Thursday, July 5, 2012

News and some links


Unfortunately, I will not be able to attend to AHS this year, due to some unexpected financial and academic (damn single molecules!) problems. I was looking forward to meet most of the “paleosphere” and discuss about science with very bright people. I hope I can make it in 2013.

I have been very busy working on the lab, teaching biochemistry and cell biology in Med School and doing a post-graduate diploma in basic and clinical immunology. Because of this, I have had little time for reading carefully studies which are not related to my thesis, and in my short spare time I wanted to let my brain “rest” a little bit, although I have read some (but not as much as I would like to). Accordingly, I haven’t had any time to update the blog. Nonetheless, I have an almost finished post on dietary fats and immune function, which may raise some controversy in the “low carb” community. I want to finish and publish this post once for all (it has been almost finished for almost one month now). I might add some information that I was keeping for my AHS talk (see bottom of the post).

In the meantime, and to make something productive out of this post, here are some studies I have found interesting lately (not all related to the topic of my blog, by the way).


Inducing myocardial infarction (MI) in NOD mice (non-obese diabetic mice) triggers the development of severe post-infarction autoimmune syndrome characterized by lymphocyte infiltration to the myocardium, infarct expansion, and autoantibody and Th1 type response to cardiac alpha-myosin. 83% of a sample of post-myocardial infarction T1D patients showed positive tests for autoantibody against cardiac proteins. 


Post-infarcted myocardium from NOD mice showed extensive lymphocytic infiltrates resembling those from  pancreas islets,  and extended into the non-infarcted myocardium (left image). Normal mice (B6) showed no infiltration nor expansion, but normal infarct healing and scar formation (right image).   © 2012 American Association for the Advancement of Science. All Rights Reserved.

Maternal western diet causes inflammatory milk and TLR2/4-dependent neonatal toxicity

Wild-type (WT) female C57B6J mice were fed with normal chow or chow supplemented with extra cholesterol, fat and sucrose (Western diet, WD) for 2 weeks before breeding with WT male mice, and were maintained on chow or WD during pregnancy and lactation. Pups from mice fed a WD exhibited alopecia* (see figure), increased body weight, thicker lipid layer in the skin (increasing the lipid/skin weight ratio), skin leukocyte infiltration and increased expression of inflammatory genes (IL-1b, TNFa, COX-2, iNOS, MCP-1) in the skin, intestines and liver.

WD-milk promotes alopecia.  Copyright © 2012 by Cold Spring Harbor Laboratory Press 

These deleterious effects were related to the composition of the WD, which contained more saturated fatty acids (SFA) than the chow diet (48.9% vs. 17.6%) **, as well as to the composition of the milk from WD fed mice, which was 24% higher in fat than control. Milk triglycerides from WD-milk had a higher percentage of long chain fatty acids (elongation ratio (C18:0/C10:0) was increased 1.9-fold) and saturated fatty acids (saturation ratio (C18:0/C18:2) was 2.8-fold higher). 

Furthermore, the inflammatory effects induced by the increase in long-chain saturated fatty acids in maternal milk was in part mediated through the production of ceramides and glucosylceramides, as well as lactosylceramide, which was capable of inducing the expression of the aforementioned inflammatory genes in RAW264.7 cells (mouse macrophage cell line). Deletion of TLR2/4 abolished or decreased some of the toxic effects observed, suggesting that TLR-signaling is involved (deletion of adaptor molecule Myd88 also reduced toxicity). Germ-free mice showed an increased toxicity for WD milk, suggesting a protective effect of the normal gut microbiota. 

Time-Restricted Feeding without Reducing Caloric Intake Prevents Metabolic Diseases in Mice Fed a High-Fat Diet

Fasting improves metabolic alterations induced by high-fat diets in mice. Copyright © 2012 Elsevier Inc. All rights reserved.

When fed a high-fat diet, C57BL/6J mice develop inflammatory symptoms and ultimately, metabolic disease. What Du, et al. wanted to know if circadian metabolic oscillations influence the response of mice to a HFD. They used a restricted feeding regimen, which has shown to reset some circadian rhythms. They found that, although eating the same calories overall, mice eating all of the calories from the HFD during an 8-hour window do not developed the symptoms exhibited by mice eating a HFD ad libitum (see figure). In other words, eating the same unhealthy diet at the same level of calories, but restricting it to an 8-hour window, practically abolishes all the deleterious effects***. This effects were mediated by preventing the abnormalities in the circadian rhythm induced by high-fat feeding.

Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis


Iliev, et al. show that although bacteria are a very important (and the major component) for the gut microbiota, there are also other organisms which play an essential role in the host's health. This is the case for some fungi, which interact with the host through dectin-1 receptors, inducing a Th17-type immune response. Mice lacking dectin-1 are susceptible to DSS-induced colitis, and show an abnormal inflammatory response. Inducing colitis altered the mycobiome, and treatment with fluconazole (antifungal) ameliorated inflammation. Analysis of the mouse fungal microbiome revealed that 65.2% of the sequences identified corresponded to Candida tropicalis, an opportunistic pathogen. Their results showed that dectin-1 restricts its body localization to the intestinal lumen (so there is no invasion of inflamed tissues). Colitis also increased the proportion of Candida and Trichosporon (opportunistic pathogens) and reduced the levels of Saccharomyces (non-pathogenic). Supporting their hypothesis, they found an haplotype in the CLEC7A gene (human dectin-1 gene) which was correlated with severe forms of ulcerative colitis, but not with non-severe manifestations.

