Tag Archives: Genetics/Epigenetics

Posts about genetics and epigenetics.

Oh, noes, GMOs!

Oh, noes, GMOs!

GMO-corn

Everyone calling vociferously for labeling GMOs on the internet seems to go silent when they are asked specific questions about why, and how much labeling they’re actually asking for. Turns out, they usually don’t know how genetic modification is done, how many different kinds of modifications there are, how much actual potential harm there is or isn’t, or, quite frankly, how digestion works. (If it worked the way some alarmists believe it does, I’m afraid we might have to turn to cannibalism!)

Labeling something “Contains GMOs” is not only uninformative and misleading, but will add an average of $500 to each American’s food bill if it were to be instituted. Also, in order for a label to be useful and valid, it would need to be much more detailed. So I would like to break it down a little more realistically.

BT TOXINS!!

Bacillus thuringiensis is applied liberally on organic crops to control pests. Catalogs that sell Bt to home gardeners describe it as “Bacillus thuringiensis (Bt) is a natural occurring, soil-borne bacteria that has been used since the 1950s for natural insect control.” (Planet Natural) and “Bt is a naturally occurring bacteria with many powerful insect-specific strains. Like other biologicals, Bts biodegrade in sunlight and may require reapplication. Bt for Caterpillars & Worms: Safe for the user and the environment, Bacillus thuringiensis v. kurstaki is a pest control mainstay for organic vegetable growers.” (Grow Organic) You would not find any food in the supermarket that would be labeled “Genetically modified with Bt,” because those crops are not used to feed people, but for animal feed and other industrial uses. You would, however, find lots of foods labeled “Sprayed with Bt,” at least if labeling were honest.

So why is it that Bt is safe and organic when sprayed in large quantities (where it drifts and affects insects that are not feeding on the crops, including some beneficial species) but suddenly becomes “Bt toxin” when it is engineered into the crop and affects only the pests that feed on the crops? The EPA has done thorough testing on Bt (http://www.epa.gov/pesticides/biopesticides/pips/regofbtcrops.htm) and assured that GM crops with the gene that produces the Bt protein are not in foods meant for human consumption, even though humans do not have the body chemistry that allows Bt to be absorbed.

Big Organic wants to have its cake and eat it, too. In order to continue using Bt itself for pest control, but demonize it as a toxin when it’s made by the plant itself, the very sites that make these statements do some unscientific speculation as to how this is so and present it as factual. Were they to admit that Bacillus thuringiensis is Bacillus thuringiensis and is harmful only to specific species (not human) that are directly exposed to it, they would not be able to continue their hypocritical campaign to use and sell it while simultaneously representing it as a life-threatening dangerous substance.

ROUNDUP READY!!

You would see “Roundup Ready,” but that would be pretty uninformative, also, because many crops that are not “Roundup Ready” are treated with Roundup, because it is an effective dessicant. For example, a wheat farmer would use it to kill and dry his entire field so that all the wheat would be usable, and would not need careful (and expensive) sorting to ensure that a few green grains wouldn’t rot an entire silo of harvested wheat. So, GMO or not, a label saying “glyphosate exposed” would be much more useful. That, however, would be a decision one should make based on environmental concerns rather than personal ones, because glyphosate is toxic to humans in such large doses that you would need to drink about three gallons of it straight to get sick.

THERE’S FISH GENES IN MY TOMATOES!!

There would also have to be a label for trans-species modification. Scientists take a gene for a trait from one species (usually another species of something that we also eat, so we’re eating that gene already, just in some other food) and insert it into another. You would need to do some serious mental gymnastics to see how this would be harmful. You would also have to start giving up a lot of foods, organic or otherwise, because this is also used to protect crops against diseases that would wipe them out. Bananas and papayas and oranges would no longer exist, or might go extinct in the future, without the modifications that allow them to resist the fungi that kill them. You might also want to check out foods that contain other foods, and perhaps stop using recipes. Your Manhattan Clam Chowder has fish genes and tomato genes. . .

THERE’S FISH GENES IN MY FISH!!

The last label would be a cross-species modification. This is when a gene for a particular trait is taken from one species and transferred to a related species – like the gene from one type of salmon that triggers larger size to a smaller sized salmon. Again, if you were to avoid foods with this label, you would need to deny yourself foods that have been cross-bred and hybridized by man for thousands of years, which would be everything we eat. It’s the same process, but accelerated and without the negative characteristics of traditional manipulation by sexual selection.

Look at what we’ve done to purebred animals – hip dysplasia in German Shepherds, seizures in Boxers and Spaniels. . .When we tried to breed a rot-resistant potato by hybridization, we ended up with a potato that was kinda poisonous. Genetic modification is working on a rot-resistant potato that won’t make you sick.

GENES FROM THE SAME PLANT!!

Golden rice was created by moving a gene that produces Vitamin A from the leaves and stem of the plant to the grain. This is a technology that may be applied to other species later on. People destroying entire crops of golden rice because it’s GMO is an example of uninformed hysteria. So we’d need a label for this at some point.

If all you want is a nice, simple label that says “Contains GMOs” so you can make buying decisions without thinking, then stick with buying things that say “GMO-Free.” The GMO labeling being proposed by the Organic Foods Industry is not designed to inform or help people make healthy decisions, but to direct buyers to their own products. If you want labels that actually give you useful information, they’re going to be on almost every item in the store, and it’s going to cost all of us. And if you really want to know what’s in your GM food, check the EPA, the ISAAA’s GM Approval Database, and consumer information from the FDA.

