It Is Imperfection - Not Perfection - That Is The End Result Of The Program Written Into That Formidably

Checking Cancer At Its Origin..

In a first, the lab led by Leonard Zon at Boston Children’s Hospital has visualised the emergence of the primary melanoma cell in transgenic zebrafish that harbour the human oncogenic BRAFV600E mutation in melanocytes. This cancerous state is characterised in maturing fish by the formation of neural crest progenitors [NCPs], which are the predecessors of melanocytes and are only seen in the embryonic stage of healthy zebrafish.

The Zon lab placed the human mutated oncogene, BRAFV600E (a characteristic of benign human nevi/moles) under the control of a melanocyte-specific promoter and introduced it into the zebrafish. Generations of this transgenic fish were engineered such that they were also deficient in functional p53 (loss of function mutation). They used previous findings that in healthy zebrafish, a gene called crestin is expressed only in the embryonic NCPs and never throughout maturity, but is re-expressed selectively in melanomatous cells during adulthood. crestin was cloned adjacent to a reporter, enhanced green fluorescent protein [EGFP] for live imaging purposes.

The developmental phases of the fish, that were by now triple transgenic (for human BRAFV600E, p53 LOF and crestin:EGFP) were observed by live imaging; ~21 days after fertilisation, the expression of crestin:EGFP localised precisely to the (future) melanoma sites, and the very first triple-transgenic (individual) cells that went on to form larger masses of cells were also observed. To summarise, melanoma formation was observed in three stages: individual fluorescent cells, followed by these cells multiplying to form groups of <50 cells, and lastly these groups forming raised lesions. This consistently held true, with all 30 observed individual cells turning into 30 lesions. These results are illustrated in Figure 1.

Figure 1. In the top left box, a single cell is visualised as it multiplies into a group of melanoma cells (top right). The bottom images show the raised melanoma lesion as observed by the naked eye and by live imaging. The green fluorescence emitted from EGFP indicates that it is localised only to the melanoma (as is crestin expression), that is, it has not metastasised elsewhere. 

These pre-cancerous cells were also shown to be self-sustaining and tumourigenic: when fish scales containing the mutant cells were transplanted to another part of the same fish (auto-transplant) or to another fish (allo-transplant) that was also exposed to radiation, the cells proliferated in the new site, as well as penetrated the hypodermis underneath (Figure 2).

Figure 2. The fluorescence indicates a single scale being auto-transplanted elsewhere on the same fish. As the days progress, the patch expands as well, and after day 33, the cells penetrate deeper into the hypodermis and thrive independently, and excising the transplanted scale proves futile. 

Role of Transcription Factor sox10

sox10 is a master TF in NCP and its over-expression has been correlated with increased crestin expression, and accordingly, sox10 over expression in the transgenic melanocytes accelerated the melanoma onset. Following the logical train of thought that sox10 promotes melanoma progression, it was then targeted by CRISPR-Cas9 and inactivated in the transgenic cells. This resulted in a delayed onset of melanoma (180 days) compared to the controls (133 days). sox10 is also known to be expressed in most human melanoma cell lines. Moreover, the DNA element that acts as the binding site for Sox10 is also found in a hyper-acetylated [H3K27Ac], super-enhancer state. This is an epigenetic alteration and may prove a useful target in therapy (ex. HAT inhibitors).

Summary

The key finding clears up a hitherto ambiguous association between a reversion to stem/progenitor cell-like status and cancer: it indicates that the apparent devolution of a specialised cell to a primitive cellular state is not a consequence of cancer progression, but that it is an hallmark of pre-cancerous cells that may contribute to tumour progression. The rarity of melanoma formation among the mutant cells also suggests that the double mutant [BRAFV600E; p53 LOF] is not the only factor to influence the onset. Experimentally, crestin expression was a definitive prelude to formation of nevi which transformed into full-fledged raised melanomas in that spot.

This discovery has two chronological applications: first, of the many susceptible melanocytes harbouring the mutated oncogene, we can find out which are most likely to enter the melanoma state. Peaks in the expression profile of sox2, or a couple other TFs, dlx2 and tfap2, can prove to be a telltale pre-melanoma signature and thus be used in diagnosis. Secondly, by doing so, these can be better targeted early on before they’ve disseminated and become virtually untreatable.

Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J, et al. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science. 2016;351[6272]:aad2197–aad2197.

Next week I’ll give a presentation on the Researchers Night at Eötvös Loránd University, Hungary with the title: “Chemistry of light and the light of chemistry”.

During this presentation one of my favorite dyes will be also presented: Nile Red. However, just as usual, the 1000 USD/gram price was a bit over our budget, so I had to make it.

The raw product was contaminated with a few impurities, but a fast purification, by simple filtering the mixture through a short column helped a lot and ended up with a +95% pure product.

At first I concentrated the product from a dilute solution on the column as seen on the first pics. It’s interesting to see, that it has a different fluorescence in solution (faint orange fluorescent)  and while it’s absorbed on the solid phase (pink, highly fluorescent).

