SLAM: cross-species gene finding and alignment with a generalized pair hidden Markov model

Comparative-based gene recognition is driven by the principle that conserved regions between related organisms are more likely than divergent regions to be coding. We describe a probabilistic framework for gene structure and alignment that can be used to simultaneously find both the gene structure and alignment of two syntenic genomic regions. A key feature of the method is the ability to enhance gene predictions by finding the best alignment between two syntenic sequences, while at the same time finding biologically meaningful alignments that preserve the correspondence between coding exons. Our probabilistic framework is the generalized pair hidden Markov model, a hybrid of (1). generalized hidden Markov models, which have been used previously for gene finding, and (2). pair hidden Markov models, which have applications to sequence alignment. We have built a gene finding and alignment program called SLAM, which aligns and identifies complete exon/intron structures of genes in two related but unannotated sequences of DNA. SLAM is able to reliably predict gene structures for any suitably related pair of organisms, most notably with fewer false-positive predictions compared to previous methods (examples are provided for Homo sapiens/Mus musculus and Plasmodium falciparum/Plasmodium vivax comparisons). Accuracy is obtained by distinguishing conserved noncoding sequence (CNS) from conserved coding sequence. CNS annotation is a novel feature of SLAM and may be useful for the annotation of UTRs, regulatory elements, and other noncoding features.     

Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.     

High and low frequency transcutaneous electrical nerve stimulation inhibits nociceptive responses induced by CO2 laser stimulation in humans

The aim of the study was to evaluate the effects of transcutaneous electric nerve stimulation (TENS) on CO(2) laser evoked potentials (LEPs) in 16 normal subjects. The volar side of the forearm was stimulated by 10 Hz TENS in eight subjects and by 100 Hz TENS in the remainder; the skin of the forearm was stimulated by CO(2) laser and the LEPs were recorded in basal conditions and soon after and 15 min after TENS. Both low and high frequency TENS significantly reduced the subjective rating of heat stimuli and the LEPs amplitude, although high frequency TENS appeared more efficacious. TENS seemed to exert a mild inhibition of the perception and processing of pain induced by laser Adelta fibres activation; the implications of these effects in the clinical employment of TENS remain to be clarified.     

Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells

Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.     

Molecular cloning and characterization of actin genes from Echinococcus granulosus

An Echinococcus granulosus genomic library has been screened with a mouse β-actin cDNA probe. Two clones carrying DNA fragments of about 15 kb, possibly derived from the same genome region, have been isolated. This 15-kb genomic region includes 2 actin-related sequences (EgactI and EgactII) separated by about 4 kb. The nucleotide sequences of both genes were determined. The EgactI sequence presents no introns, but an intron of 591 bp was observed in the EgactII sequence. The genes potentially encode 375 and 376 amino-acid-long actins, respectively, with a homology of 85.3%. The deduced amino acid sequences from both genes were compared to the actin sequences from other organisms, showing similarities ranging from 63.5% to 90.6%. The nucleotide sequence of a partial actin cDNA clone has been determined. The deduced amino acids sequence showed a homology of 90.3% and 88.0% in relation to the EgactI and EgactII sequences respectively, suggesting the existence of at least one more actin gene in E. granulosus. This hypothesis is reinforced by the number of bands detected in the Southern blot analysis. Experiments based on the amplification of DNA segments using 3′-specific actin primers indicate that the EgactI gene is transcribed in protoscoleces.     

TNF revisited: TNF-independent antitumor activity in sera of mice sensitized with Propionibacterium acnes and challenged with lipopolysaccharide

Sera of mice sensitized with bacteria and subsequently challenged with lipopolysaccharide promote hemorrhagic necrosis of tumors in vivo and display cytotoxic activity against tumor cells in vitro, which has been attributed to the induction of tumor necrosis factor (TNF). Here, we describe the induction of a previously unrecognized antitumor activity in such sera, which is distinct from TNF but displays tumor-specific cytocidal activity in vitro as well as potent tumor-regressing activity in vivo. Biochemical analysis of this activity yielded a molecular mass of approximately 150 kDa, closely resembling a novel tumoricidal factor of murine macrophages (Mphi) termed MTC 170 (Mphi tumor cytotoxin, approximate molecular mass 170 kDa), which we have previously proposed to constitute a major effector pathway for the destruction of tumor cells by activated Mphi.     

Detection of apple chlorotic leaf spot virus using a 5' nuclease assay with a fluorescent 3' minor groove binder-DNA probe

The development of a real-time 5' nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3' minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. The covalent attachment of the minor groove binder moiety at the 3' end of the probe increased the probe/target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.