Monthly Archive for September, 2010

Take home message of the 2010 Amebiasis Montreal Meeting: beware of who you kiss…

The Entamoeba community is a small and collegial one.  Everyone knows everyone and everyone else wants to collaborate, and learn and do more to tackle down this neglected among neglected diseases.  For many, the thought of an amoeba brings to memory Garry Larson’s The Far Side amorphous characters watching TV and dealing with domestic issues…but what a few know is that the WHO considers amebiasis one of the major health problems in developing countries surpassed  only by malaria and schistosomiasis for death caused by a parasitic infection.

Amoeba Real Life...

TIGR/JCVI has had a long-standing relationship with Entamoeba histolytica and other related species. Started by Brendan Loftus back in the day, followed by Neil Hall and continuing by yours truly, with the Entamoeba GSCID project, we have provided this eager community with what they are in great need of: genome sequences. And they are appreciative of that, for sure.

The meeting was small and intense, and started with a one day workshop on clinical aspects of the disease, not only the enteric disease, but the oral disease. It was good to mingle with dentists and see their point of view and the devastating reality of clinical cases of periodontal disease…and periodontal disease affects, at one point of another, the majority of the adults. And Entamoeba gingivalis is always there…but not always diagnosed.  Why? We assumed bacteria, bacteria, and bacteria. Treat the bacteria with antibiotics…but ignore the amoebas. Patients do everything right when it comes to oral hygiene, but the disease persists, the bone continues to be destroyed, and teeth are lost…secondary consequences such as fatigue, diabetes, heart disease, renal dysfunction, low birth weight, and a myriad of other diagnoses are known to be related to oral health and periodontal disease, but rarely in the context of this amebiasis infections. Interesting cases of entire families being affected by this parasite were presented (it is highly contagious). Dr. Bonner, the organizer of the conference is a big advocate of microscopes in the dentist practices as the primary diagnostic method for periodontal disease and identification of amebiasis, and he is surely determined to speak to the world about that. And he also wants the genome done.

Entamoeba community, Montreal 2010

During the core of the Meeting, Neil Hall and myself were the only ones on the genomics side of things: we presented on SNP analysis done so far on the strains we have sequenced, in their case using SOLiD, on our case just 454 (for now). Both talks were very well received and the community is eager to see more. Both pieces of work will be used in a global SNP analysis, particularly focusing on a family of proteins known to be involved in virulence, the Gal/GalNac lectins. These proteins are one of the main targets for vaccine development, led by Dr. Bill Petri, who is in the process to start full speed with that endeavor. Also on this topic, Dr. Jonathan Ravdin (Dean and Executive Vice President of the Medical College of Wisconsin) presented the results on an intranasal Gal-lectin subunit vaccine on experimental Entamoeba histolytica in baboons, showing its protective effect against enteric colitis, and the promising future of this vaccine target for humans.

For amebiasis “a la mode”, the usual suspect topics of the conference involved Entamoeba histolyica signaling pathways, classic protein characterization of large families, proteases and invasion, and large genotyping studies of outbreaks, as well as mechanism of pathogenesis.  One interesting talk was on the generation of cyst like forms in vitro for Entamoeba histolytica, a true breakthrough, since there is no model or encystations so far, and the possibility to obtain this structures in vitro will certainly open up a whole new world of studies that before were confined to Entamoeba invadens, a very distant parasite of lizards that does encyst in vitro.

A modest interest into drug discovery was present. There are so far two kind of drug therapies for amebiasis, luminal amoebicides (paromomycin, diloxanide furoate and iodoquinol) for intestinal disease, but ineffective against organisms in tissue and metronidazole and other derivatives,  for invasive disease. However, resistance to these drugs is easily achievable at clinically acceptable drug levels. James McKerrow (UCSF) group presented a high throughput screening of small molecules using an FDA-approved library of drugs and known bioactive compounds, and they identified six compounds with similar or better activity than metronidazole, in vitro. Other group from Mexico is focusing on probiotics and natural compounds such as Astrophitus capricorne (cactus), Jatrhropa dioica and Eucalyptus camaldulensis.

