Beating cardiac cells now in petri dishes

Our body can replace as many cells as it desires, but not for all organs. Liver on one side has most and brain on other side least regeneration capability out of all organs. Cardiac cells are also hardly regenerated and pose a grave threat in case of cardiac arrest.

heat beat

Back in 2011 a direct reprogramming of skin cells that bypasses i-PS led to a totally new paradigm. Sheng Dings group from Scripps Research Institute reprogrammed stem cells in such a way that laborious process of going through embryonic-stem cell generation was evaded. In 2013 Marko Mihovilovics group in Vienna claimed to have created certain chemicals that can regulate the differentiation process of progenitor cells and turn them into cardiac cells.

A team of researchers recently generated cardiac cells that can beat and are observable under microscope only. These cardiac cells created from tissues like skin and blood have the talent to multiply. Normal cardiac cells after they wear and die are irreplaceable or regenerate very poorly. Due to this reason cardiac attacks are often fatal. One of the main disadvantages of any organ or tissue replacement is the denial by immune system. However these beating cardiac cells unlike embryonic stem cells are obtained from patient itself and hence have least chances of being getting rejected. Though more advancement in this area is needed, but this achievement by Glen Tibbits group (Canada) can have a great impact on personalized or tailored treatment in near future.

GPS system of brain- The Nobel Prize 2014 in Physiology or Medicine

Karolinska Institute [Sweden] has chosen the Year 2014 Nobel Prize award in Physiology/Medicine to be awarded to a pair of researchers from Norway [May-Britt Moser and Edvard I. Moser] and John O’Keefe an American born British researcher. The Columbia University’s Louisa Gross Horwitz Prize was also awarded to this trio last year 2013. These scientists solved a riddle which had puzzled researchers for over centuries-how does ones brain generate a mental map of surrounding space and how is such map used to navigate in an environment or how can one navigate his/her way through a compound environment?

May-Brit-Moser

Understanding the mechanisms behind the extraction and representation of environmental signals by the brain is one of the hot and emerging areas of neuroscience. The fundamental principles that govern our cognition and navigation are still to be fully understood. Most of us when new to any area often use GPS for locating the places. In an analogous fashion brain uses GPS system to help us and other organisms to navigate from place to place. But how exactly does our brain accomplish such smart-phone kind of activity was for the first time deduced/elaborated by the efforts of the team mentioned above.

This geo-positioning system of our brain [which actually uses some distinct cells for cognitive functions] stores evidence from every place we visit or happen to be at any point of time and then retrieves the same information from memory to help us navigate or pilot in the 3D world.

The beginning of this miraculous discovery started when Dr. O’Keefe found that certain nerve cells of the hippocampus region of brain of a rat stored information that is visible and or invisible. Such cells were called as “place cells’’ and abet navigation system by backing up the seen as well as unseen data from various environments. Such piling up of inner maps is actually the soul or library of the navigational system of our brains.

The vital add up to this finding was the discovery of “grid cells” by Mosers in the year 2005 in the entorhinal cortex of the brain. In fact “grid cells” are in blood-relation with “place cells” and the functions of navigation system are distributed among them. “Place cells” act as a tape recorder while “grid cells” decode that message to allow coordination and positioning for the system. It is like opening the application HERE Maps or setting up the destination in a HERE Drive application on a windows phone and in smooth way you steer to your target location. Grid cells and place cells thus seem to pile up spatial mental maps of the environment and then exactly compute the path to be followed.

This Nobel Prize highlights the importance of Brain-Maps in navigation system. Since Alzheimer’s patients suffer from the navigation difficulty in their environment, more research might really help in a noble cause to treat such patients in near future.

Transparent Mouse. Believe it or not?

You might have seen transparent mouse used to point icons on laptop/computer, a transparent cell phone or a transparent screen, but transparent mouse (rodent), no way! Read how researchers created a transparent mouse for you.

transparent

Mice form and important part of research now-a-days. Most of the experiments are carried on mice because of their striking similarity to humans in most matters. In-fact mice can be manipulated and used to treat human diseases.

Transparent mice offer complete anatomical view. One can see all of the organs including brain. Although there have been several studies where in individual organs were made transparent, but this paper is the first one to describe whole body tissue clearing.

The researchers have proposed a method for whole-body clearing by delivering clarifying agents. Passive clarity technique (PACT), perfusion-assisted agent/release in-situ (PARS) and Refractive index matching solution (RIMS) are the advanced techniques/chemicals used for tissue extraction. These techniques have been successfully utilized by the researchers to craft optically transparent organisms. Not only can one see through the organs, but can also analyze things at sub-cellular, cellular and single-molecule levels.

