Category Archives: General

Jun25

Wednesday Links

Posted on by
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On the topic of GMOs, Gilles-Eric Séralini’s paper linking glyphosate to tumors in rats, which was retracted last year because of methodological and statistical flaws, has been re-published in a journal with apparently less exacting standards.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.

May24

What has been happening. . .

Posted on by
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.

Apr21

Learning from Research, The Results.

Posted on by
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
Allele – An 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.