CRISPR Editing In Blood Stem Cells

CRISPR editing in blood stem cells; abstract:

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.

Strains Of Misfolded Proteins And Alzheimer's Disease

Neurodegenerative diseases tend to involve tangled misfolded proteins, and for some diseases “strains” are already known.  Here we see that the same applies for amyloid and tau proteins in Alzheimer’s disease, a finding that can be helpful for targeted, personalized medicine.  Abstract:

Most neurodegenerative diseases are proteinopathies, which are characterized by the aggregation of misfolded proteins. Although many proteins have an intrinsic propensity to aggregate, particularly when cellular clearance systems start to fail in the context of ageing, only a few form fibrillar aggregates. In Alzheimer disease, the peptide amyloid-β (Aβ) and the protein tau aggregate to form plaques and tangles, respectively, which comprise the histopathological hallmarks of this disease. This Review discusses the complexity of Aβ biogenesis, trafficking, post-translational modifications and aggregation states. Tau and its various isoforms, which are subject to a vast array of post-translational modifications, are also explored. The methodological advances that revealed this complexity are described. Finally, the toxic effects of distinct species of tau and Aβ are discussed, as well as the concept of protein 'strains', and how this knowledge can facilitate the development of early disease biomarkers for stratifying patients and validating new therapies. By targeting distinct species of Aβ and tau for therapeutic intervention, the way might be paved for personalized medicine and more-targeted treatment strategies.

Egg whites and tomatoes dish

Here is an easy and low-carbohydrate dish that can be served for breakfast, lunch or dinner. For breakfast, add a few nuts. For lunch and dinner, serve with a cucumber-dill salad.

Ingredients:
1 carton egg whites (32 oz)
2 cans diced tomatoes (I use Hunt's 14.5 oz each)
2 – 3 cups crumbled feta cheese
optional spices: black pepper, winter savory, hot red pepper flakes  (I do not use salt since the feta cheese is salty enough)


Directions:
Preheat oven to 400ºF and oil a 12 x 14-inch pan. Mix all ingredients, pour into the pan and bake for 40 - 45 minutes. The baking time would depend upon the size of the pan (depth including).

Cytoplasmic DNA And Metastasis

Chromosomal instability results in DNA in the cytoplasm, which is of course not normal, DNA being normally found only in the nucleus.  This misplaced cytoplasmic DNA triggers a pathway that simulates cell signaling that can promote metastasis.  This could in theory be targeted to inhibit metastasis (keep in mind that cancer mortality is for the most part due to metastasis).  Abstract:

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Colon Cancer Mutations Associated With The Gut Microbiota

An extremely important study on colon cancer concludes: “We found statistically significant associations between loss-of-function mutations in tumor genes and shifts in the abundances of specific sets of bacterial taxa, suggestive of potential functional interaction.”  Further, the correlation between bacterial types and tumor mutations allowed for predictions about interactions between the mutations and the downstream signaling pathways affected by these mutations.  One can expect future work to more clearly delineate the molecular mechanisms of interaction between the microbiota and cancer mutations.  In the meantime, once again – for the thousandth time it seems – the importance of the gut microbiota is emphasized.  Abstract:

Variation in the gut microbiome has been linked to colorectal cancer (CRC), as well as to host genetic variation. However, we do not know whether, in addition to baseline host genetics, somatic mutational profiles in CRC tumors interact with the surrounding tumor microbiome, and if so, whether these changes can be used to understand microbe-host interactions with potential functional biological relevance. Here, we characterized the association between CRC microbial communities and tumor mutations using microbiome profiling and whole-exome sequencing in 44 pairs of tumors and matched normal tissues. We found statistically significant associations between loss-of-function mutations in tumor genes and shifts in the abundances of specific sets of bacterial taxa, suggestive of potential functional interaction. This correlation allows us to statistically predict interactions between loss-of-function tumor mutations in cancer-related genes and pathways, including MAPK and Wnt signaling, solely based on the composition of the microbiome. In conclusion, our study shows that CRC microbiomes are correlated with tumor mutational profiles, pointing towards possible mechanisms of molecular interaction.