After all the product was on the solid phase, I added another solvent and washed down the pure, HIGHLY FLUORESCENT product. Everything else, what was mainly products of side reactions, stuck at the top of the column as seen on the second pics and the gifs.

Also here is a video from the whole process in HD: https://youtu.be/W0Lk5jkd_B0

 Poison in the Brain: Toxic Structures in Neuronal Nuclei of Alzheimer’s Patients 

Spherical structures in the nucleus of nerve cells, so-called nuclear spheres, are suspected to trigger Alzheimer’s disease. A team headed by Dr Thorsten Müller from the research group Cell Signaling in Neurodegeneration has for the very first time demonstrated the presence of the presumably toxic protein aggregates in the human brain. The researchers from Ruhr-Universität Bochum have published their article in the journal Neurobiology of Aging.

The team compared brain samples from Alzheimer’s patients with those of the healthy individuals in the same age group. The result: in the samples taken from Alzheimer’s patients, the number of nuclear spheres was much higher than in those taken from healthy study participants.

Moreover, the group from Bochum analysed in what way nuclear spheres are generated. It was demonstrated in experiments with cell cultures that the amyloid precursor protein (APP) plays a crucial role in this process. APP has long been associated with Alzheimer’s disease. The researchers observed that nuclear sphere generation preferably takes place, if the amyloid precursor protein carries no phosphate group in a specific amino acid. An APP cleavage product, moreover, is contained in the nuclear spheres.

“Extensive nuclear sphere generation in the human Alzheimer’s brain” by Katharina Kolbe, Hassan Bukhari, Christina Loosse, Gregor Leonhardt, Annika Glotzbach, Magdalena Pawlas, Katharina Hess, Carsten Theiss, and Thorsten Müller in Neurobiology of Aging. Published online August 18 2016 doi:10.1016/j.neurobiolaging.2016.08.016

This year’s Halloween special wraps up the chemistry behind making a mummy: http://wp.me/p4aPLT-26m

(Image caption: An fMRI scan shows regions of the brain that become active when devoutly religious study participants have a spiritual experience, including a reward center in the brain, the nucleus accumbens. Credit: Jeffrey Anderson)

This is your brain on God

Religious and spiritual experiences activate the brain reward circuits in much the same way as love, sex, gambling, drugs and music, report researchers at the University of Utah School of Medicine. The findings were published in the journal Social Neuroscience.

“We’re just beginning to understand how the brain participates in experiences that believers interpret as spiritual, divine or transcendent,” says senior author and neuroradiologist Jeff Anderson. “In the last few years, brain imaging technologies have matured in ways that are letting us approach questions that have been around for millennia.”

Specifically, the investigators set out to determine which brain networks are involved in representing spiritual feelings in one group, devout Mormons, by creating an environment that triggered participants to “feel the Spirit.” Identifying this feeling of peace and closeness with God in oneself and others is a critically important part of Mormons’ lives — they make decisions based on these feelings; treat them as confirmation of doctrinal principles; and view them as a primary means of communication with the divine.

During fMRI scans, 19 young-adult church members — including seven females and 12 males — performed four tasks in response to content meant to evoke spiritual feelings. The hour-long exam included six minutes of rest; six minutes of audiovisual control (a video detailing their church’s membership statistics); eight minutes of quotations by Mormon and world religious leaders; eight minutes of reading familiar passages from the Book of Mormon; 12 minutes of audiovisual stimuli (church-produced video of family and Biblical scenes, and other religiously evocative content); and another eight minutes of quotations.

During the initial quotations portion of the exam, participants — each a former full-time missionary — were shown a series of quotes, each followed by the question “Are you feeling the spirit?” Participants responded with answers ranging from “not feeling” to “very strongly feeling.”

Researchers collected detailed assessments of the feelings of participants, who, almost universally, reported experiencing the kinds of feelings typical of an intense worship service. They described feelings of peace and physical sensations of warmth. Many were in tears by the end of the scan. In one experiment, participants pushed a button when they felt a peak spiritual feeling while watching church-produced stimuli.

“When our study participants were instructed to think about a savior, about being with their families for eternity, about their heavenly rewards, their brains and bodies physically responded,” says lead author Michael Ferguson, who carried out the study as a bioengineering graduate student at the University of Utah.

Based on fMRI scans, the researchers found that powerful spiritual feelings were reproducibly associated with activation in the nucleus accumbens, a critical brain region for processing reward. Peak activity occurred about 1-3 seconds before participants pushed the button and was replicated in each of the four tasks. As participants were experiencing peak feelings, their hearts beat faster and their breathing deepened.

In addition to the brain’s reward circuits, the researchers found that spiritual feelings were associated with the medial prefrontal cortex, which is a complex brain region that is activated by tasks involving valuation, judgment and moral reasoning. Spiritual feelings also activated brain regions associated with focused attention.

“Religious experience is perhaps the most influential part of how people make decisions that affect all of us, for good and for ill. Understanding what happens in the brain to contribute to those decisions is really important,” says Anderson, noting that we don’t yet know if believers of other religions would respond the same way. Work by others suggests that the brain responds quite differently to meditative and contemplative practices characteristic of some eastern religions, but so far little is known about the neuroscience of western spiritual practices.

