quarta-feira, 15 de abril de 2015

Thioredoxin Superfamily

Hey you !

Are you ready to learn a bit more about a gene superfamily?

Today we will talk about Thioredoxins (TRX), that is class of small proteins characterized for a short sequence of four amino acids (Cys–Xxx–Xxx–Cys)* at their active site. TRX were identified in 1964 in bacterium Escherichia coli, and ever since it has been widely studied. 

Thioredoxins are important molecules involved mainly in the control of cellular reduction/oxidation (redox) balance. Therefore, they are related to oxidative stress and different types of cancer cases, usually showing an elevated expression. The two cysteines (Cys) residues presents in the conserved active site sequence work as an intracellular reductase by a dithiol/disulfide exchange and are responsible for their redox activity. TRX also is associated with others biological processes as gene expression and signal transduction in all organisms.

In our analysis Pterygoplichthys transcriptome, we found 3 differents types of TXN (TMX, TXN and TXNDC) but they have practically the same functions. The basic difference is the local where are expressed and found in cell. The most abundant of those types in the transcriptome of Pterygoplichthys was TXNDC, with 13 distinct transcripts.

Got interested in Thioredoxins? Learn more with this nice review: Multiple catalytically active thioredoxin folds: a winning strategy for many functions .

*Cys - amino acid cysteine

Xxx - some amino acid

quarta-feira, 8 de abril de 2015

A little about Loricariidae...


Hey guys,

I'm here to write about the Loricariidae family, that is part of the Siluriformes order. In Brazil, fish of this family are known as Cascudo and are characterised by having bony plates covering their bodies, a ventral mouth with papillae on the lips and body characteristically depressed.


Photo from a great paper from our colleague Nathan Lujan.
http://www.biomedcentral.com/1471-2148/12/124


This fish family has wide distribution in the neotropics and a great ecological importance, as they have a crucial role in nutrient cycling in neotropical aquatic ecosystems. In addition, they also have great socioeconomic importance. They are important food source for Amazonian communities and, with its exotic beauty, internationally used for ornamental purposes, as aquarium fish. However, this often represent a threat to these species due to overexploitation and international fish traffic of endangered species. In fact, the international trade of those and other Brazilian fish species are regulated by IBAMA, our environmental agency.

Now you all know a little more about those cute fish which we work. In others posts in this blog, we'll talk about some species that are part of our project. I hope you like this publication, till next post!

quarta-feira, 1 de abril de 2015

Nuclear receptors

Today we will talk about one of the gene superfamilies that we are working; the Nuclear Receptors (NRs).

The NRs are transcription factors, which act as a translator of environmental stimuli to the molecular language of the cells. They function as receptors of hormones and others endogenous and exogenous molecules like vitamin D and several pollutants. By interacting with regulatory regions of target genes to perform its various functions in homeostasis, playing important roles in the regulation of cell growth, development, basic metabolism of metazoans. The NRs have the ability to directly bind to DNA and regulate gene expression of other genes.

All nuclear receptors share a similar organisation classified in six homolog regions, but just two of these regions are very conserved: the DNA-binding domain (DBD) (region C) and the ligand-binding domain (LBD) (region E). The most conserved is the region C that contain two zinc fingers that binds to specific sequences of DNA. These zinc fingers are used to find the P-box that characterises this region and confers specificity to a target region of  the DNA.
Region E is also conserved, but has more aminoacids changes than region C, therefore is classified as a moderately conserved sequence.


http://en.wikipedia.org/wiki/Nuclear_receptor

In our first analyzed transcriptome, the Pterygoplichthys anisitsi, we found 13 different transcripts (mRNA) of NRs and we had succeed in the identification of the domains C and E in all these mRNA. Sure things; we accomplished this after reading tons of papers to learn about the Nuclear Receptors. So, we will continue working hard for more posts like this.









I hope you enjoyed. See you soon!

domingo, 29 de março de 2015

2015 PEER Latin America & Caribbean Meeting

A wonderful meeting was held in Lima from March 22 to 26. Sponsored by USAID, the principal investigators of PEER projects in Latin America and Caribbean region presented their achievements, had a great chance to start new collaborations, and learned more about other funding opportunities and about communicate science, technology & innovation to the general public.

It is hard to highlight specific moments, because most of them was really instructive. The flash talks, however, were particularly challenging for most of us. The mission was to communicate the progress and impacts of your work in just three minutes, using the model of FameLab. The training for the flash talks took three of the four days of our gathering. Six PIs were voted to present their talks at the US embassy in Lima. These final talks were recorded and I hope to share the link for the videos and pics soon.