If you want to see why the studies being cited as proof that GMOs are dangerous are not valid evidence, here are a few links. Academics Review looks at a large selection of studies and explains what they actually found and whether those findings are accurate. The Seralini rat tumor study was so deeply flawed that even a low-impact journal retracted it out of embarassment – lots of scientific explanation and criticism is collected at David Tribe’s blog. Skeptical Raptor breaks down the information in a recent meta-analysis of 1,783 studies, including at least 600 independently funded, which found no tangible dangers and many benefits of GM crops.

(Image source Also a good article!)

Where I Go For Science

Where I Go For Science

A friend of mine asked me for a few links to science sites so she could learn a little more, so I set to copying and pasting my bookmarks for her. Now I know why I lose so much time sitting at the computer. Most of these sites are life sciences, so sorry about the lack of Chemistry and Physics and such. Here’s the list. . .

Sites in my WordPress Reader, loosely arranged by subject:

Skepticism/Critical Thinking
Science or Not?
I fucking hate pseudoscience
Edzard Ernst
Why Evolution is True
Doubtful
Violent Metaphors

Brain Stuff
Neurobollocks
Left Brain Right Brain
Mind Hacks
Neurologica Blog
Wiring the Brain
Science Over a Cuppa
Gabriela Tavares
BPS Research Digest

Medicine
Science Based Medicine
Science-Based Pharmacy
Science-Based Life
Drug Monkey

Genetics/Epigenetics
Bits of DNA
Code for Life

Vaccination/Disease
Skeptical Raptor’s blog
Shot of Prevention
The Poxes Blog

Other. . .
Inspiring Science
Double X Science
Bishop Blog

Not on wordpress:

Not Exactly Rocket Science Not only a lot of interesting articles on Biology, but a weekly roundup of interesting links. (You can also visit The Loom and Only Human from here, plus some others, but these three are my favorites.)
In The Pipeline Chemistry, but a lot of it related to Pharmaceuticals.
Skeptical Medicine A critical look at both conventional medicine and pseudoscience.
Scitable Nature Publishing Group’s educational site.

Aggregators:

Phys.org
Research Blogging
Science News (limited access for free, but still a lot of good science.)
Science Seeker (you can filter what you see by checking the subject boxes to the right.)

I’m always checking for new places, especially those that would be good for people who are not scientists, but want to understand. I’ll take suggestions for anything that’s not behind a paywall or too difficult for non-academics!

Wednesday Links

Wednesday Links

genebrain

Genetic research has a meaningful place in psychiatry, as a major study has just found out. Thomas Insel of the NIMH blogs about the impact of a study on schizophrenia and explains its importance. 108 gene regions, put together, show a significant increase in the risk for the condition, and with 37,000 affected participants and over a hundred thousand controls, this is pretty big. Thank goodness several hundred million dollars have just been donated to psychiatric research.

What is complex about complex disorders? A paper by Kevin Mitchell explains what’s involved in finding the genes that contribute to polygenic disorders like ” schizophrenia, autism, depression, asthma, epilepsy, diabetes, rheumatoid arthritis, hypertension, coronary artery disease, obesity, Crohn’s disease, Alzheimer’s and Parkinson’s disease, multiple sclerosis and probably hundreds of other conditions”. Perhaps some of these will be discovered now that more funding is available!

Is “reductionism” in behavioral genetics a boon or curse? asks if and when reductionism is a bad thing. In behavioral genetics, most scientists are looking for complex genetics behind complex traits, but they need to be careful of how their public statements can be read. The author points out, “There is a difference between methodological reductionism, a tool, and philosophical reductionism, a guiding principle.”

Evan Thompson on core theories of neurophenomenology and time-consciousness opens, “Evan Thompson, one of the authors of 1991′s The Embodied Mind: Cognitive Science and Human Experience, in 2010 authored a sweeping, dare I say even magisterial, account of how science and philosophy should understand consciousness, embodiment, evolution, and neuroscience.” The piece that follows is brief but covers a lot of ground – and makes me interested in reading the book.

An interesting neurological phenomenon is auditory pareidolia – She’s Hearing Voices talks about this symptom that’s common in certain mental disorders and how even ordinary people can be prompted to hear things that aren’t there. In schizophrenia and OCD and certain types of depression and personality disorders, this may be a magnification of what is normally an adaptive trait, IMO.

Shakespeare, Vermeer, and the “Secrets” of Genius takes the almost revolutionary position that practice does not necessarily make perfect – sometimes you have to be born with talent.

Most of Us Still Don’t Get It: Addiction Is a Learning Disorder questions the idea that we have genes or areas in our brain that predispose us to certain addictions. I read it and thought that perhaps all addiction could be characterized as a salience disorder, because it takes the position that it’s a maladaptive state of a survival trait. Just read.

Wednesday Links

Wednesday Links

Sorry this is short. Time just got away from me. Enjoy!

Why all medical professionals need to study evolution. I think everyone should, period.

Excellent piece on gender disparities in the study of Autism by Virginia Hughes. This applies to ADHD, too, and it would be nice to see something this well-written on that.

Dorothy Bishop points out the shortcomings in a neuroimaging and genetics study, and in doing so, tells you some things you should be able to find in a good one.

Continuing on the potential pitfalls of neuroimaging studies, here’s a longread that explains in detail what happens when images are taken and analyzed for study. It should give you some perspective next time you see an article claiming that scientists have found something amazing in the brain that explains a huge chunk of cognition or emotion.

