Loading...
12 results
Search Results
Now showing 1 - 10 of 12
- Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCRPublication . Prevost-Boure, Nicolas Chemidlin; Christen, Richard; Dequiedt, Samuel; Mougel, Christophe; Lelievre, Melanie; Jolivet, Claudy; Shahbazkia, Hamid Reza; Guillou, Laure; Arrouays, Dominique; Ranjard, LionelFungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen (R) method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1/FF390. This in silico analysis of the specificity of FR1/FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1/FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C:N ratio and land use in determining fungal abundance in soils.
- Hessian-polar context: a descriptor for microfilaria recognitionPublication . Al-Tam, Faroq; dos Anjos, Antonio; Shahbazkia, Hamid R.This paper presents a new effective descriptor for microfilaria. Since microfilaria is a thin elastic object, the proposed descriptor handles it locally. At each medial point of the microfilaria, the local structure of the microfilaria votes for a given shape. Accumulating these votes in the polar domain yields a rich descriptor. Experimental results show the effectiveness of the proposed approach when compared to a set of different well-established methods.
- Midgut microbiota of the malaria mosquito vector Anopheles gambiae and Interactions with plasmodium falciparum InfectionPublication . Boissiere, Anne; Tchioffo, Majoline T.; Bachar, Dipankar; Abate, Luc; Marie, Alexandra; Nsango, Sandrine E.; Shahbazkia, Hamid R.; Awono-Ambene, Parfait H.; Levashina, Elena A.; Christen, Richard; Morlais, IsabelleThe susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.
- A user-friendly software to easily count Anopheles egg batchesPublication . Mollahosseini, Ali; Rossignol, Marie; Pennetier, Cédric; Cohuet, Anna; Anjos, António dos; Chandre, Fabrice; Shahbazkia, Hamid RezaStudies on malaria vector ecology and development/evaluation of vector control strategies often require measures of mosquito life history traits. Assessing the fecundity of malaria vectors can be carried out by counting eggs laid by Anopheles females. However, manually counting the eggs is time consuming, tedious, and error prone. Methods In this paper we present a newly developed software for high precision automatic egg counting. The software written in the Java programming language proposes a user-friendly interface and a complete online manual. It allows the inspection of results by the operator and includes proper tools for manual corrections. The user can in fact correct any details on the acquired results by a mouse click. Time saving is significant and errors due to loss of concentration are avoided. Results The software was tested over 16 randomly chosen images from 2 different experiments. The results show that the proposed automatic method produces results that are close to the ground truth. Conclusions The proposed approaches demonstrated a very high level of robustness. The adoption of the proposed software package will save many hours of labor to the bench scientist. The software needs no particular configuration and is freely available for download on: http://w3.ualg.pt/∼hshah/eggcounter/.
- A user-friendly software to easily count Anopheles egg batchesPublication . Mollahosseini, Ali; Rossignol, Marie; Pennetier, Cedric; Cohuet, Anna; Dos Anjos, António; Chandre, Fabrice; Shahbazkia, Hamid RezaBackground: Studies on malaria vector ecology and development/evaluation of vector control strategies often require measures of mosquito life history traits. Assessing the fecundity of malaria vectors can be carried out by counting eggs laid by Anopheles females. However, manually counting the eggs is time consuming, tedious, and error prone. Methods: In this paper we present a newly developed software for high precision automatic egg counting. The software written in the Java programming language proposes a user-friendly interface and a complete online manual. It allows the inspection of results by the operator and includes proper tools for manual corrections. The user can in fact correct any details on the acquired results by a mouse click. Time saving is significant and errors due to loss of concentration are avoided. Results: The software was tested over 16 randomly chosen images from 2 different experiments. The results show that the proposed automatic method produces results that are close to the ground truth. Conclusions: The proposed approaches demonstrated a very high level of robustness. The adoption of the proposed software package will save many hours of labor to the bench scientist. The software needs no particular configuration and is freely available for download on: http://w3.ualg.pt/(similar to)hshah/eggcounter/.
- Eukaryotic richness in the abyss: insights from pyrotag sequencingPublication . Pawlowski, Jan; Christen, Richard; Lecroq, Beatrice; Bachar, Dipankar; Shahbazkia, Hamid R.; Amaral-Zettler, Linda; Guillou, LaureBackground: The deep sea floor is considered one of the most diverse ecosystems on Earth. Recent environmental DNA surveys based on clone libraries of rRNA genes confirm this observation and reveal a high diversity of eukaryotes present in deep-sea sediment samples. However, environmental clone-library surveys yield only a modest number of sequences with which to evaluate the diversity of abyssal eukaryotes. Methodology/Principal Findings: Here, we examined the richness of eukaryotic DNA in deep Arctic and Southern Ocean samples using massively parallel sequencing of the 18S ribosomal RNA (rRNA) V9 hypervariable region. In very small volumes of sediments, ranging from 0.35 to 0.7 g, we recovered up to 7,499 unique sequences per sample. By clustering sequences having up to 3 differences, we observed from 942 to 1756 Operational Taxonomic Units (OTUs) per sample. Taxonomic analyses of these OTUs showed that DNA of all major groups of eukaryotes is represented at the deep-sea floor. The dinoflagellates, cercozoans, ciliates, and euglenozoans predominate, contributing to 17%, 16%, 10%, and 8% of all assigned OTUs, respectively. Interestingly, many sequences represent photosynthetic taxa or are similar to those reported from the environmental surveys of surface waters. Moreover, each sample contained from 31 to 71 different metazoan OTUs despite the small sample volume collected. This indicates that a significant faction of the eukaryotic DNA sequences likely do not belong to living organisms, but represent either free, extracellular DNA or remains and resting stages of planktonic species. Conclusions/Significance: In view of our study, the deep-sea floor appears as a global DNA repository, which preserves genetic information about organisms living in the sediment, as well as in the water column above it. This information can be used for future monitoring of past and present environmental changes.
