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

Elucidation of localization of long non coding RNA in different subcellular compartments

Research has proven the role of noncoding RNA transcripts in various cellular processes which includes telomeric maintenance and chromosome silencing. Although in past decade researchers have identified many long non protein coding RNA transcripts, but the functional role of most of them is still in its infancy.

The sub-cellular localization of lncRNAs is of utmost importance in order to elucidate or account for their functional utilities. Researchers have tried to explore lncRNAs abundance in various compartments within the cellular systems. About 17 percent of lncRNAs are known to occur in nuclear compartment, were as only 4 percent occur in cytosol. This observation is for some of the RNAs that are thought to be involved in nuclear structural organization and gene-expression regulation, e.g., MALAT1 and NEAT1. Moreover ribosomal association of cytosolic lncRNAs is also known. Despite having all this knowledge about lncRNAs, there is a lack of data that could really support this relative lncRNA species abundance in compartments like nucleus, cytosol and ribosomes.

A team of researches at the Netherlands has used sub-cellular RNA-seq and ribosomal fractionation methods in combination with microarray technique to elucidate the exact compartmental locations of the lncRNAs.

From the different sub-cellular sample fractions the authors have considered three transcript types only: sncRNAs, lncRNAs and protein-coding transcripts and excluded miRNAs. In overall the expressed transcript set contained 7734 number of genes, out of which 7206 genes were protein-coding, 152 genes were lncRNAs and 376 genes were sncRNAs. However despite having different compositions of sub-cellular samples, the authors conclude that lncRNAs are existent in each of them. Despite these findings some questions raised, that how the individual transcripts distribute among different sub-cellular sample fractions? And how these lncRNA species behave in a different way as judged against normal coding transcripts? In order to find answers to these queries an investigation was done to find the correlation of the distribution of every lncRNA across the sub-cellular fractions and every coding transcript. Experimentation revealed that the association is complex. However authors have tried to simplify it by using a clustering model wherein a total of eleven individual clusters were made. The first ten clusters (I-X) contained genes showing specific sub-cellular localization, but cluster XI did not demonstrate enrichment anywhere in any of the samples. Nuclear enrichment was found for clusters I, II and III; ribosome-free cytosol compartmentalization for clusters IV and V; and ribosome enrichment for clusters VI to X. Cluster III contained majority of sncRNAs that suggested shuttling of these RNAs between cytosol and nucleus. In terms of percentage about 30% of the lncRNAs were found in ribosome-free cytosol and 38% in ribosome-enriched clusters.

Apart from this some known lncRNAs like MALAT1, NEAT1 and TUG1 were found localized in nuclear fraction; RPPH1, RN7SL1 and DANCR were found in cytosolic compartments and H19 and TUG1 in ribosomal fractions. Although high levels of TUG1 occur in nucleus, but authors have found appreciable levels in ribosomal fractions also. This diverse localization of various lncRNAs in different sub-cellular compartments implies that an extensive array of functions is associated with lncRNAs than is known so far. So in overall for the functional characterization of these individual lncRNAs the data presented in this very study can be of priceless use.

Reference: Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes. Sebastiaan van Heesch, Maarten van Iterson, Jetse Jacobi, Sander Boymans, Paul B Essers, Ewart de Bruijn, Wensi Hao, Alyson W MacInnes, Edwin Cuppen and Marieke Simonis. Genome Biology 2014, 15:R6  doi:10.1186/gb-2014-15-1-r6

Role of WASP and N-WASP in B Cell Receptor Signaling.

After the elimination of infection, termination of immune response is necessary. This process can occur only when B-cell activation is shut down otherwise autoimmunity can occur. Recently researchers from University of Maryland and Harvard Medical School USA tried to analyze the process involved in inactivation of B-cells after infection has been cleared from the body. The main focus was on Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP). Wiskott-Aldrich syndrome is X-linked disorder and results in immune dysregulation. WASP is entirely expressed in hematopoietic cells and N-WASP in neuronal cells.

Which molecules are involved in activation or inhibition of N-WSAP and how the latter affects B-cell activation were some key areas of enquiry and research in this study. The role of N-WASP in BCR (B-cell receptor) activation was analyzed by devising an experiment that activates human B cells and mouse BCR in a similar way. Results of this experiment revealed that at places where BCRs interact with antigen similar to WASP, transient activation of N-WASP occurs. In other words BCR stimulation causes N-WASP activation following WASP activation. Earlier studies lead this team to hypothesize that N-WASP has a compensatory role in WASP KO B-cells. For investigating this hypothesis KO mice were used. Results revealed that for antigen-induced BCR clustering both N-WASP and WASP are critical. Also B-cell morphology and B-cell spreading are affected.  However N-WASP in the absence of WASP, supports B-cell spreading and in presence of WASP, B-cell contraction.

The effects of N-WASP and WASP KO on B-cell morphology led the team of researchers to dig into the BCR signaling.  Experiments revealed that attenuation as well as stimulation of BCR signaling involves N-WASP. Earlier study from the same authors had shown that BCR signaling and clustering are in a two-phase relationship. Since cNKO had effects on both B-cell contraction and clustering, the researchers thought of N-WASP regulating signaling via cluster modulation of surface BCRs. Data from experiments showed role of N-WASP in promoting growth of BCR micro-clusters into the central cluster by down-regulating the BCR signaling system. Infact authors of this paper have clearly demonstrated that N-WASP has role in both positive and negative regulation of BCR signaling. However negative regulation suggested that B-cell self tolerance could also be affected by N-WASP. For clarification serum levels of anti- dsDNA and anti-nuclear DNA antibody were measured in cKNO (N-WASP deleted) mice and were found to be elevated, suggesting a clear role in self-tolerance. Moreover activation of BCR induces receptor internalization which in turn involves reorganization of actin. To elaborate the role of N-WASP and WASP in internalization and co-localization and immmunoflorescence studies were done with a marker named as LAMP-1 and surface-labeled BCRs respectively.

WASP and N-WASP involvement in activation of BCR led to understanding of their relationship. The experimental data suggests that both of them regulate each other negatively during activation of B-cells. However, BCR signaling inversely regulates both WASP and N-WASP activation.

On the whole the results have shown that B cells lacking N-WASP protein are activated for extended periods of time than the normal B-cells. Also mice with B-cells deficient in making N-WASP show increase in number of self-reactive B cells.

Open Access Article Under Creative Commons Attribution License.

References:

Chaohong Liu, Xiaoming Bai, Junfeng Wu, Shruti Sharma, Arpita Upadhyaya, Carin I. M. Dahlberg, Lisa S. Westerberg, Scott B. Snapper, Xiaodong Zhao, and Wenxia Song. N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling. PLoS Biol. doi:  10.1371/journal.pbio.1001704.