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