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.


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.)

What has been happening. . .

What has been happening. . .

And why I’ve been away so much. This started about three weeks ago, when my Mom went in to see the oncologist for her biopsy results, and the staff didn’t think that her shortness of breath and loss of consciousness was simply stress. They sent her to the emergency room, and I got a call that if I didn’t come to pick up my Dad, he’d be picked up by a long-term care facility and they might not release him to my Mom because she was so sick. Talk about incentive. I hadn’t finished doing laundry, so I went down with whatever I had to wear, my medications, and an aerobed.

Turns out, she’d been having these problems for a while (and, of course, downplayed them so nobody would be concerned) and it was a pulmonary embolism. A small one, but I don’t think size really matters much. Thank goodness they insisted. But here’s where the fun begins. You see, Dad has been declining mentally for several years, and his condition is another thing Mom has been downplaying. The reasons are numerous and complex, and I’m not going to get into too much detail because that’s outside of this narrative. Since Mom’s got atrial fibrillation and has been on blood thinners, which she stopped so she could get her biopsy (Non-Hodgkin’s lymphoma, treatable with chemo) it became kind of complex to get her clotting factor right at the same time as they broke up the clot in her lung – and while she was there, they wanted to install a chemo port and give her her first treatment.

This meant over a week in the hospital for her.

This meant over a week of caring for my Dad by myself.

At this point, my new meds had not kicked in, I was still having panic attacks, and I was trying to process a whole bunch of information without the benefit of Adderall, either. And my Dad’s dementia is. . .bad. Looking at the description of a seven-stage progression, he’s between five and six, and awfully close to entirely stage six. After just a couple of days with him, taking him back and forth to the hospital and then being yelled at later for not having told him Mom was in the hospital, not being able to do anything except have the same conversations with him over and over without him getting upset that I wasn’t engaging him, being awakened at all hours of the night and early in the morning either because he was wandering the house (sometimes on his way outdoors) or waking me up to ask where Mom was, I was really on edge. I texted my sister and asked if she could relieve me for the weekend, and, bless her heart, she showed up on Thursday night. Not only did it help me out enormously, but I now had someone else to corroborate my story about his condition.

Things were relatively OK, but then Mom called me on a Saturday to tell me that Dad was in the hospital – this was his second time, but his first one (a couple of weeks prior to Mom’s for the embolism) she didn’t tell me anything until he was home. This time, she really shouldn’t have been driving, but she would have if we hadn’t come down, so hubby and I headed down on Sunday to take her to visit Dad. We got her a wheelchair to take her around the hospital, because she needed it. Dad was really out of it, sleeping slouched in a chair when we arrived, so we went and got lunch. When we came back, nothing had changed, and Mom found that she couldn’t wake him up, that he muttered a few incoherent things, and we realized that his arms and legs were ice cold.

When we called the nurse and they realized that they couldn’t get his blood pressure, we were shooed out of the room, and soon there were more doctors and nurses than could even fit. They moved him to the bed, and tried to get a chest x-ray, but he was uncooperative and physically fighting them off. They figured that in addition to the one infection he had, he probably also had pneumonia, so they began an IV drip of antibiotics for that. He was conscious when we finally got back into the room, but nothing he said made any sense.

Once we knew he was out of danger (because we were pretty worried for a while) we brought Mom home. I called her the next day, and she had spoken to him, and he was doing better, but still thought he was being held in a jail for something. She had no idea if she actually had someone to give her a ride to her second chemo appointment, so I figured I would drive her myself and then go to the hospital to see if they could just care for him for a couple more days – because even this had not been enough for her to actually get a home health aide or a visiting nurse. I knew that there was no way that she could care for him and keep him out of danger while recovering from an infusion.

So this is where it gets dramatic.

I slept badly, of course, and set off a little before 6:30AM to pick her up. I went in with her to speak with the oncologist and we discussed, frankly, the reality that she would not be able to care for Dad by herself, and I think that hearing it from the doctor lent it credibility that it didn’t have coming from me. She agreed that as much as she wanted Dad to be able to stay at home with her, it was in her best interest that he be cared for around the clock somewhere else for at least a little while when she was feeling like crap.