- P-TRAP: a Panicle Trait Phenotyping toolPublication . Al-Tam, Faroq; Adam, Helene; Dos Anjos, António; Lorieux, Mathias; Larmande, Pierre; Ghesquiere, Alain; Jouannic, Stefan; Shahbazkia, Hamid RezaBackground: In crops, inflorescence complexity and the shape and size of the seed are among the most important characters that influence yield. For example, rice panicles vary considerably in the number and order of branches, elongation of the axis, and the shape and size of the seed. Manual low-throughput phenotyping methods are time consuming, and the results are unreliable. However, high-throughput image analysis of the qualitative and quantitative traits of rice panicles is essential for understanding the diversity of the panicle as well as for breeding programs. Results: This paper presents P-TRAP software (Panicle TRAit Phenotyping), a free open source application for high-throughput measurements of panicle architecture and seed-related traits. The software is written in Java and can be used with different platforms (the user-friendly Graphical User Interface (GUI) uses Netbeans Platform 7.3). The application offers three main tools: a tool for the analysis of panicle structure, a spikelet/grain counting tool, and a tool for the analysis of seed shape. The three tools can be used independently or simultaneously for analysis of the same image. Results are then reported in the Extensible Markup Language (XML) and Comma Separated Values (CSV) file formats. Images of rice panicles were used to evaluate the efficiency and robustness of the software. Compared to data obtained by manual processing, P-TRAP produced reliable results in a much shorter time. In addition, manual processing is not repeatable because dry panicles are vulnerable to damage. The software is very useful, practical and collects much more data than human operators. Conclusions: P-TRAP is a new open source software that automatically recognizes the structure of a panicle and the seeds on the panicle in numeric images. The software processes and quantifies several traits related to panicle structure, detects and counts the grains, and measures their shape parameters. In short, P-TRAP offers both efficient results and a user-friendly environment for experiments. The experimental results showed very good accuracy compared to field operator, expert verification and well-known academic methods.
- New approach for segmentation and quantification of two-dimensional gel electrophoresis imagesPublication . Dos Anjos, António; Moller, Anders L. B.; Ersboll, Bjarne K.; Finnie, Christine; Shahbazkia, Hamid R.Motivation: Detection of protein spots in two-dimensional gel electrophoresis images (2-DE) is a very complex task and current approaches addressing this problem still suffer from significant shortcomings. When quantifying a spot, most of the current software applications include a lot of background due to poor segmentation. Other software applications use a fixed window for this task, resulting in omission of part of the protein spot, or including background in the quantification. The approach presented here for the segmentation and quantification of 2-DE aims to minimize these problems. Results: Five sections from different gels are used to test the performance of the presented method concerning the detection of protein spots, and three gel sections are used to test the quantification of sixty protein spots. Comparisons with a state-of-the-art commercial software and an academic state-of-the-art approach are presented. It is shown that the proposed approach for segmentation and quantification of 2-DE images can compete with the available commercial and academic software packages.
- Iterative illumination correction with implicit regularizationPublication . Al-Tam, Faroq; Dos Anjos, António; Shahbazkia, Hamid RezaThis paper presents a retrospective algorithm for correcting the uneven illumination field in microscopy images. The illumination field is iteratively made uniform using an increasing sequence of bivariate polynomials. At each iteration, the least squares problem of fitting a 2-D polynomial to a sampled image is solved by using QR decomposition with column pivoting, where image samples are obtained by dynamic programming or watershed transform. This incremental scheme allows the smoothness constraint of the estimated bias field to be implicitly satisfied. The proper number of iterations is determined by an automatic stopping criterion. The experimental results show the effectiveness of the proposed approach when compared to a set of different well-established methods.
- INsPECT, an open-source and versatile software for automated quantification of (Leishmania) intracellular parasitesPublication . Yazdanparast, Ehsan; Dos Anjos, António; Garcia, Deborah; Loeuillet, Corinne; Shahbazkia, Hamid R.; Vergnes, BaptisteIntracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra-from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (49,6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the "gold standard" microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites.