The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

ContextThe efficient sequestration of hemoglobin by the red blood cell membrane
and the presence of multiple hemoglobin clearance mechanisms suggest a critical
need to prevent the buildup of this molecule in the plasma. A growing list
of clinical manifestations attributed to hemoglobin release in a variety of
acquired and iatrogenic hemolytic disorders suggests that hemolysis and hemoglobinemia
should be considered as a novel mechanism of human disease.Evidence AcquisitionPertinent scientific literature databases and references were searched
through October 2004 using terms that encompassed various aspects of hemolysis,
hemoglobin preparations, clinical symptoms associated with plasma hemoglobin,
nitric oxide in hemolysis, anemia, pulmonary hypertension, paroxysmal nocturnal
hemoglobinuria, and sickle-cell disease.Evidence SynthesisHemoglobin is released into the plasma from the erythrocyte during intravascular
hemolysis in hereditary, acquired, and iatrogenic hemolytic conditions. When
the capacity of protective hemoglobin-scavenging mechanisms has been saturated,
levels of cell-free hemoglobin increase in the plasma, resulting in the consumption
of nitric oxide and clinical sequelae. Nitric oxide plays a major role in
vascular homeostasis and has been shown to be a critical regulator of basal
and stress-mediated smooth muscle relaxation and vasomotor tone, endothelial
adhesion molecule expression, and platelet activation and aggregation. Thus,
clinical consequences of excessive cell-free plasma hemoglobin levels during
intravascular hemolysis or the administration of hemoglobin preparations include
dystonias involving the gastrointestinal, cardiovascular, pulmonary, and urogenital
systems, as well as clotting disorders. Many of the clinical sequelae of intravascular
hemolysis in a prototypic hemolytic disease, paroxysmal nocturnal hemoglobinuria,
are readily explained by hemoglobin-mediated nitric oxide scavenging.ConclusionA growing body of evidence supports the existence of a novel mechanism
of human disease, namely, hemolysis-associated smooth muscle dystonia, vasculopathy,
and endothelial dysfunction.

https://jamanetwork.com/journals/jama/articlepdf/200611/JCR50001.pdf

The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease.

The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties.

Free heme toxicity and its detoxification systems in human

Rhodnius prolixus is amongst the most important vectors of Trypanosoma cruzi in the Americas, putting thousands of people at risk of contracting Chagas Disease. This insect is also one of the most important models in insect physiology, especially regarding the blood-feeding process. However, studies on R. prolixus genetics lagged, and our understanding on the regulation of gene expression is incipient. Transcriptomes have the power to study the expression of thousands of genes in a single experiment. A comprehensive R. prolixus transcriptome was performed in 2014, sequencing RNA from different tissues (anterior gut, midgut, posterior gut, rectum, ovaries, fat body, maphigian tubules, and testicles). However, on that occasion, only the gut transcriptome was deeply analysed. Here we evaluated the results of the testicles transcriptome of R. prolixus with the objective to find and understand genes that could have an important role in male reproduction. We found, that from the 25,673 transcripts assembled in the whole transcriptome, 5,365 have a testicle specific expression pattern. As expected, amongst the most abundant families of transcripts, are those related to spermatogenesis and male fertility, such as myosins, actins, and dyneins. To our surprise, lipocalins, serine protease inhibitors (serpins), and lysozymes also were highly abundant in testicles. The role of these classes of genes are well known in other tissues, such as salivary glands and gut, but very little is known on their role in male reproduction (and we proposed here a few hypothesis that could be tested to address the role of these genes in male fertility). It would be interesting to study further the role of these genes on R. prolixus male fertility. Finally, as a reflection of the lack of knowledge on triatomine genetics, we found that almost half of the transcripts in R. prolixus testicles have no similarities to any other genes on reference databases. Our study shows that we still have a lot to know and to understand about reproduction in triatomine, especially in males. Besides the large number of genes without described function (possibly novel genes), there are those in which the function is known for other tissues, and we can only guess, at best, the role and importance of such genes for triatomine male fertility.