I got Mom settled in with her pillow and blanket and book, and headed out to the hospital. I went to the nurses’ station, trying to keep out of his sight so I could speak to them without upsetting him. They shocked the heck out of me by announcing that they had been calling all morning because it was time to release him! I explained that this was really, really, really bad timing, because my Mom would be hooked up to chemo drips until at least 4PM, and I couldn’t take him to the oncologist’s office OR leave him home alone. Well, they told me, the papers had already been signed, so I’d have to take him and do one or the other. No room for negotiating.

The social worker was at the desk, and I asked her if he could stay. No. Are there any short-term places he can go? No, he doesn’t qualify for short-term rehab, so he’d have to go into long-term care, and then he’d be there permanently. I didn’t want to place him somewhere permanently, and I especially didn’t want to be the one responsible for placing him permanently, against my Mom’s wishes. I asked if I could speak to the doctor who signed the release. (n.b., at this point, my Dad has not seen me, but he no longer has a guard in the room – he’s all by himself, seated on a pad that sounds an alarm every time he gets up, at which point, nurses rush in and make him sit down again.)

The doctor comes out, and I have to say, I have not been treated so condescendingly or disrespectfully by a doctor in close to 20 years. I’m not naming the hospital or the doctor – I’m going to write to them, I don’t need them to have a bunch of people descending angrily upon them, because my anger should be just about all they can handle! I tried to explain to him that Mom was getting chemo all day, I live an hour and a half drive away, Dad’s dementia makes it impossible for her to care for him while she’s dealing with her own treatments, and isn’t there some way he can just keep Dad there for even one more day? He gives me the same line about either I take him home, or he gets committed permanently and there’s no way he’ll ever come home, and he’s a professional gerontologist and I should know that Dad’s mental condition will decline rapidly if he goes into a home and I’ll be responsible for giving him a death sentence.

That’s when I threw out the names of the other doctors with whom I had consulted who agreed that he needed nursing care while mom was sick (GP as well) and suddenly he’s all “Oh, I know them. Good doctors. Well, I wish you luck,” and then walked away with a smile as if he had not just implied that I don’t care if my father dies in a nursing home.

Believing I had no choice and needing to do something quickly, because Dad was beginning to get really angry with the alarms and the nurses and such, I arranged for a liaison from the home closest to my parents’ house to start the admission process. Dad had seen me at this point, so I had to sit with him for a bit, but I needed to call Mom and it’s impossible to make a phone call with Dad there because he gets upset if you’re talking but not to him. Of course, he tried to follow me out, and the alarm went off, and the nurses came, and he was fighting and yelling.

Down the hall, I tried Mom’s cell phone, but she didn’t have it or it wasn’t on, so I called the main number for the oncologist. The receptionist passed the message, and shortly after, I was talking to Mom about what I had been forced to do and why – then she passed the phone to one of the nurses and the oncologist’s social worker. They were pretty furious, because I’d been lied to. Yes, my only option was a nursing home, but it wasn’t a prison. We could take him out any time.

Trying to explain this to my Dad was an awful experience. He didn’t get the concept – of anything. He forgot who I was. He was angry because he’d been kept alone in this room for so long and didn’t understand why this alarm kept going off and why nobody would let him walk around. He was tired of waiting around to go visit whomever he thought he was visiting, because he didn’t remember that he was the patient. When the rep from the nursing home arrived, she was wonderful. It was obvious that she understood how to handle people with memory issues, and had the patience of a saint. She figured out the one thing that caught my Dad’s full attention – he wanted to take care of Mom. She told him that he was going to need to build up his strength so he could do that, so he was going to stay in this place and do physical therapy every day until he was ready to be Mom’s caregiver. And when she saw that I would tell him what was happening and then he’d get mad because nobody had told him this was happening, over and over again, she told me that the hospital social worker needed to see me so I should say goodbye to him. It got me out of the room, and Dad accepted it – but nobody actually wanted to talk to me. She just knew that was the only way to disengage.

I went back and stayed with Mom until her chemo was done. I hadn’t had anything to eat, so I was given some crackers and coffee by the oncology nurse. When the chemo was done, I went out to pick up Mom’s prescriptions while she packed clothes for Dad. I got back, and she was on the phone with Dad, explaining to him that he needed to get strong so he could take care of her. The moment they hung up, my sister called. I may have been a bit abrupt (sorry, Jen!) but I had been up since 4:30, had eaten nothing but those crackers since 6AM, and still had to drive to the home and drop off the clothes and pick up dinner from the diner.