There was a scientific dust-up last week in which a journal had to retract a good number of papers because of problems with peer review. Nature suggests a double-blind system. Unfortunately, this isn’t much different from what’s supposed to be happening now, and it’s flawed. Nature even makes note of the bias in the current system, so I’m wondering why they are recommending this.

Kids who are raised by same-sex parents actually do pretty well.

Biodiversity is key to our survival. Scientific American shows us maps where biodiversity exists at high levels – right in the same spots that are threatened by global warming.

I love my pets, too, but this is kind of gross:

Wednesday Links

Wednesday Links

reality check

Debunking!

In the wake of pretty much every outbreak of every vaccine-preventable disease, comments on the news articles fill up with people who still think that vaccines cause autism. One article keeps getting referred to, “22 Studies that Prove Vaccines Cause Autism.” I’m not going to link, it doesn’t need any more hits, because it already shows up on the first page of many searches on vaccines. Instead, I’m going to direct you to Liz Ditz’s excellent rebuttal.

Foodbabe proves over and over that she’s all style and no substance. The Foodentists dissect her attack on Lean Cuisine and the Grocery Manufacturers Association with many facts about GMOs that she apparently doesn’t know – or chooses to ignore.

On the topic of GMOs, Gilles-Eric Séralini’s paper linking glyphosate to tumors in rats, which was retracted last year because of methodological and statistical flaws, has been re-published in a journal with apparently less exacting standards. I’m thinking along the lines of “repeat a lie often enough and it becomes the truth.”

SFARI tells us that autism is not the only neurodevelopmental disorder that’s on the rise. The numbers may actually be a good thing, because it means that more people are getting needed treatment.

You know that study that said watching porn shrinks your brain? Well, maybe not so much. Christian Jarrett at Wired talks about the study’s many shortcomings.

Business Insider has an interesting piece on the Myers-Briggs personality test. By the way, I’m ENFP.

Sometimes things are partly true, or true but misrepresented. In those cases, we don’t need debunking, we need. . .

Critical Thinking

I got a little gut-punch here, because I hate neuroscience hype, but I also did a few little happy dances reading about optogenetics. I pick on optogenetics, but… and Moving on from optogenetic frustrations are actually not too far from the mark, though. I think it is possible to get excited about a new method without looking at it as a be-all and end-all breakthrough. . .as long as you look at the research and stay away from the media version.

Another thing that gets oversold is brain imaging. Again, cool, but not as magical as it’s portrayed sometimes. Lots of times. Virginia Hughes talks realistically about the limits and potential of neuroimaging.

A longread (28 pages) on critical thinking. I have to admit, it’s still open in another tab as I write this. Written from a legal viewpoint, as in how something would stand up in court when exposed to scrutiny, but relevant in a general sense as well.

I often take issue with people who are strict “nurturists” because they are so unspecific about what “environment” is and what it does. Genetics and epigenetics are mechanisms that are, while still being incompletely understood, more logical and straightforward than the more nebulous claims of environmental influence. Many of the people I’ve run across take a Lamarckian viewpoint, or even imagine evolution as a personal change (more akin to Pokemon evolution than anything we see in biology!) So I read Developmental Plasticity and the “Hard-Wired” Problem all the way through, and was pleasantly surprised to see a thoughtful and detailed approach to the “Nature vs. Nurture” question. I don’t know how convinced I am, but it’s more than I’ve been by anyone else presenting this argument.

Genetics/Epigenetics

If you wish to make a gene from scratch explains that, well, it’s not really as easy as that.

Cath Ennis explains how epigenetics works in two parts.

Video – Pallas Cat kittens

Somehow not as freaky when they’re kittens, and funny to see domestic cat behavior in response to the intrusion of the camera.

Learning From Research – The Discussion

Learning From Research – The Discussion

It’s been a while, and I’ve had a lot of stuff going on both in my life and in my mind, but I’m determined to finish this thing. Previous posts:

Part 1
Part 2

This is the section in which everything that was talked about before is kind of recapped and explained and, well, justified. I approached this in a much simpler format, because that’s really all it needs. My comments are bolded.

DISCUSSION

It was first demonstrated here that the fidelity of replicating methylation patterns of CGIs in the promoter regions is significantly higher than that of CGIs outside the promoter regions. (CGIs in promoter regions replicate themselves more accurately than the ones outside of promoter regions.) It was also demonstrated here that methylated genomic regions show much higher fidelity than unmethylated genomic regions. (If the genes are methylated, they tend to stay methylated, if they’re unmethylated, they can become methylated.) These showed that maintenance methylation of hemimethylated CpG sites into fully methylated CpG sites at DNA replication was highly reliable, while unmethylated CpG sites tended to be methylated by de novo methylation. (Methylation sticks.) It is well-known that exogenous DNA is exposed to a de novo methylation pressure (Doerfler et al. 2001; Bird 2002), and a similar methylation pressure seems to be working on the endogenous DNA. (Unmethylated sites are vulnerable to methylation from outside sources.) To maintain the unmethylated status of CGIs, protection mechanisms from the de novo methylation pressure seem to be necessary. (Unmethylated CGIs need something that protects them from methlyation or they’re vulnerable to it.) Since the MPERs were significantly lower in CGIs in the promoter regions than in CGIs outside the promoter regions, the presence of a protection mechanism(s) specific to the promoter regions, in addition to a mechanism(s) common to all CGIs, was indicated. (Promoter region CGIs probably have stronger protection against methylation of unmethylated regions, because they resist methylation better than non-promoter-region CGIs do.) Although the details of the mechanisms are still unknown, binding of transcriptional factors, such as Sp1, has been indicated as a promoter-specific mechanism (Han et al. 2001). (Hint, hint – this is something someone might want to look into, guys, ‘cuz our grant has been spent! Heh.)

