Humberto Ortiz-Zuazaga

The khmer software package: enabling efficient nucleotide sequence analysis

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at https://github.com/dib-lab/khmer/.

Gene expression profiling of intestinal regeneration in the sea cucumber

BackgroundAmong deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers are excellent models for studying organ regeneration since they regenerate their digestive tract after evisceration. However, echinoderms have been sidelined in modern regeneration studies partially because of the lack of genome-wide profiling approaches afforded by modern genomic tools.For the last decade, our laboratory has been using the sea cucumber Holothuria glaberrima to dissect the cellular and molecular events that allow for such amazing regenerative processes. We have already established an EST database obtained from cDNA libraries of normal and regenerating intestine at two different regeneration stages. This database now has over 7000 sequences.ResultsIn the present work we used a custom-made microchip from Agilent with 60-mer probes for these ESTs, to determine the gene expression profile during intestinal regeneration. Here we compared the expression profile of animals at three different intestinal regeneration stages (3-, 7- and 14-days post evisceration) against the profile from normal (uneviscerated) intestines. The number of differentially expressed probes ranged from 70% at p < 0.05 to 39% at p < 0.001. Clustering analyses show specific profiles of expression for early (first week) and late (second week) regeneration stages. We used semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) to validate the expression profile of fifteen microarray detected differentially expressed genes which resulted in over 86% concordance between both techniques. Most of the differentially expressed ESTs showed no clear similarity to sequences in the databases and might represent novel genes associated with regeneration. However, other ESTs were similar to genes known to be involved in regeneration-related processes, wound healing, cell proliferation, differentiation, morphological plasticity, cell survival, stress response, immune challenge, and neoplastic transformation. Among those that have been validated, cytoskeletal genes, such as actins, and developmental genes, such as Wnt and Hox genes, show interesting expression profiles during regeneration.ConclusionOur findings set the base for future studies into the molecular basis of intestinal regeneration. Moreover, it advances the use of echinoderms in regenerative biology, animals that because of their amazing properties and their key evolutionary position, might provide important clues to the genetic basis of regenerative processes.

Hippocampal gene expression profiling in spatial discrimination learning.

Learning and long-term memory are thought to involve temporally defined changes in gene expression that lead to the strengthening of synaptic connections in selected brain regions. We used cDNA microarrays to study hippocampal gene expression in animals trained in a spatial discrimination-learning paradigm. Our analysis identified 19 genes that showed statistically significant changes in expression when comparing Nai;ve versus Trained animals. We confirmed the changes in expression for the genes encoding the nuclear protein prothymosin(alpha) and the delta-1 opioid receptor (DOR1) by Northern blotting or in situ hybridization. In additional studies, laser-capture microdissection (LCM) allowed us to obtain enriched neuronal populations from the dentate gyrus, CA1, and CA3 subregions of the hippocampus from Nai;ve, Pseudotrained, and spatially Trained animals. Real-time PCR examined the spatial learning specificity of hippocampal modulation of the genes encoding protein kinase B (PKB, also known as Akt), protein kinase C(delta) (PKC(delta)), cell adhesion kinase(beta) (CAK(beta), also known as Pyk2), and receptor protein tyrosine phosphatase(zeta/beta) (RPTP(zeta/beta)). These studies showed subregion specificity of spatial learning-induced changes in gene expression within the hippocampus, a feature that was particular to each gene studied. We suggest that statistically valid gene expression profiles generated with cDNA microarrays may provide important insights as to the cellular and molecular events subserving learning and memory processes in the brain.

Population genomics of parallel hybrid zones in the mimetic butterflies, H. melpomene and H. erato

Hybrid zones can be valuable tools for studying evolution and identifying genomic regions responsible for adaptive divergence and underlying phenotypic variation. Hybrid zones between subspecies of Heliconius butterflies can be very narrow and are maintained by strong selection acting on color pattern. The comimetic species, H. erato and H. melpomene, have parallel hybrid zones in which both species undergo a change from one color pattern form to another. We use restriction-associated DNA sequencing to obtain several thousand genome-wide sequence markers and use these to analyze patterns of population divergence across two pairs of parallel hybrid zones in Peru and Ecuador. We compare two approaches for analysis of this type of data-alignment to a reference genome and de novo assembly-and find that alignment gives the best results for species both closely (H. melpomene) and distantly (H. erato, ∼15% divergent) related to the reference sequence. Our results confirm that the color pattern controlling loci account for the majority of divergent regions across the genome, but we also detect other divergent regions apparently unlinked to color pattern differences. We also use association mapping to identify previously unmapped color pattern loci, in particular the Ro locus. Finally, we identify a new cryptic population of H. timareta in Ecuador, which occurs at relatively low altitude and is mimetic with H. melpomene malleti.