The study is the first initiative of the Religious Brain Project, launched by a group of University of Utah researchers in 2014, which aims to understand how the brain operates in people with deep spiritual and religious beliefs.

This team of early female astronomers created the star classification system we use today.

In the late 19th century, astronomy was a growing field. At the time, Edward Pickering, the director of the Harvard College Observatory, was working to create a classification system for stars by capturing the light from these distant celestial objects onto photographic glass plates. A team of female assistants and astronomers meticulously maintained and analyzed these delicate negatives. In her new book, The Glass Universe: How the Ladies of the Harvard Observatory Took the Measure of the Stars, Dava Sobel shares the stories of these female “human computers” and how their work helped to advance the field of astronomy and the role of women in science.

This team of astronomers included Williamina Fleming, who was once Pickering’s maid but eventually became a supervisor to the group and went on to identify hundreds of variable stars. And Henrietta Swan Leavitt’s observations about the luminosity of stars would shape later ideas about the expanding universe.

Listen to the interview here.

[Photos courtesy of The Glass Universe]

Stephanie Kwolek was born #OTD in 1923. She’s most famous for inventing the bulletproof polymer Kevlar: wp.me/p4aPLT-3dv

Today is the International Day of Women and Girls in Science, so let’s write women back into science history. Check out the gallery here.

What Happens in the Brain During Unconsciousness?

Researchers are shining a light on the darkness of the unconscious brain. Three new studies add to the body of knowledge.

When patients undergo major surgery, they’re often put under anesthesia to allow the brain to be in an unconscious state.

But what’s happening in the brain during that time?

Three Michigan Medicine researchers are authors on three new articles from the Center for Consciousness Science exploring this question — specifically how brain networks fragment in association with a variety of unconsciousness states.

“These studies come from a long-standing hypothesis my colleagues and I have had regarding the essential characteristic of why we are conscious and how we become unconscious, based on patterns of information transfer in the brain,” says George A. Mashour, M.D., Ph.D., professor of anesthesiology, director of the Center for Consciousness Science and associate dean for clinical and translational research at the University of Michigan Medical School.

In the studies, the team not only explores how the brain networks fragment, but also how better to measure what is happening.

“We’ve been working for a decade to understand in a more refined way how the spatial and temporal aspects of brain function break down during unconsciousness, how we can measure that breakdown and the implications for information processing,” says UnCheol Lee, Ph.D., physicist, assistant professor of anesthesiology and associate director of the Center for Consciousness Science.

Examining different aspects of unconsciousness

The basis for the three studies, as well as other work from the Center for Consciousness Science, comes from a theory Mashour produced during his residency.

“I published a theoretical article when I was a resident in anesthesiology suggesting that anesthesia doesn’t work by turning the brain off, per se, but rather by isolating processes in certain areas of the brain,” Mashour says. “Instead of seeing a highly connected brain network, anesthesia results in an array of islands with isolated cognition and processing. We have taken this thought, as well as the work of others, and built upon it with our research.”

In the study in the Journal of Neuroscience, the team analyzed different areas of the brain during sedation, surgical anesthesia and a vegetative state.

“It’s often suggested that different areas of the brain that typically talk to one another get out of sync during unconsciousness,” says Anthony Hudetz, Ph.D., professor of anesthesiology, scientific director of the Center for Consciousness Science and senior author on the study. “We showed in the early stages of sedation, the information processing timeline gets much longer and local areas of the brain become more tightly connected within themselves. That tightening might lead to the inability to connect with distant areas.”

In the Frontiers in Human Neuroscience study, the team delved into how the brain integrates information and how it can be measured in the real world.

“We took a very complex computational task of measuring information integration in the brain and broke it down into a more manageable task,” says Lee, senior author on the study. “We demonstrated that as the brain gets more modular and has more local conversations, the measure of information integration starts to decrease. Essentially, we looked at how the brain network fragmentation was taking place and how to measure that fragmentation, which gives us the sense of why we lose consciousness.”

Finally, the latest article, in Trends in Neurosciences, aimed to take the team’s previous studies and other work on the subject of unconsciousness and put together a fuller picture.

“We examined unconsciousness across three different conditions: physiological, pharmacological and pathological,” says Mashour, lead author on the study. “We found that during unconsciousness, disrupted connectivity in the brain and greater modularity are creating an environment that is inhospitable to the kind of efficient information transfer that is required for consciousness.”

How these studies can help patients

The team members at the Center for Consciousness Science note that all of this work may help patients in the future.

“We’re looking for a better way to quantify the depth of anesthesia in the operating room and to assess consciousness in someone who has had a stroke or brain damage,” Hudetz says. “For example, we may assume that a patient is fully unconscious based on behavior, but in some cases consciousness can persist despite unresponsiveness.”

The team hopes this and future research could lead to therapeutic strategies for patients.

“We want to understand the communication breakdown that occurs in the brain during unconsciousness so we can precisely target or monitor these circuits to achieve safer anesthesia and restore these circuits to improve outcomes of coma,” Mashour says.