The home was nicer than some, not as nice as others, but the staff was good, and nobody was restrained. Dad wanted me to take him on a tour, and this was just not an option at this point. Fortunately, a staffer was approaching us, and I asked her if she could show him around. She agreed, and I said my goodbyes. Picked up food. Ate. Drove home, got there about 10:30.

A couple of days later, I called Mom. She’s talked with Dad, and he thinks this place is pretty luxe, and he’s doing physical therapy so he can come home. She, meanwhile, has been able to sleep whenever she needs to and for as long as she wants, and get sick without Dad trying to “help” her. She didn’t want him to be taken away from home, but for the moment, things seem to be working out, and that takes a huge weight off my mind.

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.

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.

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:


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!

Epigenetics Made Easy, Part 2

Epigenetics Made Easy, Part 2

Let’s reiterate from the previous post, just in case you need a recap:

All cells are made from other cells; we start with a few that are the same, and as the number of cells increases, they begin to differentiate and become cells for specific body parts.

DNA is the blueprint for the final product (a sexually mature adult human being, for illustration purposes.) RNA is a segment of DNA that begins the process of cell differentiation, but the mechanism that actually creates the proteins that build cells is the epigenetic process, which depends upon histones interpreting the genetic instructions.

Once we are full grown, our bodies are almost always replacing cells rather than making new ones, and the new cells may not be exact duplicates of the cells that created them.


histone modification

Yes, there is such a thing as histone modification. Yes, gene expression (in the form of cells that follow specific genetic instructions) can be changed during the epigenetic process. Yes, it’s possible for some of these changes to become heritable (passed on from parent to offspring.) But let me explain what’s reasonable and rational about these possibilities.

Histone Modification

You’ve heard of this, but usually in the form of “you can change your DNA by doing this thing or eating that thing” which is, essentially, not true. Histone modification takes place on a cellular level, and changes in different ways depending on what the chemicals that can modify histones are doing. I’ll save the technical terms and illustrations for another time. Baby steps.

What happens is that while a cell is preparing to replicate itself, a chemical can make the histones do something differently from the way they were instructed, and that makes the resulting copied cell different from the cell that created it. Right now, we have some very specific examples of changes that depend on specific chemical exposures (some from external environment, some from internal environment.) DNA is huge. We have a hundred trillion or so cells in our bodies. The genome is almost infinitely diverse. There are very few examples right now of direct cause and effect, and each one we discover in the future will be just as limited.

The number of possibilities alone makes it pure speculation to assume that a food given to a pregnant mouse that changes her babies’ fur color and body shape is going to do the same thing for a fully-grown adult, or even something similar!

Now the add another layer of complexity, these are the things that can happen when you modify the histones in a cell:

*a beneficial gene is suppressed
*a detrimental gene is suppressed
*a beneficial gene is activated
*a detrimental gene is activated

So if someone claims that a food or something “methylates” your genes (besides being wrong) it could easily be a bad thing!

Changing Gene Expression

I mentioned the prenatal modification above, and that’s because it’s an important thing to study. Why? Because in order for histone modification to have any observable and verifiable effect, it needs to happen early. Think about it. If you modify the histones of a four or eight celled creature, then a lot more cells are going to be made not according to plans. In an adult, modifying a single cell, or even a few cells, out of all the cells in our bodies, is going to have minimal impact. In order to change gene expression in an adult, exposure needs to be intense enough or prolonged enough to influence a large number of cells.

I like to use the example of skin, partly because it’s a cell type that’s replaced frequently, and partly because we can see a lot of the possible changes to it. It’s a good way to illustrate that an environmental factor can produce a change that does not alter gene expression, and how the level of exposure can make a difference in whether an epigenetic change is even possible.

If you go out into the sun, your skin changes color. It could get burned, it could get tanned. But when those darker skin cells make their replacements and die, the replacements are your original skin color. You have exposed yourself to an environmental factor that has an obvious effect on your body, but it doesn’t change your gene expression. Why? Because the exposure was not prolonged enough that the visible change was messing around with histones while the replacement cell was being created.

On the other hand, if you’re out in the sun all the time so that your skin is constantly in a damaged state, then those cells are more likely to be in that damaged state when they’re replicating themselves. This could still even be temporary, but it could change gene expression so that the replacement cells are cancerous, for example. (Cancer is epigenetic – but it could be caused by environment *or* part of the plan all along.) So you need to expose the same group of cells to the same environmental factor for long enough that most of the cells begin reproducing with the alteration in gene expression. . .and that is not guaranteed to be a good thing, so don’t buy into the hype.

