quinta-feira, 6 de agosto de 2015

Transcription machinery

Transcription is the initial step in gene expression. The number of genes or the genome size are not related to the complexity of an organism, but instead the ability to express a particular set of genes over a specific period of time and in different tissues allows organismal acclimatization to environmental changes. For this reason, it is extremely important the study of transcription machinery to better understand the evolutionary process.

We have sequenced several transcripts involved in the transcription process in all the 16 Loricariidae species investigated so far. The table below shows the sequenced transcripts according to their classification. Note the abundance of transcription factors. 



A fish? A shark? A cat? Or a dog? Just call me Squali


Knock, knock.

Who is there?







Squaliforma emarginata.

Squali who?

Squaliforma emarginata, a fish of the Loricariidae family. Yes, I have this shark-ish name, but I am indeed a fish, more precisely a catfish

OMG, Squalidae is a family of dogfish sharks! What are you?

I am a catfish that looks like a dogfish shark.

---------------------------------------------

There are around to 13 species of Squaliforma genus, all native from South America. This one was sampled in Manaus, Amazon state, Brazil for our work.

Among the fisherman in Manaus and in portuguese, this fish is known as "bodó de praia", which in a free translation would be "beach suckermouth armoured catfish". The popular name reflect the habit of this to live on sandy beaches along the banks of Solimões river.

The ''bodó de praia'' is trade as ornamental fish.

I hope you enjoyed! 
Bye

First of all, biosafety

Hello!

Biosafety is crucial!

Work in a laboratory frequently involve the use substances that are toxic, flammable, acidic and nasty stuffs. It is very important to be careful and prepared for possible issues. Let's recall some useful steps to avoid accidents in our work environment and always work with security.



Biosecurity is a set of actions to prevent, minimize or eliminate risks to the health of humans, animals and environment that are inherent to laboratory duties. Chief among those actions is the dressing code! Always wear your lab coat, gloves, long pants and closed shoes. The laboratory environment also asks for appropriate behaviour. Do not run, eat, drink or smoke. Always work with careful and attention. These steps will ensure your safety and also the quality of your results.

Be careful with the products, substances, samples, reagents. It is important to know how to handle each of these compounds and to utilize the appropriate safety equipments, like laminar flow cabinets (to volatiles and gases, for instance, since it promotes air recirculation and prevents the person inhale the toxic product). For example, we use TRIzol reagent, which is composed by Phenol and Chloroform, toxic chemicals used for RNA\DNA extraction. We only open Trizol inside laminar flow cabinets. Solvents, infectious substances, mutagenic or carcinogenic chemicals also deserve close attention.  All must be properly disposed of, according to its danger.

Others precautions as in handling fire, laboratory glassware (beaker, graduated cylinder, Erlenmeyer flask), liquid nitrogen and exposure to radiation should be thought. For instance, when we use the ultraviolet light to see the results of our PCRs, we must be careful with the direct exposure to it. U.V. light is a ionising radiation, and can damage the DNA and ultimately lead to cancer development.

To finalize, each laboratory has its particularity and different levels of biosafety are required. Watch out the biosafety level of your laboratory and stay on the safe side. 

Take care!



Don't trust manufacturer and pay attention

Hello!

Today we will give you a friendly advice.

Last month, Maithê and I (Paula) were making some reactions for real-time PCR. As usual, we planned the entire experiment with our advisor. We calculated all reagents that we would to use and we schematised the plate for the real-time PCR. Everything looked fine!

However, one of the necessary primers suddenly vanished when we were pipetting the plate!

This reagent was used just a few times, Maithê and I didn't know what was wrong. There should be plenty of primer left! We went to talk to our advisor and after some thought, the mystery was solved. 

Solution: We followed the manufacturer's instructions!

Yes, that is right, the problem was follow the manufacturer's instructions.

Making a long story short, they recommend to use 50 uL final volume, but use 25 uL to calculate the number of reactions the kit can perform. Easy to compute, if you use the recommended volume, you will be able to do only half of the reactions they advertise. 

At least, they recognised their mistake and are sending a new primer for replacement.

Lesson for the undergrad students: we can prevent these situations and alike by paying attention to the used reagents. We could (and should) have realised this primers was ending just by looking to the remaining volume. This would avoid bigger problems, such as the delay we are experiencing and waste of reagents.

The piece of advice? Do not trust manufacturer and pay close attention to the volume of all reagents you need.

See you.

quarta-feira, 10 de junho de 2015

Sulfotransferases

Hi guys,
Today I'm gonna write about sulfotransferases family.
As their name suggest, Sulfotransferases are transferase enzymes that catalyses the transfer reaction of a sulfate group from a donor molecule to an acceptor alcohol or amine. These enzymes add sulfate groups in proteins after translation.
In one of the transcriptomes, from the Pterygoplichthys anisitsi (see our previous post), we found the above members of Sulfotransferases:

We can see that two groups are more abundant: group 1, the carbohydrate sulfotransferases, that are responsible for transfer sulfates to carbohydrate groups in glycoproteins and glycolipids; and the group 2, SULT, that refers to sulfotransferases, are responsible to catalyse the conjugation reaction between sulfate and hormones, neurotransmitters, drugs, and xenobiotics. Its distribution in tissues is different, just as the substrate specificity. Always good to remember, those transcripts were all sequenced just in the liver tissue. It is possible (and most probable) that this fish possess even more sulfotransferases that were not expressed in the liver.