Montreal View from Le Crystal Hotel

To finalize, because the list of interesting things can be just too much, a wonderful piece of work that will hopefully provide an extensive framework for further studies of host susceptibility to E. histolytica comes from Bill Petri’s lab. They have performed a small hairpin RNA (shRNA) screen to identify human factors crucial for E. histolytica cytotoxicity. Using a mammalian shRNA knockdown library they did nine rounds of selection using 1:5 parasite:host and 1:50 parasite:host ratios and resistant clones after 6 rounds of selection, were sequenced using Solexa. This way, they identified a number of host gene families including kinases, surface receptors and ion channels that may be important for susceptibility to the parasite, and of course, they are working on that…

But the take home message that I have imprinted in my brain is…do not kiss your dog.  After three years of age, ALL DOGS have periodontal disease. And inevitably they have Entamoeba gingivalis and possibly other species as well…and they are one of the sources of infection to humans. Dog kisses owner, owner kisses lover, wife, husband, and kids…and the amoeba conquest of the world continues…

For your delight, two movies on the topic: Mark Bonner, the meeting organizer (in French) and a film of a patient with periodontal disease biofilm. Enjoy!

Entamoeba histolytica research presented at the Molecular Parasitology Meeting

Entamoeba histolytica causes invasive intestinal and extraintestinal infections, known as amoebiasis, in about 50 million people and still remains a significant cause of human death in developing countries. However, for unknown reasons, fewer than 10% of E. histolytica infections are symptomatic (causing symptoms such as diarrhea, dysentery or liver abscess). The J. Craig Venter Institute is among the institutions awarded the NIAID Genome Sequencing Centers for Infectious Diseases (GSCID) contracts to provide high-quality genome sequencing and high-throughput genotyping of NIAID Category A-C priority pathogens.

Photo of Entamoeba histolytica

Entamoeba histolytica in the trophozoite stage.

A GSCID project led at JCVI by Dr. Elisabet Caler includes performing whole-genome sequencing of Entamoeba phenotypic variants from symptomatic, asymptomatic and liver abscess-causing strains chosen to include a range of clinical manifestations and taken from human cases, as well as strains grown under different conditions. Our objective is to develop a genome-wide landscape of Entamoeba diversity to understand how sequence variations in the parasite relate to pathogenicity (ability to cause disease) and clinical outcome.

The Molecular Parasitology Meeting held at the Woods Hole Oceanographic Institution, Woods Hole, MA last week provided a window into the exciting science of Parasitology.  The keynote speaker, Fotis Kafatos, spoke on “Major Challenges to Global Health in the Tropics and Beyond–Insect Vectors of Malaria and Other Parasitic or Viral Diseases.”  Dr. Kafatos stressed that a multi-pronged approach to the control of malaria is necessary to prevent the devastating loss of life that malaria causes.

Woods Hole Oceanographic Institution

A view of Woods Hole Oceanographic Institution.

The many excellent papers and posters provided an overview of the field, including   Plasmodium falciparum, Toxoplasma gondii, the trypanosomes, Giardia lamblia, Trichomonas vaginalis, Entamoeba histolytica, Schistosoma species, Babesia bovis, and associated vectors.  Topics spanned basic biology, drug design, sequencing and host-pathogen interactions.

I presented an overview of the Entamoeba sequencing project at the meeting.   Discussions as a result of the presentation included questions about the details of sequencing and handling the next-generation sequencing data.   We had animated discussions about methods for assembly of the DNA sequences, including reference-guided vs de novo assembly.   Many attendees were impressed with JCVI’s open-source METAREP metagenomic tool (J. Goll, et al., Bioinformatics 2010).  Determination of the best methods for the analysis of differences in the clinical isolates generated much discussion.  Entamoeba researchers see the sequences as a great resource and are looking forward to being able to mine the data.  One, from India, was very excited that he was going to have about 15 times the resources he has had in the past, since he has had only had one genome to mine up until now.

The Molecular Parasitology Meeting was an excellent venue for scientific exchange.  The Entamoeba histolytica GSCID project will help us understand the pathogenicity of Entamoeba histolytica, and has the potential to save lives in developing countries.

Virtual Comparative Metagenomics

We have created an open virtualization format (OVF)  package of JCVI’s Metagenomics Reports (METAREP)- a high performance comparative metagenomics analysis tool. The software runs on a web server, retrieves data from two different database systems and uses R for statistical analysis. The new OVF package bundles all these 3rd party tools and is configured to run out of the box in a virtual machine.

Screenshot of the virtual box appliance import wizard. The wizard allows you to specify the CPU and memory usage of the virtual machine on which METAREP will run on.

To run a virtual version of METAREP on your machine, follow these steps

  1. download the METAREP OVF package from our ftp site [download] .
  2. unzipp the OVF package
  3. download and install Oracle’s Virtual Box, a OVF compatible virtualization software [download]
  4. Start Virtual Box
  5. Click File/Import Appliance and select the OVF file.
  6. Adjust RAM/CPU usage using the Appliance Import Wizard (see image)
  7. Start VM
  8. Double-Click on the METAREP firefox link on the VM desktop
  9. Log into METAREP with username=admin and password=admin

This virtual machine appliance is the first step in developing a fully cloud-enabled analysis platform where users can easily launch the application wherever is most convenient: on their personal desktop or in the cloud where they can scale-out the appliance to suite their needs.