Note: more about this meeting in the text from one of the panelists, Juan Casasbuenas from SciDev.com. Picture was bored from Juan's post and is a courtesy from USAID.

quinta-feira, 29 de janeiro de 2015

Next Generation Sequencing vs Sanger Sequencing

Hi all,

Long time, no see.

We are very sorry for the long time to update the blog. We have been working like crazy since we had a big methodological change on our aproch to reach our final goal. 

Recapitulating, our main aim was to sequence the CYP1 and AHR genes of 100 Siluriformes species and this would be done by the Sanger method DNA sequencing. However we had some issues, with the primer, for instance. Basically, we were able to amplify cyp1a in just six of the 30 species we had sampled. For practical purposes, this was not good. However, it suggested that the molecular diversity of cyp1a in loricariidae fish is much greater than what we expected, which in turn is an excellent news. Our sequencing method was modified from the traditional Sanger method to one of the Next Generation Sequencing (NGS) methods.

Now we are sequencing the liver transcriptome of 40 individuals from 37 different species using the Illumina Technology Hiseq2500 (Next Generation Sequencing) at the Brazilian National Cancer Institute (INCA).

A major advantage of this new approach is that it is not based on specific primers. Now we will obtain the molecular data without the bias caused by primers. Besides, it will generate much more raw data to analyse than our previous method. Consequently, we will get the sequence of not only the two desired genes, CYP1A and AHR, but from all genes that were expressed at the time of the sampled fish liver. In fact, this method is generating more raw data than we will be able to analyse during this peer grant. During this grant period, we are focusing our attention in a particular set of genes involved in the responses of the organisms to chemical compounds. Other genes will be analysed later. Another advantage is the price per base pair which is cheaper than Sanger, as we can see in the table below.


Method
Sanger
NGS
Read Length
Up to 1.100 bases
2x 100pb
Read \Run
96
2 billions
Time\Run
1 hour
<1-6days
Capacity
~1Mb
50-1000Gb
Price\Run
~$480
~$9.600

So, I will talk about the processes before and during the preparation of these libraries. First of all, we had to talk with the responsible for the Illumina Hiseq 2500 in INCA to know if it would be possible to use this sequencer, when it would happen and if the technologist, Carolina Furtado, could teach us to prepare our samples. After it was solved, we could start to work hard.

Well, this is a summary of what has being going on!

We wish you enjoy it and hope to write more often.



See you soon!

Preparing the libraries for the Next Generation Sequencing

Today let´s talk a little about Next Generation Sequencing.

After we decided for this sequencing method, we had to prepare all samples to be sequenced; which includes RNA extraction, verification of RNA quality using a method more reliable than the one we used before, construct the cDNA libraries and evaluate its quality and quantity.

Based on the nanodrop quantification, we performed another kind of analysis using the Bioanalyzer, which is a more precise equipment to assess the quality of the material that has been extracted. Using this method, a RNA Integrity Number (RIN) is generated, this is a number assigned by the software that considers also the presence of degradation products. Although Illumina recommend a RIN higher than eight for transcriptome sequencing, we sete our threshold in six due to sample particularities. By doing this, we assume the risk of getting transcriptomes biased for the 3' end of the transcripts. However, most of our samples had RIN between 7.5 - 8.0  and our first results indicates high coverture of 5' end.

Initially, we select the mRNA with special beads that contains oligo dT, this way the material will be purified and only the mRNA will stay in the well plate. After this, we fragment the RNA in a delicate step of 3 minutes at 94 ° C in the thermocycler.The longer this step takes, the more fragmented the RNA gets. This time give us fragments of about 300pb.

The next step is to synthesise the cDNA in two different reactions. First strand first and then, in another reaction, the second strand. This way we end up with double strand cDNA, and not with the hybrid cDNA used for regular molecular biology applications. When the cDNA is ready, we have to  repair their Ends to ensure that each cDNA molecule has a blunt end and contains a 5 'phosphate and an end 3'OH free.

The adapters binding step is crucial to sequencing as it is when we give an identity for each sample.  By doing this, we can sequence several different samples in the same lane. However, it is vital to take note about which adapters were used in each samples, so at the time of sequencing, we won't put samples with the same adapter in the same lane. Then, it is made a short (15 cycles) PCR enrichment of DNA fragments, where the molecules with adapters at both ends are selected and the DNA sample is amplified. In this PCR primers binds the adapters.

Finally, we need to run the libraries in bioanalyzer to check their sizes and perform qPCR for precise quantification. The size and quantity informations are important for the normalisation, when all samples are prepared to have the same number of molecules, so all the samples at the same lane will have equal chances to be sequenced. Then the samples are finally ready to be sequenced with illumina Hiseq 2500.