Subtype analysis of Blastocystis isolates in Swedish patients.

Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5'-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P=0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.

Surveillance for Listeria monocytogenes and listeriosis, 1995-2004.

Canadian cases and outbreaks of illness caused by Listeria monocytogenes between 1995 and 2004 were assessed. Isolates (722 total) were characterized by serotyping, and pulsed-field gel electrophoresis (PFGE) was performed to provide a means of detecting case clusters. Rates of listeriosis remained fairly consistent during the period of study, and patient characteristics were similar to those seen in studies of other populations. Most isolates were obtained from blood and cerebrospinal fluid, although during some outbreak investigations isolates were also obtained from stools. Serotype 1/2a predominated in isolates from patients in Canada, followed by serotypes 4b and 1/2b. Outbreaks caused by L. monocytogenes that occurred during the period of study were caused by isolates with serotypes 1/2a and 4b. A retrospective analysis of PFGE data uncovered several clusters that might have represented undetected outbreaks, suggesting that comprehensive prospective PFGE analysis coupled with prompt epidemiological investigations might lead to improved outbreak detection and control.

Thermodynamic, structural, and nanomechanical properties of a fluorous biphasic material.

The dynamics of the amphiphilic semifluorinated F(CF2)12(CH2)12H (F12H12) alkane that undergoes two condensed phase transitions have been investigated by Brillouin light spectroscopy, shear rheometry, small- (SAXS) and wide-angle (WAXS) X-ray scattering, and thermodynamic PVT measurements. The solid (I)-solid (II) transition (Ts) is marked by a stronger temperature dependence of the sound velocity in phase II and by a 2 orders of magnitude drop of the shear modulus. Between the Ts and the melting transition (Tm), the presence of two phonons implies a coexistence of solid (II) and amorphous (liquid) regions in the submicrometer range at thermal equilibrium as revealed by the SAXS pattern of a single reflection superimposed on a very broad amorphous halo. This intriguing finding of a transient, very slow (over 10 h) solid/liquid coexistence within phase II is rationalized by a two-stage mechanism for melting of the smectic phase (II) of F12H12. A refinement of the known packing motifs for the two solid-state structures is proposed.

Cryptic diversity in intestinal protists.

In the past few years our understanding of genetic variation within and between species of intestinal parasitic protists has changed significantly. New species names have been assigned and others have been dropped in response to new data. In this review, I summarise these findings and discuss their implications for future studies. In several cases the findings suggest that caution needs to be exercised to prevent premature conclusions being reached.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.

A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity.

BACKGROUND: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). RESULTS: To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). CONCLUSIONS: These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family.

Pretreatment depression severity in breast cancer patients and its relation to treatment response to behavior therapy.

OBJECTIVE: Major depressive disorder is prevalent in breast cancer patients. There is a paucity of research on variables associated with depression severity and the link between depression severity and response to psychotherapy. To provide optimal mental health services to breast cancer patients, examining correlates of depression severity and its relation to treatment response is critical. METHOD: In the context of a randomized trial of behavior activation and problem-solving therapy for depressed breast cancer patients, this study evaluated demographic (marital status, age, education), psychosocial (social support, environmental reward, anxiety, number of coexistent anxiety disorders), and cancer-related (bodily pain, length of diagnosis, cancer stage) variables associated with pretreatment depression severity. Second, the relation of pretreatment depression severity with posttreatment and 12-month response and remission was assessed. RESULTS: For pretreatment depression severity, the overall regression model accounted for 40% of the variance, F(5, 74) = 9.87, p < .001. Less environmental reward and greater somatic anxiety were significantly and uniquely associated with depression severity. Depression severity was unrelated to treatment remission but was a significant moderator of treatment response at posttreatment and 12-month follow-up; individuals with higher depression severity were more responsive to therapy. For patients treated with behavior activation, environmental reward significantly mediated the relationship between pre- and posttreatment depression. CONCLUSIONS: Consistent with behavioral models of depression, less environmental reward and greater anxiety might influence depression severity in breast cancer patients. Data support the efficacy of behavior therapy for breast cancer patients, particularly those with more severe depression.

Mindfulness-based therapy and behavioral activation: A randomized controlled trial with depressed college students.

