SSAHA: a fast search method for large DNA databases

We describe an algorithm, SSAHA (Sequence Search and Alignment by Hashing Algorithm), for performing fast searches on databases containing multiple gigabases of DNA. Sequences in the database are preprocessed by breaking them into consecutive k-tuples of k contiguous bases and then using a hash table to store the position of each occurrence of each k-tuple. Searching for a query sequence in the database is done by obtaining from the hash table the "hits" for each k-tuple in the query sequence and then performing a sort on the results. We discuss the effect of the tuple length k on the search speed, memory usage, and sensitivity of the algorithm and present the results of computational experiments which show that SSAHA can be three to four orders of magnitude faster than BLAST or FASTA, while requiring less memory than suffix tree methods. The SSAHA algorithm is used for high-throughput single nucleotide polymorphism (SNP) detection and very large scale sequence assembly. Also, it provides Web-based sequence search facilities for Ensembl projects.     

Angiogenesis in the neoplastic sequence of Barrett's oesophagus. Correlation with VEGF expression

The purpose of this study was to determine the angiogenic profile of human oesophageal adenocarcinoma. This study was carried out on a large series of surgically resected Barrett's adenocarcinomas and associated preneoplastic lesions. Vascularization was quantified by microvessel counting and measurement of the percentage microvessel surface area after immunohistochemistry using the CD34 antibody. The expression of vascular endothelial cell growth factor (VEGF) was also examined by immunohistochemistry. Results were correlated with clinico-pathological data and prognosis. Vascularization, assessed by both microvessel counting and measurement of the microvessel surface, was statistically higher in superficial cancers than in others. Higher vascularization was correlated with a lower rate of lymph node and distant metastasis, as well as with better survival. However, when superficial carcinomas were excluded from the study, microvessel count failed to provide any significant prognostic information. Irrespective of the inclusion or exclusion of superficial tumours, the expression of VEGF was correlated with a higher vascularisation but did not provide prognostic significance. It is concluded that high angiogenic properties are acquired in precancerous lesions and early cancers in Barrett's oesophagus. Vascularization as assessed by both microvessel counting and measurement of the microvessel surface is not informative for prognosis in infiltrative Barrett's adenocarcinomas. The expression of VEGF is correlated with vascularization, but has no independent prognostic relevance.     

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus,a newly emergent paramyxovirus

E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of α-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials. Received January 23, 2003; accepted February 28, 2003 Published online April 28, 2003     

Malaria of importation in the child: epidemiological, clinical and therapeutic analysis. Apropos of 70 cases observed in a pediatric hospital in Paris

Seventy children from 7 months to 15 years old have been treated for malaria at Hospital Trousseau (Paris) during years 1987 and 1988. Thirty nine of them were living in France usually. The infection was one chiefly in Africa (68 cases), and by P. falciparum in 78% of children. The digestive symptoms were frequent (40/70); splenomegaly was observed in 40 children and hepatomegaly in 31. Anemia was present in 59 cases and mild thrombopenia for 31 cases. The C. reactive protein raised in 92% of cases. The diagnosis was late in 31 patients. Only one cerebral malaria case was observed. The chemoprophylaxis was unfitted or absent in 74% of children living in Paris. The chloroquino-resistance was clinically present in 17 cases and the mefloquine was more often used during 1988 year.     

Newly recognized forms of colitis: collagenous colitis, microscopic (lymphocytic) colitis, and lymphoid follicular proctitis

Based on pertinent data from the literature and on their personal experience, the authors present a clinicopathological review of collagenous colitis, microscopic (lymphocytic) colitis, and lymphoid follicular proctitis. The following aspects of these three newly recognized forms of colitis are emphasized: clinical features, light microscopy, histological diagnostic problems, immunological aspects, coexisting diseases, treatment, etiology, and pathogenesis. Special attention is paid to possible overlap between collagenous colitis and microscopic (lymphocytic) colitis.     

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.     

Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Effect of rat soleus muscle overload on neuromuscular transmission efficacy during continuous and intermittent activation

Increased neuromuscular activity is known to provoke morphological and functional adaptations at the neuromuscular synapse. Most of these changes have been documented following endurance exercise training programmes. In this study, the effect of rat soleus muscle overload produced by tenotomy plus voluntary wheel-cage activity on neuromuscular transmission efficacy was investigated. The overload protocol increased miniature endplate potential (MEPP) and endplate potential (EPP) amplitudes by 17 and 19%, respectively (both P < 0.01), and increased MEPP frequency by 86% (P < 0.01). EPP amplitude rundown during continuous trains of activation was attenuated by approximately 10% in the overloaded group (P < 0.01). Also, during intermittent activation, the overload protocol attenuated EPP amplitude rundown, mainly by enhancing EPP amplitude recovery by approximately 10% during the quiescent periods (P < 0.01). Although the present results show that both the degree and direction of adaptation are similar to what has been observed at rat soleus neuromuscular junctions following an endurance training protocol, there are important nuances between the results, suggesting different mechanisms through which these changes may occur.