The Authors of the paper mention that,

“PARS opens up the possibility of whole-organ and whole-organism mapping with high phenotypic content. With this in mind, quick, low-resolution scanning of large tissue blocks can direct investigators to restricted areas worthy of slow, high phenotypic content analysis, including smFISH: a method that preserves fluorescent markers long-term is particularly valuable in this respect.

Possible applications of PARS include:

1. Improved screening throughput, by eliminating the need to section individual tissues.

2. Improvement in existing therapies and can lead a way for novel new therapeutic strategies.

3. Screening of cancerous and tumor cells.

4. Accurate postmortem quantification.

5. More advancement in neuroscience.

6. Treatment of diseases like autism and chronic pain.

7. Locate hidden viruses in the tissues and monitor metastasis of cancer.

For some of the really cool pictures and images taken by the team of this paper please check out the original paper. Wonderful, you will see that it looks more decorated art than images.

Citation:

Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

Bin Yang, Jennifer B. Treweek, Rajan P. Kulkarni, Benjamin E. Deverman, Chun-Kan Chen, Eric Lubeck, Sheel Shah, Long Cai, Viviana Gradinaruemail

DOI: http://dx.doi.org/10.1016/j.cell.2014.07.017

Stem Cell Controversy-How an acid bath leaves you a skeleton??

Two papers describing the stimulus-triggered acquisition of pluripotency (STAP) Stem cells, which received mass attention, earlier January this year, have been retracted from the scientific journal Nature after about six months of publication. The titles of the papers are “Stimulus-triggered fate conversion of somatic cells into pluripotency” (H. Obokata et al. Nature 505, 641–647; 2014) and “Bidirectional developmental potential in reprogrammed cells with acquired pluripotency” (H. Obokata et al. Nature 505, 676–680; 2014).

http://upload.wikimedia.org/wikipedia/commons/thumb/6/65/Human_embryonic_stem_cells.png/250px-Human_embryonic_stem_cells.png

Human embryonic stem cells that are not yet differentiated.

These papers described a versatile strategy for producing stem cells without any amendment of genetic material-DNA. The papers claimed STAP requires neither nuclear transfer nor introduction of any transcription factors. The process of producing STAP stem cells as explained in the paper is simple. Regular cells from any body part are taken and then exposed to stress, by dipping them in an acid bath. This process seemed to be rich and resourcefull in nature and promised stem cells exploitation in disease treatments.

Researchers tried to reproduce these STAP cells for months, but all in vain. The protocols and data described in the papers have been proven to be manipulated/misrepresented or non-reproducible.

What Authors to say about it?

A statement describing the details of the errors, at least in five points has been outlined by the authors of the papers.

“We apologize for the mistakes included in the Article and Letter. These multiple errors impair the credibility of the study as a whole and we are unable to say without doubt whether the STAP-SC phenomenon is real. Ongoing studies are investigating this phenomenon afresh, but given the extensive nature of the errors currently found, we consider it appropriate to retract both papers.”

What Nature reveals about it?

“We at Nature have examined the reports about the two papers from our referees and our own editorial records. Before publishing, we had checked that the results had been independently replicated in the laboratories of the co-authors, and we regret that we did not capture the authors’ assurances in the author-contributions statements.”

While highlighting the policy of retraction, Nature also mentions an important note that goes like this-

The papers themselves have now been clearly watermarked to highlight their retracted status, but will remain hosted on Nature’s website, as is consistent with our retraction policy. (In our opinion, to take down retracted papers from journal websites amounts to an attempt to rewrite history, and makes life needlessly difficult for those wishing to learn from such episodes.)”

How reputation of authors can get you a Nature publication.

The authors of the papers published were almost all reputed in their respective fields. Teruhiko Wakayama is a well known internationally known Mouse cloning pioneer. Hitoshi Niwa is an internationally respected stem cell researcher at RIKEN CDB. Charles A. Vacanti is a well known tissue engineer at Brigham and is the corresponding author of these papers. C.A. Vacanti in an official letter to Nature on 30th May this year agreed to retract the papers and was last of the authors to do so.

It seems that Nature has overlooked the things in the papers due to these big names on front. Peer-review system seems to be ineffective and slow.  It no doubt is clear that we need more filters and scrutiny for screening the manuscripts to prevent future controversies and misleading research. Science is all build upon trust and if one researcher for the sake of increasing his/her impact points misleads other researchers then he/she has no right to be in the scientific community. It is about wasting time, effort, money etc and all this makes you look a crook in eyes of others. The pictures below shows lead author Haruko Obokata after queries were raised about her paper.

