RSS has done exemplary rescue and relief work during national disasters. If anything, its critics accuse it of being ultra-nationalist. Thus, to question its nationalist credentials might appear outrageous.
Yet this question needs to be debated in all seriousness and all fairness. Given the salience of the RSS in our national public life today, this is a pressing question. We worry, rightly so, about the impact of Islamic fundamentalist groups and Maoist insurgents on our nation. We debate, as we should, the challenge posed by separatism in Kashmir and Nagaland to our nationhood. But we no longer debate with any seriousness the challenge posed by the RSS and its associates to the project of nation-building the Indian nation. The question is about the theory and practice of the RSS as an organisation and its relation to the Indian nation, its past, present and future.
The nation and the past
Let's begin with some indisputable facts about its past. Right from its inception in 1925, the RSS was not in any way active during the national movement. In fact, its associates such as the Hindu Mahasabha actively opposed the national movement. It is also a well-documented fact that V.D. Savarkar, whose ideology inspired the RSS's founders and who remains its icon, was released from Cellular Jail in the Andaman and Nicobar Islands after he wrote four mercy petitions to the Viceroy pledging loyalty to the British empire. After his release, he lived off a stipend from the British government and obeyed faithfully the conditions it had imposed on him. Syama Prasad Mookerjee, another Hindu Mahasabha leader, actively collaborated with the British during the Quit India movement while the RSS kept aloof from this biggest anti-colonial uprising. The two-nation theory was propagated by Hindu nationalists, much before the Muslim League. And it is no secret that Nathuram Godse was once an RSS member and was very much a part of its extended family when he murdered Mahatma Gandhi. Bluntly put, the RSS made zero, if not negative, contribution to the national struggle. But that is not sufficient to dub it anti-national today.
The role of the RSS after Independence is more relevant here. How did the RSS contribute to the project of nation-building? Sadly, the answer is again in the negative. The RSS was among the few organisations in independent India that refused to honour some of the key symbols of the Indian republic: the national flag, the national anthem and, of course, the Constitution of India. It speaks volumes that the head of the RSS has to clarify, nearly seven decades after the promulgation of the Constitution, that his organisation believes in it, something explicitly contradicted by his predecessor. Notwithstanding its recent claims to the contrary, the RSS does not quite subscribe to any of the key tenets of the Constitution: socialism, secularism, federalism and, indeed, democracy.
In practice, far from being a part of the solution, the RSS was always a part of the problem that India faced in its difficult journey of nation-building. The legacy of Partition and the challenge of bringing together immense diversities posed an unprecedented challenge to the nascent Indian nation. During this delicate phase, the RSS was at best an irresponsible denominational pressure group for the Hinduisation of the Indian state, opposing any and every concession to minorities and advocating a hawkish foreign policy. At worst the RSS became a fulcrum of organised subversion of the constitutional order, as in the demolition of the Babri Masjid in 1992. If constitutional patriotism is the heart of national political life, the RSS has repeatedly stood in opposition to the nation.
More than anything else, it is the theory and practice of its nationalism that shows the RSS to be a European import, out of sync with Indian nationalism. The RSS subscribes to the now outdated European model of nation-state which assumed that the cultural boundaries of a nation must match the political boundaries of a state. In Europe it meant a uniform race, religion, language and culture as the defining features of a nation. In India it meant Hindu-Hindi-Hindustan, the slogan coined by Savarkar. India's home-grown nationalism challenged this European model and its futile and bloody quest for matching cultural and political boundaries. Instead, Indian nationalism was about creating political unity in conditions of deep diversity of culture, religion and language.
Paradox of its workings
Today, as a rapidly diversifying world seeks to learn from the Indian model, the RSS clings on to an alien, borrowed and fractious understanding of nationalism. Worse, its model of separatism of the majority is clearly the biggest obstacle for Indian nationalism. Isn't it odd that an organisation that claims to work for national integration has, or has had, little time and energy for an amicable resolution of some of the issues that challenge our national unity? These include intractable regional disputes (the Karnataka-Tamil Nadu and Punjab-Haryana water disputes), intra-regional tensions (demand for Telangana or Vidarbha), language issues (Punjabi-Hindi, Kannada-Marathi) or differences with racial and ethnic dimensions (violence against migrants from the Northeast in Bengaluru, Hindi speakers in Mumbai).
The RSS version of nationalism comes into play only when there is a religious angle to any issue. It is not that they care for Hinduism either. The RSS ideologues have little knowledge of or interest in Hindu traditions. In fact, the version of Hinduism that it seeks to impose is itself a parody of orthodox Islam and orthodox Christianity and against the basic spirit of Hinduism, let alone the spirit of humanism that informs all religions. Unfortunately, the principal focus of the RSS has been to foment Hindu-Muslim differences, division and hatred. Since Hindu-Muslim violence poses the biggest single threat to national unity today, those who work for the exacerbation of Hindu-Muslim tension must be seen as anti-national, and guilty of treason.
The secessionists challenge the territorial integrity of India. The left-wing extremists challenge the writ of the Indian state. The challenge posed by the RSS is much deeper: it challenges the very idea of India, the swadharma of the Republic of India. If this is not anti-national, what is anti-national?
I am not for a ban on the RSS. Its theory and practice represent a cultural-political malady that needs a deeper cure rather than a ban. It originates in an inferiority complex of a modern Hindu, made worse by a westernised, deracinated form of our secularism. This might sound odd, but what the RSS needs is exposure to Indian culture and its multiple traditions, greater appreciation of culturally more confident Indians such as Tagore and Gandhi and a deeper understanding of Hinduism itself. If it introspects rather than hold an outreach at Vigyan Bhavan, I am sure its Sarsanghchalak would recommend to the RSS what Gandhiji suggested to the Congress party: dissolve itself.
Yogendra Yadav is the President of Swaraj India
https://civilservicesstrategist.com/author/css/
भारतीय संगीत की उत्पत्ति और विकास
भारतीय इतिहास के स्त्रोत
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