Major Depressive Disorder (MDD) manifests in 20-30% of college students, with increased incidence in recent decades. Very limited research has assessed the efficacy of evidence-based interventions for MDD in college students. Mindfulness-Based Therapy (MBT) and Behavioral Activation (BA) are two interventions with significant potential to meet demands of college counseling clinics and effectively treat college students with MDD. This study utilized a randomized controlled research design (n = 50) to examine the efficacy of four-sessions of abbreviated MBT and BA relative to a wait-list control condition with depressed college students. Intent-to-treat data analyses on depression outcome measures suggested both treatments were superior to the control group. There were significant pre-post treatment improvements across measures of depression, rumination, stress, and mindfulness, gains largely maintained at 1-month follow-up. Neither active treatment effectively reduced somatic anxiety. Both treatments generally had moderate-strong effect sizes relative to the control group, and based on depression response and remission criteria, 56-79% of patients exhibited clinically significant improvement. Based on reliable change indices, 75-85% experienced clinically significant reductions in depression. There was strong therapist competence and adherence to treatment protocols and high patient satisfaction with both interventions. Study limitations and implications for the assessment and treatment of depressed college students are discussed.

Precise identification of cleavage sites involved in the unusual processing of trypanosome ribosomal RNA.

The large subunit ribosomal RNA (LSRNA) of Trypanosoma brucei is unusual in being cleaved at multiple sites to yield six stable fragments of RNA. We report here the complete nucleotide sequence of two regions of the ribosomal DNA repeat unit. The first sequence includes all of the processing sites involved in the generation of one of the small LSRNA fragments. The second region encodes the trypanosome 5.8 S RNA. By RNA sequencing and S1 nuclease mapping, we have identified the processing sites involved in the generation of both of these small RNAs. On the basis of predicted secondary structure models, we infer that all the cleavages apparently occur near the junction of single- and double-stranded regions. The sites involved in the novel LSRNA processing show a clear symmetry with respect to a conserved region of ten base-pairs. No such signals are evident for the processing sites that generate the 5.8 S RNA.

Contractile properties of the adductor pollicis in obese patients on a hypocaloric diet for two weeks.

The muscle function of 21 obese patients on a hypocaloric diet (450 kcal/day) was studied. On days 1, 7, and 12 the maximal isometric force and contractile properties (force:frequency relationship, relaxation rate, and fatigability) of the adductor pollicis were measured. The patients lost a mean of 5.7 kg but there was no significant change in the muscle strength or contractile properties. Twenty normal and three malnourished patients were also studied; the latter demonstrated abnormalities of muscle function. The use of such tests to monitor changes in nutritional status must be treated with caution.

Differences in mucosal appearances and in relapse rates in duodenal ulceration treated with sucralfate or cimetidine.

A total of 46 patients with duodenal ulcer were randomly assigned, without the knowledge of the investigators, to treatment with cimetidine 200 mg three times daily and 400 mg at night or sucralfate 1 g four times daily followed by one year of maintenance treatment with cimetidine 400 mg at night or sucralfate 1 g twice daily, respectively, in those patients with healed ulcers. The endoscopic healing rates and relapse rates during the maintenance period were similar, four relapses occurring in each group. All four relapses in the sucralfate group occurred at 12 months and only two were symptomatic. All the cimetidine relapses were symptomatic, two occurring at six months, one at nine months, and one at 12 months. Following the one year maintenance period, 13 cimetidine patients and 11 sucralfate patients were followed up for 36 months. During the first two years, nine of 13 (69 percent) cimetidine-treated and two of 11 (18 percent) sucralfate-treated patients had relapses. During the third year, three more sucralfate-treated patients and one more cimetidine-treated patient had relapses, making a total of 10 of 13 (77 percent) and five of 11 (45 percent) in the cimetidine and sucralfate groups, respectively. Duodenal biopsy specimens obtained before and after healing and after one year of maintenance were examined by light and electron microscopy. The sucralfate group showed greater improvement after one year of maintenance therapy than did the cimetidine group, although the appearances in either group were not predictive of subsequent relapse. The results show that relapses are less frequent and occur later after sucralfate therapy and also that the morphologic appearances are more normal after treatment with sucralfate than after treatment with cimetidine.

Extensive genetic diversity in Blastocystis hominis.

Blastocystis homonis is a common human parasite of uncertain role in human disease. Approximately equal numbers of reports implicate it and exonerate it as a pathogen. Genetic diversity in B. hominis was investigated using riboprinting to study sequence variation in the small subunit ribosomal RNA genes of 30 randomly selected isolates. Extensive sequence variation was discovered in B. hominis ribosomal RNA genes and this species consists of at least seven morphologically identical but genetically quite distinct organisms. If only a subset of the B. hominis variants have the potential to cause disease in humans this might explain the disparate findings reported. Future clinical studies must take the heterogeneity of B. hominis into account.

The Laredo strain and other 'Entamoeba histolytica-like' amoebae are Entamoeba moshkovskii.