/pdf/analysis-of-the-testicle-s-transcriptome-of-the-chagas-3um0zakmsh.pdf

Analysis of the testicle’s transcriptome of the Chagas disease vector Rhodnius prolixus

Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.

/pdf/non-immune-traits-triggered-by-blood-intake-impact-vectorial-2x9w202kzc.pdf

Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence

In the past decade, metagenomics studies exploring tick microbiota have revealed widespread interactions between bacteria and arthropods, including symbiotic interactions. Functional studies showed that obligate endosymbionts contribute to tick biology, affecting reproductive fitness and molting. Understanding the molecular basis of the interaction between ticks and their mutualist endosymbionts may help to develop control methods based on microbiome manipulation. Previously, we showed that Rhipicephalus microplus larvae with reduced levels of Coxiella endosymbiont of R. microplus (CERM) were arrested at the metanymph life stage (partially engorged nymph) and did not molt into adults. In this study, we performed a transcriptomic differential analysis of the R. microplus metanymph in the presence and absence of its mutualist endosymbiont. The lack of CERM resulted in an altered expression profile of transcripts from several functional categories. Gene products such as DA-P36, protease inhibitors, metalloproteases, and evasins, which are involved in blood feeding capacity, were underexpressed in CERM-free metanymphs. Disregulation in genes related to extracellular matrix remodeling was also observed in the absence of the symbiont. Taken together, the observed alterations in gene expression may explain the blockage of development at the metanymph stage and reveal a novel physiological aspect of the symbiont-tick-vertebrate host interaction.

/pdf/coxiella-endosymbiont-of-rhipicephalus-microplus-modulates-2hrq573f.pdf

Coxiella Endosymbiont of Rhipicephalus microplus Modulates Tick Physiology With a Major Impact in Blood Feeding Capacity

dsRNA synthesis Double stranded RNAs for MAL (dsMAL) and NOX4-art (dsNOX4-art) were produced from PCR-amplified fragments using the HiScribe T7 In Vitro Translation Kit (New England Biolabs). Amplicons for dsMAL were produced using plasmid templates and for dsNOX4-art by reverse transcriptase PCR (RT-PCR) products, from sugar-fed female cDNA. Two rounds of PCR were performed to amplify NOX4-art. The first PCR round was performed with primers containing a short adaptor sequence at the 5′ end (tggcgcccctagatg). The primers used for the first round of PCR were NOX4-artFwd 5′ tggcgcccctagatgAGTGGCACCCGTTTACAGTC 3′ and NOX4-artRev 5′ tggcgcccctagatgTTTGGGACCACAACT GAACA 3′. The PCR cycles utilized were 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 57 °C for 45 s and 72 °C for 45 s

/pdf/erratum-to-evolutionary-origin-and-function-of-nox4-art-an-1q79yxu4nt.pdf

Erratum to: Evolutionary origin and function of NOX4- art, an arthropod specific NADPH oxidase.

Triatomine insects had a fundamental role in the establishment of essential concepts of insect physiology, especially thanks to the use of Rhodnius prolixus as a model for basic research in the last century. The major unique feature that made triatomines excellent models is their strictly hematophagous way of life, as molting in nymph stages and oogenesis in the adult are triggered by a blood meal. Therefore, development and reproduction are strictly controlled in an on/off fashion by the arrival of the voluminous blood meal at the digestive channel. However, these insects are less popular in the science arena due to the scarcity of genetic and molecular data. The last few years, however, have manifested a change in the field due to the appearance of large datasets of genomic, transcriptomic, and phylogenetic molecular information, as well as a series of new methodological advances. Here, we tried to make a brief connection between the classical outline of blood digestion physiology in triatomines and this new science scenario, trying to identify critical information that is still lacking.

Pedro L. Oliveira

Papers

Analysis of the testicle’s transcriptome of the Chagas disease vector Rhodnius prolixus

Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence

Coxiella Endosymbiont of Rhipicephalus microplus Modulates Tick Physiology With a Major Impact in Blood Feeding Capacity

Erratum to: Evolutionary origin and function of NOX4- art, an arthropod specific NADPH oxidase.

Blood Digestion in Triatomine Insects