Future virtual machine images will be certified to run on other virtualization software platforms. Stay tuned.

If you like to learn more about METAREP and talk to the developers,  join us  at  Lucene Revolution Conference in Boston (October 7-8  2010). We will present a lightning talk about METAREP  the first day of the conference 5pm  (see agenda).

Links:

JCVI’s METAREP Instance

METAREP Flyer

METAREP Manual

METAREP Source Code

Italy- Sites and Sailing

Saturday July 31st

When I last wrote we had finished our 10 day  sampling window in Italian waters. On Wednesday July 21st we arrived in Rome the same day Dr. Venter, Heather Kowalski, and Darwin the super boat dog had flown in from the states.  We spent 3 days in Rome, most of the time was spent doing media events, restocking the boat with supplies and trying to see as much as Rome as possible in our spare time.  On the night of July 23rd we set sail to make our way down the coast of Italy, tonight we will do the overnight sail to Greece.  I wanted to post some pictures of some of the beautiful places we have seen as we sailed in Italy.

Darwin In His Favorite Resting Position

Saint Peter's Basilica, Vatican City

Laocoonte, Vatican City

Ponza Island

Coliseum, Rome

Water Spout

Vulcano Island

View Of Vulcano Island

Porto Santo Stefano At Night

Crater Of The Volcano in Vulcano Island

Capri Island

Vatican City, Rome

10 Days of Italian Sampling Coming to a Close

Tuesday July 20th

On July 16th we finished our Straits of Messina sampling and headed into the Ionian and Adriatic Seas.  We sailed overnight and collected our Ionian Sea sample,  we continued  northeast and  on July 18th we collected our Adriatic Sea sample.  After we collected the Adriatic Sea sample we turned around and headed back to the Straits of Messina to resample the south entrance on July 19th.  We proceeded through the straits directly to Ponza Island where we anchored tonight July 20th.

CTD Profile Of South Side Of Messina Straits Taken on July 16th

CTD Profile Of South Side Of Messina Straits Taken on July 16th

CTD Profile Of South Side Of Messina Straits Taken on July 19th

We have never really resampled a site in the same exact location a few days later, the profiles above are from the same GPS coordinates and the same time of day 3 days apart.  The fluorescence, Oxygen, and salinity look very similar but there are some slight differences in temperature down the water column.  These two samples could be very interesting to compare once they have been sequenced.

Since we entered Italian waters on July11th we have collected 24 samples from 11 different sample sites.  It was a very busy 10 day but a very successful!  We collected samples in all the different bodies of water  and unique environments as we had planned.  One of the reasons we were so successful was due to weather………we had calm winds and seas the entire time which made sample collection very easy, and made overnight passages possible (we only anchored 3 nights in 12 days).  Below are some pictures of the seas we had during different legs of this trip, sometimes it felt like we were on a lake it was so calm!  Tomorrow we sail the 50 miles to pick up Dr. Venter and do some Italian media events.

Ionian Sea Sunrise

Sunrise in the Ligurian Sea

Sunset in the Tyrrhenian Sea

HMP Consortium – St. Louis Missouri

Human Microbiome Project Consortium – September 2010 – St Louis, Missouri

We received warm welcome messages from Dr George Weinstock and Dr Jane Petersen as well as a humorous welcome from Dr Larry Shapiro, Dean of Washington University Medical School. 

It was wonderful to see so many scientists come together to share the progress on their individual HMP related demonstration projects.  Our own demonstration project with Dr Zhiheng Pei, involving the esophagus microbiome and how that relates to esophageal adenocarcinoma (EA), was quite unique compared to the other projects as we were the only group to focus on the correlation between bacterial population and a form of cancer. 

With over 400 participants and 59 speakers, the conference was quite successful and very interesting.  JCVI Director Dr Karen Nelson did a wonderful job moderating one of the segments.  Dr Roger Lasken also gave a thorough presentation on his lab’s single cell approaches to genomic sequencing of uncultureable bacteria.  Johannes Goll gave a great presentation on his recent work with an open source tool called METAREP (recently published in Bioinformatics 8/26/2010), which is designed to help scientists with analyzing annotated metagenomic data.  And Dan Haft presented his interesting work with algorithmically tuning protein families from reference genomes for systems discovery. 

Overall the conference was quite interesting and informative.  I continue to wish all of the participating sequencing centers, PIs, and others involved with the HMP much success with their projects. 

Hope to see everyone in Vancouver!!!