The differential fidelities in replicating methylation patterns of CGIs in the promoter regions and those outside indicated that aberrant methylation of CGIs would occur at different rates depending upon their locations. This will be important when tumors are analyzed for the CGI methylator phenotype (CIMP), which are considered to be caused by molecular defects that allow accumulation of aberrant CGI methylations (Toyota et al. 1999). The differential fidelities shown here suggest that there are two types of CIMP, one due to a defect(s) in the protection mechanisms common to all CGIs and the other due to a defect(s) in the protection mechanisms specific to CGIs in the promoter regions. Actually, a correlation between the CIMP and the diffuse-type histology was clearly observed in gastric cancers when CGIs in the promoter regions were used for CIMP analysis (Kaneda et al. 2002b), while it was unclear when CGIs outside the promoter regions were used. (This will help us do more research that will help with cancer prediction/prevention/treatment, in case you don’t think that these findings have a worthwhile purpose of their own. When in doubt, reference cancer. For people with maybe a little less vision or curiosity. Just sayin’.)

In order for an impaired fidelity in maintaining a methylation pattern to exert any biological effect, methylation statuses of multiple CpG sites in a CGI must be altered. (One change at a single location isn’t going to make a big difference.) A significant increase of MPERs would be necessary for this, and quantitative analysis of MPERs in cells with suspected increase of MPERs is necessary. (We don’t know how many besides “more than one,” so another study would be required.) DMR of the H19 gene had a polymorphism at nt. 391 (nt. 8217; GenBank accession no.AF125183), and this served to distinguish the two alleles clearly. (This location was where we could best see what happened.) The G-allele was methylated in all of the six cultures, and the T-allele was unmethylated. The methylation patterns of the T-alleles were similar in HMEC11 and HMEC15, but were essentially variable among the six cultures. This indicated that, although the original cells in HMEC11 and HMEC15 might have had a common ancestral cell, methylation patterns in a tissue alter significantly during a human life span. (Methylation may change because of time, not necessarily because something came in and methylated stuff. No pointing at a specific environmental influence like a chemical or somesuch. Just demonstrating that it happened, and where and why it would be more or less likely to happen.)

Future clarification of what protection mechanisms are involved and how they are impaired in various diseases will contribute to understanding of aging (Ahuja et al. 1998; Issa et al. 2001) and various pathological conditions. (This is a single step in a huge process, but it puts us on a track to learning more than what we know now.)

Learning from Research, The Results.

Learning from Research, The Results.

This is the part where my brain is going to explode. I might need to break this up into more than one post.

RESULTS
Preparation of HMECs

A single HMEC in its log phase was plated, and expanded to 1.4 × 106 to 1.5 × 106 cells (Fig.1). Plating efficiency during the two transfers of plates was 67 ± 0.9(mean ± SE)%. Based on these values, the number of cells that should have been produced at the time of harvest was calculated as 3.2 × 106(1.4 × 106/0.67/0.67). This value predicted that each cell harvested underwent 21.6 generations from the initial single cell. Doubling time was 48 h.

Strategy of cell culture. A single HMEC was inoculated in a well by limiting dilution, and the cell was expanded up to approximately 106 cells. Based on the plating efficiencies during the two transfers and the actual final cell count, the number of cells that should have been produced at the time of harvest and the number of generations observed were calculated. DNA was extracted from the final cells, and used for bisulfite sequencing. Six independent cultures were performed.
Slide1

HMEC – Human Mammary Epithelial Cells. They were put into a container, allowed to reproduce, and then they were checked to see if the right number of cells were made after specific numbers of generations. There were six containers of these cells. Once enough generations had passed and there were enough cells, their DNA was tested with the bisulfate test (illustrated in my earlier post.)

Gene Selection and Their Expression Levels

Methylation statuses were determined by bisulfite sequencing for CGIs in the promoter regions of the E-cadherin,p41-Arc, SIM2, 3-OST-2, and Cyclophilin A genes; CGIs in the downstream exon/introns of theE-cadherin, p41-Arc, and SIM2 genes; CpG sites outside CGIs of the E-cadherin and p41-Arcgenes; a NM-CGI of the MAGE-A3 gene; and differentially methylated region (DMR) of the H19 gene (Fig.2A). The former five genes were selected because they had CGIs in the downstream exon/introns that met a strict criterion of CGIs, regions of DNA of >500 bp with a G+C ⋝ 55%, and observed CpG/expected CpG of 0.65 (Takai and Jones 2002). The MAGE-A3 gene and the DMR of the H19 gene were selected as a representative NM-CGI and a region critically involved in genomic imprinting, respectively. By quantitative RT-PCR analysis, their expression levels were shown to range from almost none (SIM2 and MAGE-A3) to very high (E-cadherin), with p41-Arc, 3-OST-2 andCyclophilin A being intermediate (Fig. 2B).

Structures and expressions of the genes analyzed. (A) Schematic representation of the genomic regions analyzed. Regions analyzed by bisulfite sequencing are shown by closed boxes, and designations A–L correspond to panels in Fig. 3. CGI-P: a CGI in the promoter regions; CGI-outside: a CGI outside the promoter regions; Non-CGI: CpG sites outside CGIs; and DMR: differentially methylated region. All panels are drawn to the same scale. (B) Expression levels of the seven genes in HMECs.
Genome Res. 2003 May 13(5) 868-74, Figure 2

Sorry, I can’t even. All I know from this is that they looked at the results of the bisulfite sequencing and found what they were looking for – the methylation status in the CpG Islands from promoter regions of DNA stayed almost exactly the same. Unmethylated CGIs from non-promoter regions were more likely to become methylated. I’m afraid I don’t have the ability to explain this to you or tell how accurate or flawed it may be. I’m taking the researchers’ word on it. Correct me if I’m wrong.