Heritability of Epigenetic Changes

Yep, this has been studied, too, and it does sometimes happen. The most repeatable changes happen when the fathers’ bodies have changed. I credit that to the fact that sperm are constantly being made, and things like stress hormones or chemical exposures, or starvation, can change what genes go into what chromosomes in the sperm cells at that time. Give the dads some time to recover, you get a completely different result.

Keep in mind that the normal set of instructions is the default. If you look at plants or other animals who’ve been genetically altered, a lot of times you’ll find that their offspring regress to the original, dominant form. In both human and animal studies, most of the epigenetic changes that were brought about by environmental exposure get passed down to the next generation, maybe the generation after that, and in a few cases, the third generation. Then things go back to normal.

I probably missed a few things, but I hope this is clear. Ask me stuff, tell me stuff. Thanks!

Epigenetics Made Easy.

Epigenetics Made Easy.

Tightly wrapped histones

No, not really. That’s a misleading title, but my hope here is that I can explain this in terms that are simple enough for people who aren’t scientists to understand. I’m hoping that because I’m not a professional scientist but am really, really into this stuff, the language and illustrations I use serve as a bridge for the gap in understanding.

So let’s start with the cell, and let’s use humans as an example. Even though epigenetics happens in every living thing, even plants, I want you to be able to identify personally with this so the information takes hold a little better. What do we know about genetics and conception and fetal development? Well, we start off with an assortment of genes and 46 chromosomes. We got all of them from our parents and grandparents and so on down the line, but it’s a mix between Mom’s side and Dad’s side, because her eggs start off with a random selection of 23 chromosomes (see my previous post about what random means) and his sperm also start off with a random selection of matching chromosomes.

Sperm meets egg, and there you go, 46 chromosomes in a single cell, and a complete, unique strand of DNA that has all the information needed to build a human body.

If you’ve watched videos of human development, you’ve seen how that one cell splits into two, two into four, four into eight, and then things really start to happen. In the beginning, each of those cells is exactly the same. Each time they split, they’re making another cell that’s just like they are. Remember this, because I’m going to mention it again later. . . Here’s how it looks, in case you haven’t actually seen it, in a video on in-vitro fertilization:

After this point, the cells begin to differentiate. Instead of simply reproducing copies of themselves, they start to become more specialized. They still contain all the DNA, but some of the instructions will be used, and some will be silenced. This starts with the transcription from DNA to RNA. What we used to believe (or at least what I was taught in school days in ancient times) was that the RNA was the sole messenger, containing only the information needed to make cells. That’s only kind of sort of true, and doesn’t explain a lot of confusing things that happen to human bodies. You see, it is part of the picture in cell differentiation, which is, to put it in simple terms, the process that makes one cell be a bone cell and another be a heart cell and another be a brain cell and so on. The RNA puts this in process by taking the pieces of the DNA that are needed to make a specific call and creating the proteins that manufacture that cell. With these instructions, cells continue to divide, but they’re not just making carbon copies of themselves.

We see this in fetal development because parts of the body from the brain, the eyes, the internal organs, to the fingers and toes, go from being kind of blobby and alien-looking, to functional and human-like. The manufacturing of differentiated cells continues throughout fetal development, and the differentiation is pretty much complete by the time a baby is born.

But there’s a piece missing – we know that RNA has instructions for making the proteins that manufacture differentiated cells, but it doesn’t make those proteins all by itself. This is where epigenetics comes in. The actual work of taking the orders from the RNA and making the proteins is done by histones. The DNA has the construction diagrams, the RNA is barking orders, the histones are doing the work.

This is still happening inside a cell. The cells are still dividing. It’s just that this epigenetic process is making two different cells out of one cell instead of two identical cells. The new cells aren’t coming out of nowhere, they’re coming from existing cells that are multiplying.

As we get older, we tend to go back to more of a model of cell replication. A cell duplicates itself, then dies after the new cell has been made. The epigenetic process takes place then, as well. Sometimes the cells won’t necessarily die, because we’re growing and need more cells. That’s done epigenetically, too, because the blueprint from the DNA says what the final adult product is supposed to be like, not just the infant version. As we get really older, the cells are trying to replace themselves, but they don’t do quite as good a job, and that’s an epigenetic process as well, because the instructions are getting messed up *after* the RNA. The histones just aren’t doing such a great job after a while.