Genetic Structure Affected By Maternal Care (In Mice)

Early life experiences, such as maternal care, alters the genetic structure in neurons in mice, with mobile genetic elements seemingly responsible.  Does the same apply to humans?  Abstract:

The brain is a genomic mosaic owing to somatic mutations that arise throughout development. Mobile genetic elements, including retrotransposons, are one source of somatic mosaicism in the brain. Retrotransposition may represent a form of plasticity in response to experience. Here, we use droplet digital polymerase chain reaction to show that natural variations in maternal care mediate the mobilization of long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons in the hippocampus of the mouse brain. Increasing the amount of maternal care blocks the accumulation of L1. Maternal care also alters DNA methylation at YY1 binding sites implicated in L1 activation and affects expression of the de novo methyltransferase DNMT3a. Our observations indicate that early life experience drives somatic variation in the genome via L1 retrotransposons.

The future workforce

Here is a recent story that I have come to know: a group of teenagers emerged through Internet connections. They found each other on social platforms and revealed common interests. Soon, their social media chat turned into actions. These teens have been churning out products together for some time now. There is no defined group structure among them. In fact, projects are fluid, ideas come suddenly and are acted upon quickly, their collective mind seeks and finds solutions to problems as they arise. People are free to join or leave the projects at any time.

Can other self-guided associations develop and reach professional realization? Can these groups come up with their “big picture” and work all the way to achieve the desired goals? Indeed, this model has worked many times, and has yielded successful and profitable companies. Think of the many tech start-ups that either developed into large companies or were acquired by giant corporations for a hefty pad of money.
 
Why does it work?
There is no administration to obstruct the progress and distract the working. Inept leadership can inflict destruction beyond recovery. Below are the well known versions of the leader’s modus operandi:

You can imagine that versions 1, 2 and 4 are not achieving great (or any) results. However, do not underestimate the inertia of “teams”. In some cases, most team members gratefully accept the leaders of type 1, 2 and 4.

1. A leader, who dictates both goal and means, is an OCD case and s/he completely devalues and squanders the intellectual and motivational power of the team.

2. A leader, who does not present any goal or “big picture” but monitors on every-day basis the subordinates’ actions, is an efficiency-killer and will not achieve too much in a long run. The team feels as if it is acting blindfolded.

4. A leader, who does not demonstrate any effort, has no ideas, no goals, no means, is someone who just collects a salary.

3. A true leader sketches the "big picture” and develops it further with the team. A true leader does not dictate how the goals will be achieved.
 

More advantages of the fluid groups

Out-of the box-ideas. In a fluid group, as the teenage group described above, when ideas emerge from the group, there is a spontaneous co-development process and enhancement of the idea. Almost everyone shares the ownership of the idea. There is no need of a well-established leader, who needs to convince the team in the merits of the idea. The idea emerges and develops organically. The team decides on the implementation path.
 
There is no red tape to shut down the creative process with petty details, mind-boggling loops, and useless meetings dictated by higher authorities and interests. We are all slaves to the red tape, especially if we are not self-employed.
 
Peer-to-peer teaching is most effective. Harvard knows it, now you know it. Teams must evolve by acquiring new information and skills. Information in some fields doubles within hours or days. To stay current, we need to run along with the informational flow. Some jobs and positions require “professional development” from their employees. This development is frequently based upon meetings and workshops, with an instructor in front of a group of bored adults. This is an inefficient model compared to what takes place in the fluid group described above. In the self-organized group of peers, there is a core of group members, but other members voluntarily come and go. This flow enriches the expertise of the team. The members are also all peers. They all share information as part of their usual behavior and daily communications. Learning from each other happens organically.

Failure happens frequently and is expected. In a rigid institution, failure is feared, it raises red flags and stress. And yet, there is not too much to lose when you do not have too much to begin with. Therefore, in a fluid group of teenagers with minuscule bank accounts, not too much could be lost. The stakes are much higher in our institutions and at later stages of life.
 
No one is afraid of or deterred by criticism. Typically, there is no well-established hierarchy at the beginning of any self-assembled, fluid group. There is mostly comradeship. In this mode of operation, one gives advise/tip/help, not criticism.
 
Size of the team is self-controlled. It is known that the number of performance problems increases exponentially with the size of the team. The recommended team size is four to 12. Beyond this number, cliques develop.