Bacterial Community in the Crop of the Hoatzin, a Neotropical Folivorous Flying Bird

ABSTRACT The hoatzin is unique among known avian species because of the fermentative function of its enlarged crop. A small-bodied flying foregut fermenter is a paradox, and this bird provides an interesting model to examine how diet selection and the gut microbiota contribute to maximizing digestive efficiency. Therefore, we characterized the bacterial population in the crop of six adult hoatzins captured from the wild. A total of 1,235 16S rRNA gene sequences were grouped into 580 phylotypes (67% of the pooled species richness sampled, based on Goods coverage estimator, with CACE and Chao1 estimates of 1,709 and 1,795 species-level [99% identity] operational taxonomic units, respectively). Members of 9 of the ∼75 known phyla in Bacteria were identified in this gut habitat; the Firmicutes were dominant (67% of sequences, belonging to the classes Clostridia, Mollicutes, and Bacilli), followed by the Bacteroidetes (30%, mostly in the order Bacteroidales), Proteobacteria (1.8%), and Lentisphaerae, Verrucomicrobia, TM7, Spirochaetes, Actinobacteria, and Aminanaerobia (all <0.1%). The novelty in this ecosystem is great; 94% of the phylotypes were unclassified at the “species” level and thus likely include novel cellulolytic lineages.

Gene Expression Profile of the Regeneration Epithelium during Axolotl Limb Regeneration

Urodele amphibians are unique among adult vertebrates in their ability to regenerate missing limbs. The process of limb regeneration requires several key tissues including a regeneration‐competent wound epidermis called the regeneration epithelium (RE). We used microarray analysis to profile gene expression of the RE in the axolotl, a Mexican salamander. A list of 125 genes and expressed sequence tags (ESTs) showed a ≥1.5‐fold expression in the RE than in a wound epidermis covering a lateral cuff wound. A subset of the RE ESTs and genes were further characterized for expression level changes over the time‐course of regeneration. This study provides the first large scale identification of specific gene expression in the RE. Developmental Dynamics 240:1826–1840, 2011.

Identification of Flap Structure-Specific Endonuclease 1 as a Factor Involved in Long-Term Memory Formation of Aversive Learning

We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.

Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

An increasing body of evidence suggests that mechanisms related to the introduction and repair of DNA double strand breaks (DSBs) may be associated with long-term memory (LTM) processes. Previous studies from our group suggested that factors known to function in DNA recombination/repair machineries, such as DNA ligases, polymerases, and DNA endonucleases, play a role in LTM. Here we report data using C57BL/6 mice showing that the V(D)J recombination-activating gene 1 (RAG1), which encodes a factor that introduces DSBs in immunoglobulin and T-cell receptor genes, is induced in the amygdala, but not in the hippocampus, after context fear conditioning. Amygdalar induction of RAG1 mRNA, measured by real-time PCR, was not observed in context-only or shock-only controls, suggesting that the context fear conditioning response is related to associative learning processes. Furthermore, double immunofluorescence studies demonstrated the neuronal localization of RAG1 protein in amygdalar sections prepared after perfusion and fixation. In functional studies, intra-amygdalar injections of RAG1 gapmer antisense oligonucleotides, given 1 h prior to conditioning, resulted in amygdalar knockdown of RAG1 mRNA and a significant impairment in LTM, tested 24 h after training. Overall, these findings suggest that the V(D)J recombination-activating gene 1, RAG1, may play a role in LTM consolidation.

Analytical Performance Evaluation of IPv6 and IPv4 Over 10 Gigabit Ethernet and InfiniBand using IPoIB

IPv6 is the response to the shortage of IPv4 addresses. It was defined almost twenty years ago by the IETF as a replacement of IPv4, and little by little, it is becoming more preponderant as the Internet protocol. The growth of Internet has led to the development of high performance networks. On one hand, Ethernet has evolved significantly and today it is common to find 10 Gigabit Ethernet networks in LANs. On the other hand, another approach for high performance networking is based on RDMA (Remote Direct Memory Access) which offers innovative features such as kernel bypass, zero copy, offload of splitting and assembly of messages in packets to the CAs (Channel Adapters), etc. InfiniBand is currently the most popular technology that implements RDMA. It uses verbs instead of sockets and a big effort of the community is required to port TCP/IP software to InfiniBand, to take advantage of its benefits. Meanwhile, IPoIB (IP over InfiniBand) is a protocol that has been proposed and permits the execution of socket-based applications on top of InfiniBand, without any change, at the expense of performance. In this paper, we make a performance evaluation of IPv6 and IPv4 over 10 Gigabit Ethernet and IPoIB. Our results show that 10 Gigabit Ethernet has a better throughput than IPoIB, especially for small and medium payload sizes. However, as the payload size increases, the advantage of 10 Gigabit Ethernet is reduced in comparison to IPoIB/FDR. With respect to latency, IPoIB did much better than 10 Gigabit Ethernet. Finally, our research also indicates that in a controlled environment, IPv4 has a better performance than IPv6.

Toa: A Web Based Network Flow Data Monitoring System at Scale

We present Toa, a web-based Network Flow data monitoring system (NMS). Toa consists of a collection of scripts that automatically parse network flow data, store this information in a database system, and generate interactive time line charts for network visualization analytics. The system is pseudo real time, meaning that it continuously updates the interactive charts from network flow data that is generated every five minutes. Toa also provides an interface to generate customized charts from the data stored in the database, and plugins that connect the visualization charts with the network flow data file for more in depth visualizations and analysis. The Toa web GUI presents users with the following network traffic visualization options: (1) per network label (interface, Autonomous System [AS], or network block) traffic, (2) per-port traffic for each network label, (3) network label to network label traffic, (4) customized charts from the database data, and (5) plugins for in-depth analysis of the network flow data file.