The point here is that epigenetics is part of the process of cell development that is already written out in the DNA. The way it works without interference is genetic and heritable, and every single one of the many trillions of cells in your body was created the same way. The DNA has the plans, the RNA is the subcontractor, the histones are giving the orders to the proteins based on the instructions from the higher-ups.

Keep this in mind when you hear things about the amazing effects of environment on epigenetics. Yes, this is the part where things can get screwed up, because, yes, histones can be modified. But I’m going to save that for later, because this is a lot to absorb. I hope this makes sense, and if anyone has questions or corrections, please comment – I want to hear from you.

Epigenetics – I do not think that word means what you think it does.

Epigenetics – I do not think that word means what you think it does.

And I kind of have a bone to pick with Scientists who are actually contributing to the problem. Epigenetics is an essential biological process that takes place at the molecular level. Each one of the hundred trillion or so cells in the human body was created via the epigenetic process. Nothing has to magically happen. All you need is cells, food for the cells (usually glucose, yum!) and DNA.

Unfortunately, the amazing and fascinating research into epigenetics has led to a description of epigenetics as “genes plus environment.” If you are a scientist, or even understand science, you recognize that this does not mean that some sort of environmental factor from outside the body is necessary for the epigenetic process to take place. But if you’re a layperson, that’s exactly what you might think when you hear that. In fact, for quite some time I’ve been debating with a couple of people who believe in this magical concept of epigenetics, and you scientists (whom I otherwise love dearly) are just not helping!

The agouti mouse study that showed a change in coat color (linked along with other references in this previous post) was really exciting, and the public glommed onto it because there was the evidence, right in front of their eyes. In no time at all, alt-med proponents and the general public were certain that this was the answer to everything that was wrong with us. It was a great boon for supplement manufacturers, diet book writers, food conspiracy theorists, and anyone who was looking for something to blame for what was wrong with them (or society, but usually themselves.) I mean, clearly if what a mother mouse ate changed the color of her babies’ fur, then what horrible things are all these toxins doing to our genes?!?!

The thought seems to be that epigenetics is a highly unstable process that actually depends upon the correct “environment” in order to occur, and that even an unpleasant event in childhood can somehow upset it and result in a dramatic condition that can be passed down to one’s offspring. Once a person has gotten this idea into his head, it is darn nigh impossible to get it out. Homeopathic amounts of a “toxin” can have traumatic results, even worse than actual poisoning from that substance, because epigenetics. Psychiatric and neurological conditions are inflicted upon perfectly healthy infants by insufficient parental attachment or attunement. Everything is caused by environmental disruption of the epigenetic process, and everything in the environment messes up epigenetics.

Look, the reality is that what epigenetics does is take the information that’s been put into the RNA from the DNA, turn on the genes that are needed and turns off the ones that aren’t, then sends proteins off with the instructions to make new cells. At conception, when there are only a few cells, there’s not a lot of differentiation, but as fetal development continues, these instructions become more specific. “Make fingers.” “Make retinas.” “Make heart valves.” Stuff like that. During growth, the instructions are more like “make more of these cells.” During adolescence, it’s “make these a little different.” As we age, it’s “make another one just like this,” and “eh, what was that, sonny?”

The environment comes in because it is the epigenetic process during which an environmental factor can possibly alter the process, turning a genetic instruction on that should have been off or vice versa. It’s quite likely that this is what triggers many cancers that are strongly associated with exposure to a particular substance. But the possibility that exposure can impact gene expression is not the same as the inevitability of exposure altering gene expression. And this, people, is a big problem. Scientists, please think about this when you talk about epigenetics. Non-scientists, I’m going to put an explanation of how this works in the simplest terms I can come up with in another post.

Your Inner Fish

Your Inner Fish

I loved this book, and now PBS is making a miniseries with Neil Shubin. I can’t wait.

A long time ago, right after I read it, I put up a series of posts on a forum detailing the wonderful things I had learned from it. After a while, the threads were hijacked by people who just didn’t get it – or didn’t want to get it – and they disappeared into obscurity. But I stand by what I wrote, and now that this book is back in public view, I want to share these thoughts again. This is a long read, over 4,000 words, and it’s taken from a forum thread, so there are parts that don’t flow entirely well, but I don’t want to edit or rewrite it because it captures the wonder and excitement I felt when I first read the book and I don’t want to change that.

So settle down with a nice cup of tea if you’re ready to go below the fold.

Read the rest of this entry