That is all for now, till next time!

The famous Pterygoplichthys anisitsi

Hi all,

We have been talking quite a bit about the first transcriptome sequenced during our project, the one of Pterygoplichthys anisitsi. But, who is Pterygoplichthys anisitsi (Figure 1)?

Figure 1: Pterygoplichthys anisitsi

Ptery, as we friendly call it, is a specie of Loricariid fish distributed over the basins of the rivers Paraná, Paraguay and Uruguay, all of those in the centro-south part of South America (Figure 2). It is characterized by their large dorsal fins with 9 or more fin rays, which distinguish the genera Pterygoplichthys from the genera Hypostomus, that have only 7-8 dorsal fin rays. Ptery also have the body covered with flexible bony plates, the abdomen is almost completely covered in small plates covered with large  spots irregularly and a ventral sucker mouth.


Figure 2: Area of distribution of Pterygoplichthys anisitsi. Figure from http://goo.gl/GY25Ov

Here in Brazil, Pterygoplichthys and other sucker mouth catfishes are known as ''limpa-vidros'' because small of these catfish are used for ornamental purposes, since it feed of the sludge that builds up on the walls of the aquarium.

The exemplar of P. anisitsi used in this work was kindly provided by Prof. Eduardo Almeida, from the São Paulo State University (UNESP) at the city of São José do Rio Preto. Actually, this fish was used in another work that investigate biochemical responses of Ptery to biodiesel, and that was recently published in the journal Ecotoxicology and Environmental Safety. You can see this work here.

That is all for now. Bye!

sábado, 30 de maio de 2015

RAIC - FIOCRUZ

Hey guys,

This week we (me and Paula) are participating of the RAIC (Annual Meeting of Undergraduate Students) at FIOCRUZ. It is the first time we take part on a meeting like that, in which undergrad students introduce their project to a committee that evaluate the 10 minutes student talk.
Last Friday (May 22), we attended the opening section. It was an interesting lecture about the differences of applied and basic research, and how importante it is to connect both.
We presented our work in the morning of May 28. I spoke about our sub-project with the endangered species Hypancistrus zebra (learn more about this species in our previous post), and Paula spoke about our work with the transcription machinery of Pterygoplichthys anisitsi (you will learn more about this in a near future post). Nonetheless, as in our section there was other three undergrads presenting their work, it was fantastic to hear about their projects, to know a little more about others areas, and what people think over our research.
We realised that not many researchers in our Institution are aware of the NGS potentialities. It is indeed an expensive method that demand a knowledge of bioinformatic that many people do not have (and, by the way, that we are learning). Two professors at our evaluation committee got interesting in this method and asked for our advisor email to exchange ideas. It was a nice and enriching experience.


Do you know what are Catalases?

Hi there,

Do you know what are Catalases? Where are they found? What does they do?

I will talk a little bit about them.

When we sequenced the Pterygoplichthys transcriptome, we found a lot of genes which were classified into many different families. After a primary separation, we obtained a big group of defensome genes, and among these, we have found one coding for a catalase enzyme.

Ok, but what is the importance them?

Catalase is an enzyme found in most aerobic organisms. In eukaryotic cells, it is located in the peroxisomes and it has the role of protecting the cell against the toxic effects of hydrogen peroxide (H2O2), a product from cellular metabolism. Catalase is an important enzyme for the prevention of oxidative damage, which is related to several diseases and ageing.

As any enzyme, catalase accelerate a chemical reaction; in this case, the conversion of a molecule of hydrogen peroxide to water and oxygen. It is important because converts the reactive oxygen species of H2O2 that causes oxidative stress in cells, compromising the health of the cell. So, it must be done quickly.

Simple reaction





Its structure is well know. Its active form is being compound for four polypeptide chains in a quaternary structure, so it's a tetrameric enzyme, with four heme groups. Interestingly, we found just a single transcript coding for catalase, specially because the active form of the enzyme is a tetramer. This is probably due to the very essencial and conserved function of this enzyme in aerobic organisms. You can see a diversity of crystallographic structures that are known in Protein Data Bank.

I hope you enjoyed.
See you later.

quarta-feira, 13 de maio de 2015

Hypancistrus zebra, the Zebra pleco

Hypancistrus zebra, or Zebra pleco, is just one of the 34 species of which we sequenced the liver transcriptome, but a good example of all the current threatens against Loricariidae fish. 