Establishment of How to Measure MPERs

The CGI in the promoter region of the E-cadherin gene (Fig.3A), the non-CGI region of thep41-Arc gene (Fig. 3F), the CGI in the promoter region of theMAGE-A3 gene (Fig. 3K), and the DMR of the H19 gene (Fig. 3L) were found to contain two major populations of clones. The two major populations were considered to represent the methylation pattern of the two alleles in the original single cell. The methylation patterns of the two major populations were different from each other in the six cultures, which indicated that the HMECs before cloning had diverse patterns of methylation, but the patterns were relatively conserved during the culture from a single cell to approximately 106 cells. Therefore, we measured the number of errors in the methylation pattern based upon the culture from a single cell to approximately 106 cells. An MPER of a region in a culture was calculated from the number of errors in methylation pattern as described in Methods, and an average MPER of the region was calculated from the six MPERs obtained for the six cultures.

MPERS – Mammalian Protein Extraction Reagent
AlleleAn allele is one of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. If the two alleles are the same, the individual is homozygous for that gene. If the alleles are different, the individual is heterozygous. Though the term “allele” was originally used to describe variation among genes, it now also refers to variation among non-coding DNA sequences.

So after making all those cells, they looked to see where and whether methylation status had changed.

Distribution of unmethylated and methylated CpG sites shown by bisulfite sequencing. Unmethylated and methylated CpG sites are shown by open and closed circles, respectively. (A)–(C) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the E-cadherin gene. (D)-(F) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the p41-Arcgene. (G), (H) A CGI in the promoter region and a CGI outside the promoter region of the SIM2 gene. (I) A CGI in the promoter region of the 3-OST-2 gene. (J) A CGI in the promoter region of the Cyclophilin A gene. (K) A CGI in the promoter region of the MAGE-A3 gene, which is normally methylated. (L) A CGI in the differentially methylated region of the H19 gene.

Here’s where they found the differences:

Genome Res. 2003 May 13(5) 868-74, Figure 3

To examine the effect of an arbitrary selection of the “original methylation pattern” in ambiguous cases, a permutation test was performed for the CGI in the E-cadherin promoter region of HMEC10. One of the clones #5–#14 (Fig. 3A) was hypothesized as one of the original methylation pattern, and the number of errors in the methylation pattern was calculated. The numbers ranged from 18–22, and these values were expected to result in the average MPER ranging from 0.022–0.023. Similar permutation tests were performed for the CGI in exon 2 of the E-cadherin gene of HMEC12 and HMEC15. The numbers of errors in methylation pattern ranged from 13–16 for HMEC12 and from 12–15 for HMEC15, and these values were expected to result in the average MPER ranging from 0.050–0.058. These showed that arbitrary selection of the original methylation pattern in ambiguous cases does not seriously affect the resultant average MPER.

Some changes weren’t so cut and dried, so they checked those cases and found that they weren’t significant enough to change the findings.

The efficiency of bisulfite conversion was examined by analyzing DNA with no methylation in the CGIs in the promoter region and exon 2 of the E-cadherin gene. In the CGI in the promoter region, none of the 600 cytosines at CpG sites (30 CpG sites per clone, 20 clones analyzed) remained unconverted, showing that unconversion rate was almost 0 in this region under our experimental condition. In the CGI in exon 2, one of 483 cytosines at CpG sites (23 CpG sites per clone, 21 clones analyzed) remained unconverted, showing that the unconversion rate was 0.0021. These values showed that the MPERs in CGIs in the promoter regions are 10-fold more than the unconversion rates.

The bisulfate conversion was also tested separately for control to make sure the results would be valid in the experiment. This reinforced the finding that the promoter regions stayed stable.

MPERs and Fidelities of Methylation Pattern in the Genome

The average MPERs obtained for each region are summarized in Table1. Unmethylated CGIs in the promoter regions showed MPERs between 0.018 and 0.032 errors/site/21.6 generations. In contrast, CGIs outside promoter regions showed significantly higher MPERs, ranging from 0.037 to 0.091 (P < 0.01 or 0.005). MPERs in the CGIs outside the promoter regions were more than twice as high as those in the promoter regions of the same genes. MPERs in Various Genomic Regions

NM-CGI of the MAGE-A3 gene and methylated alleles of the DMR of the H19 gene showed MPERs of 0.002 and 0.007, respectively. Any genomic regions that were normally methylated, whether or not they were in CGIs, showed significantly lower MPERs than those unmethylated. This was particularly clear when the MPER of the allele methylated at DMR of the H19 gene was compared with that of the other unmethylated allele.

Interpretation of the tables, summary of findings.

This is not as good as part one, sorry. In other news, I couldn’t watch Besharam because it sucked, so I didn’t learn any Hindi, either. One more post to go in this series. Anyone who can clarify/explain better than I can, please comment – I’d appreciate it.

Science Education – How I Would Do It.

Science Education – How I Would Do It.


Of course, this is assuming that the world was a sensible place and I was in charge of all the important decision-making. Heh.

Over time, I’ve come to realize that a lot of the things I was taught in school didn’t stick because they weren’t interesting. They weren’t interesting because they were unrelated to my life, and I couldn’t see how they could possibly be important to me. I memorized things for tests, and I did a darn good job of it, good grades, good standardized test scores, but only because I had to, not because I wanted to.