Fungi levels are an important part of the murine gut microbiota. This probably occurs also in humans.   © 2012 American Association for the Advancement of Science. All Rights Reserved.


A Single-Molecule Hershey-Chase Experiment


The mechanism by which phages transfer their DNA to bacteria has been a key question for decades. Using a single-molecule system with two different strains of lambda phage (differing in genome length), Van Valen, et al. found that the time it takes for lambda phage to transfer its DNA to a single E.coli cell takes 5 minutes (with high cell-to-cell variability and sometimes showing long pauses), compared to what it is  seen in vitro, where the process takes roughly 10 seconds. The in vivo velocity of DNA ejection seemed to be determined by the amount of DNA ejected, not by the amount of DNA left in the viral capsid (which is what has been seen in vitro). The method utilized by the authors is theoretically simple (see figure). They stained viral DNA while still in the capsid with a cyanide dye (SYTOX orange), after which excessive dye is washed out. The stained phages are then briefly bound to bacterial cells, which are pipetted into a flow chamber. After washing again with buffer, the sample is imaged with time-lapse bright-field and fluorescence microscopy. The ejection of DNA is measured by comparing the fluorescence intensity inside the phage capsid to that of the bacterial cell: a loss of fluorescence inside the capsid with a concomitant increase in the cell represents DNA translocation.

Copyright © 2012 Elsevier Inc. All rights reserved. 

This study is important for several reasons. First, it supports the notion that biological process must be seen at a single-molecule level for understanding them. When we do an experiment in bulk, we have millions of molecules at the time interacting. Any measure of a given parameter is just an average of what is being seen. But many times, we fail to see the behaviour of outliers, or in other case, we can have individual differences which are obscured by the number of molecules being analyzed in a given moment and a given space. What is more realistic is to measure single molecules and average repeated experiments. In this way, we a. reduce individual variability obscured by the average, b. observe how molecules behave inside cells (interactions are between single molecules) and c. reduce the influence of other co-solutes and molecules which might influence the process being observed. 


Fluorescence imaging of the process of DNA ejection to a single E.coli cell. Copyright © 2012 Elsevier Inc. All rights reserved. 

Second, this finding challenges what was thought about this process based on in vitro evidence: that the amount of DNA inside the capsid was the driving force for DNA ejection. This was the most logical hypothesis, given that before ejection, the repulsive forces experienced by the tight packing of the DNA would promote its ejection. Lastly, the method utilized its a novel approach that can be expanded to other systems. 

* Hair was recovered after weaning.
** It was also lower in PUFA (12.2% in WD vs. 61.8% in chow), but much higher in sucrose (152.8g/kg in WD vs. 0g/kg in chow). 
*** Nevertheless, some inflammatory cytokines were still increased compared to control.


PD. I wanted to make public my thanks to the AHS organizers for their help with paper work and answering any question I had immediately. I feel very bad for cancelling my talk, specially after their help. Also, I want to thank to everyone who supported my talk me via donations. Please, if anyone who donated wants a refund, send me an email. For anyone who was expecting my talk, I will compensate it with more information on my blog. First, I will finish and post my second post on my nutrition immunotherapy protocol, and second, I will add a new page to the blog with schematic and didactic diagrams made for my presentation. They will be available for free to everyone. 




ResearchBlogging.orgIliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker CA, Fleshner PR, Dubinsky M, Rotter JI, Wang HL, McGovern DP, Brown GD, & Underhill DM (2012). Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science (New York, N.Y.), 336 (6086), 1314-7 PMID: 22674328

Van Valen D, Wu D, Chen YJ, Tuson H, Wiggins P, & Phillips R (2012). A Single-Molecule Hershey-Chase Experiment. Current biology : CB PMID: 22727695

Hatori M, Vollmers C, Zarrinpar A, Ditacchio L, Bushong EA, Gill S, Leblanc M, Chaix A, Joens M, Fitzpatrick JA, Ellisman MH, & Panda S (2012). Time-Restricted Feeding without Reducing Caloric Intake Prevents Metabolic Diseases in Mice Fed a High-Fat Diet. Cell metabolism, 15 (6), 848-60 PMID: 22608008

Du Y, Yang M, Lee S, Behrendt CL, Hooper LV, Saghatelian A, & Wan Y (2012). Maternal western diet causes inflammatory milk and TLR2/4-dependent neonatal toxicity. Genes & development, 26 (12), 1306-11 PMID: 22713870

Gottumukkala RV, Lv H, Cornivelli L, Wagers AJ, Kwong RY, Bronson R, Stewart GC, Schulze PC, Chutkow W, Wolpert HA, Lee RT, & Lipes MA (2012). Myocardial infarction triggers chronic cardiac autoimmunity in type 1 diabetes. Science translational medicine, 4 (138) PMID: 22700956

4 comments:

  1. Its good to see you back. Looking forward to announced post.

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  2. Thanks. I am loving the animal studies that relate to IF and circadian rhythm. Looks like the "metabolic advantage" or whatever it is from normalizing cortisol timing equals or trumps the macronutrient ratio equivalent (whatever that may be) in these mice (see also http://www.ncbi.nlm.nih.gov/pubmed/22593546)
    and that diet timing equals or surpasses light-dark timing as a way to reset the circadian clock; a damn good thing as it's easier to eat a good meal than have a good sleep as planned.
    http://www.ncbi.nlm.nih.gov/pubmed/21952132
    make that two good meals...
    http://www.ncbi.nlm.nih.gov/pubmed/22587351

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    Replies
    1. Im starting to lean towards a more "flexible" approach which has its basis on rhythmicity and cellular energy partitioning.

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