A small number of Entamoeba isolates from humans, the best known of which is the 'Laredo' strain, have the ability to grow at room temperature. This peculiarity, along with other characteristics, distinguishes the strains from the human pathogen E. histolytica despite their being morphologically inseparable. In contrast, these 'E. histolytica-like' strains share several features with E. moshkovskii, which is most frequently isolated from polluted water. To examine the taxonomic relationships among these morphologically similar organisms, we have used polymerase chain reaction amplification of the small subunit ribosomal RNA gene combined with restriction fragment length polymorphism analysis, 'riboprinting'. The results clearly show that the 'E. histolytica-like' amoebae are indeed strains of E. moshkovskii, and not closely related to E. histolytica.

Ribosomal RNA genes of 'pathogenic' and 'nonpathogenic' Entamoeba histolytica are distinct.

Most infections with Entamoeba histolytica are asymptomatic. Two forms of the organism can be distinguished biochemically, and this finding has been explained by two distinct hypotheses: (1) there are two morphologically indistinguishable species, one of which causes disease; (2) there is one species which exists in two interconvertible forms, one of which causes disease. Knowledge of which hypothesis is correct has major implications for evaluation and treatment of carriers. We have studied the ribosomal RNA genes of the two forms hypothesizing that, if E. histolytica is one species, there should be no differences between them. We have found that the ribosomal RNA genes of the two forms are quite distinct, which supports the hypothesis that E. histolytica is two species.

Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns.

Ribosomal DNA (rDNA) plasmid restriction maps of 10 strains and rDNA hybridisation patterns of 61 additional strains have been used to assess inter- and intra-specific diversity and phylogenetic relationships in the genus Naegleria. The results obtained by this method largely confirm those of previous studies based on a variety of other criteria. They indicate that very little variation exists within the pathogenic species Naegleria fowleri despite its worldwide distribution and that it is closely related to the nonpathogenic Naegleria lovaniensis. Naegleria gruberi is most likely a polyphyletic grouping and care should be taken when using one strain as a reference point for this species. In addition, the two subspecies of the pathogenic Naegleria australiensis arose separately from within the range of variability encompassed by N. gruberi, as did the species Adelphamoeba galeacystis which should probably be assigned to the genus Naegleria. The species Naegleria jadini and Naegleria andersoni are not closely related to any other in the genus based on their rDNA patterns.

Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar.

The electrophoretic patterns of hexokinase and phosphoglucomutase have been widely used to distinguish Entamoeba histolytica from Entamoeba dispar isolates. Although E. histolytica and E. dispar, previously called pathogenic and nonpathogenic Entamoeba histolytica, differ clearly in sequences of many homologous genes, a conversion between the two has been reported by several laboratories, in each case showing the conversion of hexokinase (ATP, D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns. An apparent mobility shift of this enzyme may either be due to posttranslational modification or processing, or to the appearance of a new isoform encoded by a second gene. In this study we observed that the four observed bands in the isoenzyme patterns of pathogenic and nonpathogenic forms of Entamoeba were correlated with four different cDNAs, and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts. Polymerase chain reaction (PCR) experiments did not reveal hidden genes which might be responsible for conversion phenomena. These results strongly support the redefinition of pathogenic and nonpathogenic Entamoeba histolytica as two closely related species Entamoeba histolytica and Entamoeba dispar.

The evolution of Entamoeba, a cautionary tale.

In addition to its medical importance, the parasite Entamoeba histolytica has been studied by evolutionary biologists because of its unusual structural and biochemical features. Recent molecular data have revealed that many of our assumptions regarding the relationships of the organism and its relatives have been incorrect.

Colonization of the uterus by the oral protozoan Entamoeba gingivalis.

Pap smears occasionally reveal protozoa of the genus Entamoeba in the uterus of intrauterine device (IUD) users, but definitive identification of the species involved has not been possible. Using riboprinting, a technique that compares ribosomal RNA gene sequences, we present evidence that the organism is Entamoeba gingivalis, an inhabitant of the mouth. Colonization most likely occurs via orogenital contact and requires the presence of an IUD and a concomitant bacterial infection.

Salmonella typhimurium InvA expression probed with a monoclonal antibody to the C-terminal peptide of InvA.

The Salmonella typhimurium InvA protein is a component of a sec-independent secretion apparatus necessary for full virulence of the bacteria. We generated a monoclonal antibody to the C-terminal portion of the InvA protein that recognized proteins in S. typhimurium and weakly in Y. enterocolitica, but not in several other species of bacteria, including S. flexneri. S. typhimurium grown without agitation produced relatively constant amounts of membrane InvA throughout the growth cycle, whereas bacteria grown with agitation had a sharp increase in the amount of membrane InvA at late exponential phase. Levels of InvA present in Salmonella membranes under some growth conditions do not appear to correlate with levels of invasion under the same conditions.