The Zebra pleco is endemic to a stretch of Xingu River of only 100km. This region, known as the “big bend”, is impacted by the construction of the Belo Monte dam, the world's third-largest hydroelectric dam. Fish ecologists say that the habitat changes caused by the dam construction puts this species at risk of extinction. This is among the main reasons why the Zebra pleco is at the list of endangered species from the Brazilian Ministry of the Environment and its capture is forbidden. Another major cause of concern is the Zebra pleco being extremely valuable in the international aquarium trade and, therefore, the traffic of this species is a regular practice that has challenged the Brazilian Environmental Authorities and the Federal Police. Sequencing and annotating the transcriptome of H. zebra and other endemic loricariids are effective ways to catalog and preserve the genetic biodiversity of these species. This genetic information provide environmental police-makers and the Federal Police unique ways to prevent and combat the traffic of the Zebra pleco and several other Loricariidae fishes popular in in the aquarium trade. Moreover, the genetic information that will be produced can also be useful to subsidize strategies for the conservation of these species, including their reproduction in captivity. 
By the way, the Zebra pleco we used were a kind donation from Dr. Jansen Zuanon, a fish taxonomy and ecology at the Brazilian National Institute for Amazonian Research who serve as the trustee for all H. zebra apprehended by Brazilian authorities.


quinta-feira, 7 de maio de 2015

ATP-binding cassette (ABC) family

Hello!

It is time to learn about one more gene superfamily.

Today, we will briefly introduce ATP-binding cassette (ABC) family, a very important group of proteins in our metabolism.

ATP-binding cassette proteins, or just ABCs, are present in cells from prokaryotes (bacterias) to eukaryotes (including mammals). Most ABCs are transmembrane proteins responsible for the transport of many substrates across cellular membrane, putting chemical compounds in and out the cell. Examples of ABCs substrates vary at length, but include amino acids, peptides, ions and others molecules that are usually hydrophilic (polar).

The transport of substrates carried out by ABCs occurs at an energy cost. ATP molecules must be   hydrolysed, realising the energy used to bind and move the substrate across the membrane. In other words, ABC proteins perform active transport (expend energy). The ABC proteins has an  hydrophilic portion in its structure and also an lipophilic portion, which is embedded inside the cellular membrane.

ABC proteins consist principally in two distinct domains, the transmembrane domain (TMD) and the nucleotide-binding domain (NBD). The first is compound for alpha helix in membrane bilayer. When transport a substrate, the TMD change its conformation and adapts to it. And the NBD is responsible for interact with ATP molecule and make the hydrolysis to produce energy for the protein action.

There are many differents types of ATP-binding cassette, its diversity is big. In the transcriptome we have analysed so far, we found 24 differentes subfamilies of ABC!

More exciting news are about to come.

See you soon!

quarta-feira, 29 de abril de 2015

Hypostomus affinis


Hey, guys!

I'm here to introduce one of the catfishes which is part of our project, the Hypostomus affinis. In Brazil, their popular name can be Cascudo, Acari or Boi-de-Guará. It is a benthic fish and it feeds organic materials, participating in the pre-stage mineralization of organic material in the substrate.

The reproduction of these fish occurs between November and February. This species has a low fertility rate, but strong parental care with its offsprings. The ones we found here in Rio de Janeiro, at the Guandu river, reaches 30 cm in adulthood. The genus Hypostomus is widely distributed in South America. Occurrence of H. affinis are described in several regions of Brazil, but it is probably another case of cryptic species. Species of this genus are an important food source in the Amazon and Pantanal wetlands, besides being economically important in the ornamental aquarium trade.

The video below contains disturbing images, if you are a sensitive person, better not watch it. It describes (in Portuguese) how to clean this fish to be eaten.



That is all for now! Till next time! By the way, none of us have tried this fish (at least yet).

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.


segunda-feira, 31 de março de 2014

Working hard on cDNA synthesis

Hello,
Today, we, Maithê and Paula, will tell you a little about what we do with the collected samples of Loricariidae when they arrive in the lab. As previously written, after fishes has been collected and livers extracted, it's necessary to extract RNA and DNA. What do we do next with this genetic material?

Well, while the DNA is sent to the Paulo Buckup's team, our collaborators at the National Museum of Natural History, the RNA is used for synthesis of complementary DNA (cDNA). But what is cDNA, how is it done and serve for??
The cDNA is a strand of DNA complementary to a RNA molecule and made by the aid of an enzyme called reverse transcriptase. To make this happen, we need: 1 - buffer solution, to maintain the ideal pH; 2 - reverse transcriptase, which is the enzyme that will do the reverse transcription of RNA to cDNA ; 3 - dNTPs, the nucleotides used as substrate by the reverse transcriptase to synthesize cDNA; 4 - the RNA sample, that serves as a template for the reverse transcriptate; 5 - and primer to help the reaction start. We used anchored oligo dT primers.Those are a sequence of 18 to 25 Ts that bind to the poly A tail of messenger RNA (mRNA).
These reagents are present in the AppliedBiosystems' kit ''High Capacity cDNA Reverse Transcription Kit" and they are put together in ideal proportion, according to the manufacturer's protocol for cDNA synthesis, that occurs at 37 °C during 120 minutes.

So this is another step needed to achieve our goal. We hope you have enjoyed. Keep up with our blog !! Soon, we are going to write more about our adventures in the Lab.