Are these principles applied in your work teams?

More On Gut Microbiota And Colorectal Cancer

Another paper about gut microbiota and colorectal cancer is of interest.  Abstract:

Pre-clinical and clinical data produce mounting evidence that the microbiota is strongly associated with colorectal carcinogenesis. Dysbiosis may change the course of carcinogenesis as microbial actions seem to impact genetic and epigenetic alterations leading to dysplasia, clonal expansion and malignant transformation. Initiation and promotion of colorectal cancer may result from direct bacterial actions, bacterial metabolites and inflammatory pathways. Newer aspects of microbiota and colorectal cancer include quorum sensing, biofilm formation, sidedness and effects/countereffects of microbiota and probiotics on chemotherapy. In the future, targeting the microbiota will probably be a powerful weapon in the battle against CRC as gut microbiology, genomics and metabolomics promise to uncover important linkages between microbiota and intestinal health.

Again we see the paradigm that targeting the gut microbiota, to yield a more healthy bacterial profile,c can be a future anti-cancer approach.

p53 Inhibits CRISPR-Cas9 Engineering In Human Pluripotent Stem Cells

Human pluripotent stem cells are difficult to engineer with the CRSPR system compared to other cell types, which is unfortunate given the great potential in being able to modify human stem cells.  The p53 gene protects against cancer and it seems that p53 function interferes with CRISPR modification of these human stem cell types by making the genetic alterations induced by CRISPR relatively toxic to the cells.  Human stem cells can acquire p53 mutations – which can lead to cancer – and it would seem that human stem cells that have been successfully engineered with CRISPR may be at higher risk for having p53 mutations since the presence of such mutations, inactivating toxicity to CRISPR, may have been the reason the cells were successfully engineered in the first place.  This is an important finding suggesting caution in using CRISPR in such cells and also suggests the need to find novel approaches that can increase CRISPR efficiency in these cells while still maintaining p53 and its anti-cancer functions.  Abstract:

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

Environmental Fires And The Carbon Cycle

By BLM - http://www.blm.gov/photos/netpub/server.np?find&catalog=catalog&site=BLM&field=Keywords&op=contains&value=wildfire&sorton=Cataloged&&template=details.np&offset=77, Public Domain, https://commons.wikimedia.org/w/index.php?curid=17311095

The environment affects our health, and this is an article showing how fire frequency is grasslands and forests alters the carbon cycle and other aspects of the environment. There probably needs to be studies of the long-term health implications of large scale environmental change.  Abstract:

Fire frequency is changing globally and is projected to affect the global carbon cycle and climate. However, uncertainty about how ecosystems respond to decadal changes in fire frequency makes it difficult to predict the effects of altered fire regimes on the carbon cycle; for instance, we do not fully understand the long-term effects of fire on soil carbon and nutrient storage, or whether fire-driven nutrient losses limit plant productivity. Here we analyse data from 48 sites in savanna grasslands, broadleaf forests and needleleaf forests spanning up to 65 years, during which time the frequency of fires was altered at each site. We find that frequently burned plots experienced a decline in surface soil carbon and nitrogen that was non-saturating through time, having 36 per cent (±13 per cent) less carbon and 38 per cent (±16 per cent) less nitrogen after 64 years than plots that were protected from fire. Fire-driven carbon and nitrogen losses were substantial in savanna grasslands and broadleaf forests, but not in temperate and boreal needleleaf forests. We also observe comparable soil carbon and nitrogen losses in an independent field dataset and in dynamic model simulations of global vegetation. The model study predicts that the long-term losses of soil nitrogen that result from more frequent burning may in turn decrease the carbon that is sequestered by net primary productivity by about 20 per cent of the total carbon that is emitted from burning biomass over the same period. Furthermore, we estimate that the effects of changes in fire frequency on ecosystem carbon storage may be 30 per cent too low if they do not include multidecadal changes in soil carbon, especially in drier savanna grasslands. Future changes in fire frequency may shift ecosystem carbon storage by changing soil carbon pools and nitrogen limitations on plant growth, altering the carbon sink capacity of frequently burning savanna grasslands and broadleaf forests.