As I got older some of it came back – and it stuck better because I had context to put it in. Before kids and before antidepressants, I read a lot of romance novels for escape (I know. . .I’m not proud, but I had an excuse.) Soon I discovered that there was a sub-genre of Historical Fiction – and some of these authors were real history buffs who included a lot of factual information. In the context of a story, with characters and plots that engaged me, I was finally learning something about history, which had bored me to tears in High School.

Later, I started reading some of the books and papers that had been assigned back then. . .suddenly they were interesting and made sense – because I now had a context for them. The context continued to expand, and more information became part of what I knew.

_______________

For me, possibly moreso than for many people, context is essential. My ADHD mental filing system demands context and associations not only for learning, but for retrieving that learning. So when I teach people what I know, I teach it in context. I learn a lot by making mistakes, so I teach “do it this way because this other way doesn’t work,” and “we do it this way because otherwise we break this piece and the whole thing is ruined.” I teach “This part seems boring, but here are all the cool things we can do with it later.”

I also learned a lot from raising my own kids and volunteering in their schools, helping all kinds of other kids learn. You need to be able to express a single piece of information many different ways in order to get different kids to understand it. As a volunteer, I was able to sit with individual children and small groups. The kids who didn’t understand things when they were taught the same way to all 30-something students would get it if I spent some time with them and figured out what their individual contexts were.

_______________

Fast forward to the mid 90s – I started antidepressants, and then I discovered that my ADHD had not actually gone away as the experts had told my parents it would, and as my parents told me it had. Now I had a reason to learn about the brain, starting with disorders and injuries, and what they taught us about the functions of various structures. That gave me a context to learn about brain development and genetics. This led to investigating epigenetics. Along the way, it also tied in to reading medical and science blogs and books, and any time a piece of knowledge stuck to something that was relevant to something I already knew, it also became relevant.

So why do you want to listen to someone who doesn’t have a degree in science or medicine when it comes to science or medicine? Because of the way I’m learning it. That whole “Translating Science into English” thing I mentioned a few posts back. Scientists have their own language, and it’s important that they do so they speak with clarity and precision. But if you don’t have the context that they do, it’s hard to understand – and easy to misinterpret. I didn’t learn this in the linear fashion that they did.

If you were to teach me vocabulary and facts and mechanisms, I’d remember it just as well as I did in high school. But give me a study of something that relates to something that interests me, and I will look up all those words and facts and mechanisms, and they’ll make sense because they’re part of something else. They have more meaning when they’re in context.

The other thing I learned came from watching scientists argue with one another. While they’re not always polite, they always present evidence. Most of them are critical thinkers, when someone says something that is questionable, they will (sometimes very methodically and in great detail) explain the flaws in the reasoning. Following along with this taught me the scientific method and why it’s important, how to evaluate how robust the data is by looking at the size of the study, the quality of the blinding, the strength of the variables and controls, how well it integrates existing evidence (and how strong that evidence is) and, most importantly, no matter how good a study may be, it’s never PROOF. It also doesn’t prove other things that weren’t part of the study. It’s also probably not a major breakthrough.

I learned about p-values, journal impact factors, the good and bad of peer review, the pros and cons of open access. I learned that not all “evidence” is actually evidence.

_______________

The problem that many, many scientists have, though, is that they forget what it’s like to not know this. Sometimes they present what they know in a way that is off-putting to laypeople. Sometimes they present a press-release version of their findings, breathless with excitement and full of hyperbole, and that’s even worse. (That’s what we have The Daily Mail and Huffington Post for. Let them do their job.)

So if I were a science teacher, or I were designing a science education program, I’d throw out teaching the basics as freestanding facts. Get rid of the rote learning. Give the students just enough information to dive into a challenge and figure out the rest. Give the kindergarteners a bowl of cream and some food coloring and dish soap – let them play and then tell them how it works. Let the older kids listen to each others’ heartbeats, check each others’ blood pressure, draw pictures of hearts and veins and arteries, and use that to introduce the circulatory system. Make everything part of an experiment that related directly to them so that it was important. Let them figure out what’s correct and what’s incorrect as much as you can on their own by giving them questions as much as answers. Make the science interesting and integrate critical thinking into the lessons, and get them excited. This will be good for them, and good for society, because they’ll question everything – and come up with their answers based on what evidence is best supported.

Learning from Research, Slowly and Methodically.

Learning from Research, Slowly and Methodically.

I was given a challenge on Twitter, and some people dismissed me as a failure because I didn’t have the academic background to come back with a quick answer. (I also discovered that I knew the answer, but forgot the words because of post-surgical anomia. I digress.) I find that this is a problem with a lot of people with certain types of expertise. They forget what it was like back when they were first learning, and no longer have the patience to explain. I don’t think it helps that there is a shit-ton of people on the internet spouting nonsense and being taken seriously. Naturally, some of them will assume that I’m doing the same, but I really don’t want to be lumped in with them, so I’m going to show them the process I go through, and how seriously I take learning new things and separating fact from fiction.

As I said in my previous post, you guys are wicked smart, and I am very often in awe of how much you know. But one thing you’re not so good at is communicating to people outside your fields of expertise. This is why we have bad science journalism. Ask Ed Yong. However, if you want to stop all your discoveries from degenerating into misrepresentation or woo, then you need people who can translate Science into English.