Stem-master675

Nature has published an editorial about the STAP stem cell controversy and is a worthy read (Source Link 1)

Source:

http://www.nature.com/news/papers-on-stress-induced-stem-cells-are-retracted-1.15501

http://www.nature.com/nature/journal/v511/n7507/full/nature13598.html

http://www.nature.com/nature/journal/v511/n7507/full/nature13599.html

DNA integrity checkpoint is conserved from yeast to humans and is controlled by PKC

Our genetic material is very breakable and needs appropriate checkpoints to make certain the integrity of the same. Hence our cells have developed mechanisms to shield the integrity of DNA. Various cellular processes including cell integrity are mediated by protein kinase C superfamily. PKC (EC 2.7.11.13) has also some essential roles like differentiation, proliferation regulation etc. Eukaryotes carry a variety of different PKC isoforms which have been divided into conventional PKCs, Novel PKCs, atypical PKCs etc. However, Saccharomyces cerevisiae carries a single PKC, called as Pkc 1. The main function of Pkc 1 is the protection of cell wall integrity.

Protein Kinase C. Source http://upload.wikimedia.org/wikipedia/commons/thumb/a/a5/Protein_PRKCI_PDB_1vd2.png/250px-Protein_PRKCI_PDB_1vd2.png

Protein Kinase C.
Source: Wikipedia.

Authors in this manuscript have claimed that PKC is implicated in a control mechanism that is conserved from yeast to humans. Since there are many isoforms of PKC, only the delta PKC isoform is able to commence the checkpoint for DNA integrity.

Authors from the same paper had earlier established that there is a possible involvement of Pkc1 in DNA metabolism. Infact it was shown that the Pkc1 mutants have high recombination rate and hence defect in genome integrity. The authors emphasize that:

‘’we have established a clear direct connection of PKC with DNA metabolism: Pkc1 activity is required in yeast cells to activate the DNA integrity checkpoint. We have checked this effect in different independent backgrounds and, moreover, checkpoint activation was restored when a PKC1 gene was re-introduced into pkc1 mutant cells, demonstrating that the lack of checkpoint function is caused by Pkc1 inactivation.’’

Under genotoxic stress especially replicative and DSB stress, electrophoretic band shift was observed for Pkc1, suggestive of genotoxic stress regulating Pkc1 activity. But, at what cellular level is DNA integrity checkpoint controlled by Pkc1 had to be determined?

Some observations has led authors to complete the proposed model by adding that there possibly could be a feedback loop between Pkc1 and Tel1 and that PKC checkpoint control must be a general trait of eukaryotes. However establishing such fact needs more research and digging into the phenomenon. For complete study please refer to citation below:

Source: María Soriano-Carot, Inma Quilis, M. Carmen Bañó, and J. Carlos Igual
Protein kinase C controls activation of the DNA integrity checkpoint
Nucleic Acids Res. 2014 : gku373v1-gku373.

Image Source: http://upload.wikimedia.org/wikipedia/commons/thumb/a/a5/Protein_PRKCI_PDB_1vd2.png/250px-Protein_PRKCI_PDB_1vd2.png

INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites

The major health problems of developing countries constitute infectious diseases caused by intracellular parasites. Leishmania, Toxoplasma and Trypanosoma parasites are smart enough to reside inside the host cells.

Researchers working on the aspect of how such parasites proliferate intracellularly, have to do some laborious parasite counting, analyze drug [natural/synthetic] susceptibility or compare virulence properties of modified strains? High Content Screening (HCS) Technology has emerged recently and is a powerful tool for screening new compounds or for testing susceptibility of parasites to drugs. However the laborious procedure and cost of the equipments makes HCS poorly accessible for academics and research.

In this paper novel open-source, Java based software has been introduced that is quite helpful in automating the measurement of infection levels. This method is based on fluorescent DNA staining and has been named as INsPECT (Intracellular ParasitE CounTer). This software is believed to be versatile enough by the authors; to precisely detect intracellular parasites and to be the best one for parasite quantification.

The image based INsPECT is more fast, user friendly and open-source based software for quantification of parasites. The fluorescent images taken are used by the software to calculate the required data automatically by using image processing framework. The software can run based on DNA fluorescent images alone or in combination with phase contrast/Differential Interference Contrast. Such combination makes the intra- and extracellular parasites to be discriminated without any use of fluorescent membrane marker.

The out-put of the software provides one with annotated images, a report table, total cell count, total infected cell count, parasite index etc. The processing pipeline used by INsPECT software can be seen in the image below:

journal.pntd.0002850.g001journal.pntd.0002850.g002

Citation:

INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites. Ehsan Yazdanparast, Antonio Dos Anjos, Deborah Garcia, Corinne Loeuillet,  Hamid Reza Shahbazkia mail, Baptiste Vergnes mail. Published: May 15, 2014 DOI: 10.1371/journal.pntd.0002850