I was given a long, information-dense study, Fidelity of the Methylation Pattern and Its Variation in the Genome by Malcolm M. Campbell, so it’s going to take several posts to dissect, research, learn the background information, and try to explain it in an accessible way. I fully expect to be wrong several times, and encourage people to correct me – in such a way that ordinary people can “get it.” So here goes:

Abstract

The methylated or unmethylated status of a CpG site is copied faithfully from parental DNA to daughter DNA, and functions as a cellular memory. However, no information is available for the fidelity of methylation pattern in unmethylated CpG islands (CGIs) or its variation in the genome. Here, we determined the methylation status of each CpG site on each DNA molecule obtained from clonal populations of normal human mammary epithelial cells.

Methylation turns genes or pieces of genes “on” or “off”. There’s a detailed explanation of various ways it does this in the components of the whole process from DNA to cell, but it’s kind of hard to understand if you haven’t done a lot of reading beforehand. I’ll give you the link anyway.

CpG sites – the quick and dirty Wikipedia definition is this: The CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. If you don’t remember from your Biology classes, or your biology classes never taught you, your entire DNA strand consists of combinations of four nucleotides – Cytosine, Guanine, Taurine, and Adenosine. I’m not going to get into that right now, because it’s just going to confound this with too much information, but if you think about the movie “Gattaca,” you’ll notice those four letters. In a movie about genetic engineering. Because those are the four letters you see in an illustration of a piece of DNA. The researchers were looking at the parts where the cytosine and guanine were next to each other.

Specifically, they were looking at epithelial cells from normal breast tissue. The link may be a little difficult to understand, but I think if you read all the way through, you’ll at least understand some of the reasons these cells were chosen. They have a lot of unique characteristics, and they’re pretty tough.

So the idea here is that we already know that if the cytosine and guanine pair are methylated in the on position or the off position in the DNA, that they’re going to stay that way in the cells that are produced by those instructions from the DNA. What we don’t know is that if that pair is unmethylated, will the cells made from the DNA instructions also be unmethylated? IOW, if they’re not already told to be switched on or told to be switched off, will they still be in that “neutral” position? In order to test that, they took a bunch of those epithelial cells and tested each one to see if it was methylated or unmethylated so they could get them to reproduce and see what happened.

This illustration is not specific to this piece of research, but keep reading, and you’ll see how it relates.. I wanted to give you a visual aid in case you learn better that way.

Methylation pattern error rates (MPERs) were calculated based upon the deviation from the methylation patterns that should be obtained if the cells had 100% fidelity in replicating the methylation pattern. Unmethylated CGIs in the promoter regions of five genes showed MPERs of 0.018–0.032 errors/site/21.6 generations, and the fidelity of methylation pattern was calculated as 99.85%–99.92%/site/generation. In contrast, unmethylated CGIs outside the promoter regions showed MPERs more than twice as high (P < 0.01). Methylated regions, including a CGI in theMAGE-A3 promoter and DMR of the H19 gene, showed much lower MPERs than unmethylated CGIs. These showed that errors in methylation pattern were mainly due to de novo methylations in unmethylated regions. The differential MPERs even among unmethylated CGIs indicated that a promoter-specific protection mechanism(s) from de novo methylation was present.

This explains how they figured a reasonable range of variation. The “islands” of unmethylated cytosine/guanine pairs in five genes over 21.6 generations (this is statistics, not absolute numbers. You clone enough cells, you sure as heck can get six tenths of a generation.) stayed unmethylated most of the time. This came from promoter regions, which are the areas in DNA that call the shots. It’s more likely that instructions from promoter regions are going to be followed.

The unmethylated cells that didn’t come from promoter regions showed more deviations – the cells after several generations were twice as likely to be different from the originals than the ones that came from the promoter regions. The methylated cells, which, as I mentioned, already have the specific instructions to turn a gene on or off, were more likely to maintain their integrity even if they weren’t from promoter regions. The unmethylated cells didn’t’ have that instruction, and hadn’t been told to stay unmethylated (because they weren’t from promoter regions) and so they just did whatever seemed right at the time and, well, mistakes were made.

CpG methylation is known to serve as cellular memory, and is involved in various biological processes, such as tissue-specific gene expression, genomic imprinting, and X chromosome inactivation (Jones and Takai 2001; Bird 2002; Futscher et al. 2002;Strichman-Almashanu et al. 2002). These important functions of methylations are based upon the fact that the methylated or unmethylated status of a CpG site is faithfully inherited. The methylated status of a CpG site is inherited upon DNA replication by the function of maintenance methylase, represented by DNA methyltransferase 1, which is located at replication forks and methylates hemimethylated CpG sites into fully methylated CpG sites (Leonhardt et al. 1992; Araujo et al. 1998; Hsu et al. 1999). The unmethylated status of a CpG site is inherited by not being methylated upon DNA replication or any other occasions. Unmethylated CpG sites generally cluster to form a CpG island (CGI), and most CGIs are kept unmethylated (Gardiner-Garden and Frommer 1987; Bird 2002). Methylations of CGIs in promoter regions are known to cause transcriptional silencing of their downstream genes by changing chromatin structures and blocking transcription initiation (Bird 2002;Richards and Elgin 2002). There are limited numbers of CGIs that are normally methylated (normally methylated CpG islands; NM-CGIs) (De Smet et al. 1999; Futscher et al. 2002). CpG sites outside CGIs, especially those in repetitive sequences, are also normally methylated (Bird 2002).

CpG methylation is important. It is carried on pretty faithfully when cells reproduce. It’s also important that unmethylated CpG remains unmethylated, and that’s usually passed on to new cells as well. Most of the unmethylated sites form a cluster called a CpG Island, or CGI. If these unmethylated CGIs become methylated, then it changes what genetic instructions get turned on or off in future generations of cells, if they’re in promoter regions. But it’s not always bad for CGIs to be methylated, because sometimes that’s on purpose.

I’m going to hold off on the transcription and chromatin stuff for later, because I think it’ll stick better when the paper goes into more detail.

To keep the methylation pattern, maintenance of both methylated and unmethylated statuses of CpG sites during DNA replication is necessary. However, the fidelity of the methylation pattern has been analyzed only for the maintenance of the methylated status (Wigler et al. 1981; Otto and Walbot 1990; Pfeifer et al. 1990). The fidelity in maintaining the methylated status of an exogenously introduced DNA was shown to be 94% per generation per site by Southern blot analysis (Wigler et al. 1981). The fidelity in maintaining the methylated status of a CGI in the 5′ region of the PGK1 gene, which was derived from the inactive X chromosome, was estimated to be 98.8%–99.9% per site per generation by the ligation-mediated PCR method after chemical cleavage of DNA (Pfeifer et al. 1990).


We’ve already studied methylated CpG sites and found that it’s pretty consistent. Some studies attesting to that are cited. We know that keeping them unmethylated is also important, but that hasn’t been investigated to our satisfaction.

Normally unmethylated regions might show different fidelities from normally methylated regions. Even among the unmethylated CGIs, the fidelities of their methylation pattern have been suggested to be different according to their location against a gene promoter. Methylation of CGIs in promoter regions almost always leads to transcriptional silencing while that of CGIs outside promoter regions does not (Gonzalgo et al. 1998; Jones 1999). Considering the cellular expense in maintaining methylation pattern, a cell could sacrifice the fidelity of methylation pattern for CGIs outside promoter regions. In addition, by recent genomic scanning techniques for methylation changes (Ushijima et al. 1997; Toyota et al. 1999; Costello et al. 2000; Jones and Baylin 2002), aberrant methylations of CGIs in cancers are observed in a nonrandom manner (Toyota et al. 1999; Costello et al. 2000; Kaneda et al. 2002a; Kaneda et al. 2002b). It is indicated that CGIs outside promoter regions were more frequently methylated than those in promoter regions (Nguyen et al. 2001; Takai et al. 2001; Kaneda et al. 2002a; Asada et al. 2003).

Unmethylated CGIs are more likely to change than methylated ones. Unmethylated CGIs from promoter regions of the DNA pretty consistently shut down the things they’re supposed to shut down, exactly as planned. Unmethylated CGIs from outside promoter regions of the DNA are not so good at that – they’re more likely to become methylated when they’re supposed to stay unmethylated. Some of this methylation of unmethylated CGIs has been seen in cancer. So that’s one example of why we don’t want this to happen.

Here, we analyzed the methylation status of each CpG site on each DNA molecule by the bisulfite sequencing technique (Clark et al. 1994) in six clonal populations of normal human mammary epithelial cells (HMECs), for CGIs in the promoter regions, CGIs outside the promoter regions, and CpG sites outside CGIs. By analyzing the deviation from the most common two patterns, MPERs, which reflected the fidelity in replicating both methylated and unmethylated statuses, were measured.

Like a five-paragraph essay here. Restating what they’re going to do and how they’re going to do it. Remember the illustration? Bisulfite sequencing technique. (Really detailed explanation, Wikipedia explanation).

And now my brain is very, very tired. I am going to watch “Besharam” because I’m also trying to learn Hindi, and I might as well be looking at Ranbir Kapoor while I’m doing it. Heh. I will continue this in a later post. Feedback is welcome and encouraged.

Science People!

Science People!

I’ve been getting a lot of attention on Twitter for the last couple of posts, and that’s given me a lot of articles to read, blogs to keep up with, and Twitter users to follow. Some people got a little testy, and I don’t blame them, because they know more than I do. I get it.

Let me tell you something right now. I am not a professional scientist. I got my Bachelor’s degree in Spanish Language and Literature back in the early 80s, and distanced myself from science since I had to take my only B-track class in all of High School in Biology. I didn’t get it, I didn’t see the point, I put no effort in, and I sucked at it.

That’s ADHD.

But then I started reading books about the brain, and that struck a chord with me because my brain is not the nice neurotypical model. I started reading blogs and websites about the brain, and medicine, and genetics. I learned how to read published research (and occasionally got friends who would sneak me links to full text articles) and would search in the middle of searches when I found terms I didn’t understand or biological processes or mechanisms that were new to me but essential to understanding what I was reading.

This obsessive pursuit of information is also ADHD, BTW.

This means that there are gaps in my knowledge. I am not ashamed to admit that you know more than I do. Please don’t get angry with me when I’m wrong – explain to me why I’m wrong and then tell me how to understand it the right way. I don’t want to be right to win arguments or lord it over people, I want to be right because I have the correct information. You can help me with that.

Thing is, one thing I know I’m really good at is teaching other people things. I take my mistakes, the process by which I figured something out, and the way it works at the most basic level, and try to use that to explain what I know in a way so that other people can “get it.” There are several college students out there pursuing degrees in science because I got them all excited about it. They’re getting the chance I missed out on.

So, you want more minions? (MUHAHAHAHA!!) Give me comments. Help me understand. Because if you help me understand, I can help other people understand. I’m an intelligent woman, I’ll get it pretty quickly, and when I don’t, I’m not in the least ashamed to admit that I was wrong. We can have a mutually supportive and respectful interchange, and I’ll do my part to explain things in an accessible way, using the tools you give me.

Really. Comment. email. Bring it on. I love you guys!