Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1-dependent and - independent mechanisms

Cell adhesion and migration are complex processes that require integrin activation, the formation and dissolution of focal adhesion (FAs), and linkage of actin cytoskeleton to the FAs. The IPP (ILK, PINCH, Parvin) complex regulates FA formation via binding of the adaptor protein ILK to β1 integrin, PINCH and parvin. The signaling protein Rsu1 is linked to the complex via binding PINCH1. The role of Rsu1 and PINCH1 in adhesion and migration was examined in non-transformed mammary epithelial cells. Confocal microscopy revealed that the depletion of either Rsu1 or PINCH1 by siRNA in MCF10A cells decreased the number of focal adhesions and altered the distribution and localization of β1 integrin, vinculin, talin and paxillin without affecting the levels of FA protein expression. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. In addition, constitutive phosphorylation of actin regulatory proteins occurred in the absence of PINCH1. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function by regulating levels of PINCH1. However, while both Rsu1- or PINCH1-depleted cells retained the ability to activate adhesion signaling in response to EGF stimulation, only Rsu1 was required for EGF-induced p38 Map Kinase phosphorylation and ATF2 activation, suggesting an Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with an Rsu1 mutant that does not bind to PINCH1 failed to restore FAs or migration but did promote spreading and constitutive p38 activation. These data show that Rsu1-PINCH1 association with ILK and the IPP complex is required for regulation of adhesion and migration but that Rsu1 has a critical role in linking integrin-induced adhesion to activation of p38 Map kinase signaling and cell spreading. Moreover, it suggests that Rsu1 may regulate p38 signaling from the IPP complex affecting other functions including survival.     

Tracking the Primary Sources of Fecal Pollution in a Tropical Watershed in a One-Year Study

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.     

Cestodes of cestodes of Peruvian freshwater stingrays

Adult tetraphyllidean tapeworms (Platyhelminthes: Eucestoda) from the spiral intestines of 3 species of potamotrygonid stingrays (Paratrygon aiereba, Potamotrygon castexi, and Portamotrygon motoro) in the Madre de Dios river in Peru were found to host numerous cysts embedded in their parenchymal tissues. Histological sections of the cysts revealed the presence of a scolex bearing 4 suckers and an unarmed apical organ consistent with larval stages of both Cyclophyllidea and Proteocephalidea. To further elucidate their identities, partial 28S ribosomal DNA (rDNA) sequences were characterized from 3 cysts and 4 adult Rhinebothrium spp. 'host' worms and screened against all available cestode 28S rDNA data. Initial BLAST screening and subsequent alignment ruled out the possibility that the cysts were cyclophyllidean, and the cyst and adult sequences were thus aligned together with all available lecanicephalidean, litobothriidean, proteocephalidean, and tetraphyllidean sequences. Sequences from all 3 cysts were identical, and phylogenetic analysis clearly placed them among derived members of the Proteocephalidea, although no exact match was found. Sequences from the adult host worms formed 2 identical pairs and grouped together with other tetraphyllidean species from rays. These results are compared with records of hyperparasites of South American catfish cestodes. This is the first confirmed record of a proteocephalidean cestode parasitizing a tetraphyllidean cestode.     

Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling

The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α−parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.     

Diversification and species boundaries of Rhinebothrium (Cestoda; Rhinebothriidea) in South American freshwater stingrays (Batoidea; Potamotrygonidae)

Background

Neotropical freshwater stingrays (Batoidea: Potamotrygonidae) host a diverse parasite fauna, including cestodes. Both cestodes and their stingray hosts are marine-derived, but the taxonomy of this host/parasite system is poorly understood.

Methodology

Morphological and molecular (Cytochrome oxidase I) data were used to investigate diversity in freshwater lineages of the cestode genus Rhinebothrium Linton, 1890. Results were based on a phylogenetic hypothesis for 74 COI sequences and morphological analysis of over 400 specimens. Cestodes studied were obtained from 888 individual potamotrygonids, representing 14 recognized and 18 potentially undescribed species from most river systems of South America.

Results

Morphological species boundaries were based mainly on microthrix characters observed with scanning electron microscopy, and were supported by COI data. Four species were recognized, including two redescribed (Rhinebothrium copianullum and R. paratrygoni), and two newly described (R. brooksi n. sp. and R. fulbrighti n. sp.). Rhinebothrium paranaensis Menoret & Ivanov, 2009 is considered a junior synonym of R. paratrygoni because the morphological features of the two species overlap substantially. The diagnosis of Rhinebothrium Linton, 1890 is emended to accommodate the presence of marginal longitudinal septa observed in R. copianullum and R. brooksi n. sp. Patterns of host specificity and distribution ranged from use of few host species in few river basins, to use of as many as eight host species in multiple river basins.

Significance

The level of intra-specific morphological variation observed in features such as total length and number of proglottids is unparalleled among other elasmobranch cestodes. This is attributed to the large representation of host and biogeographical samples. It is unclear whether the intra-specific morphological variation observed is unique to this freshwater system. Nonetheless, caution is urged when using morphological discontinuities to delimit elasmobranch cestode species because the amount of variation encountered is highly dependent on sample size and/or biogeographical representation.     

A new genus and species of cestode (Rhinebothriidea) from Mobula kuhlii (Rajiformes: Mobulidae) from Malaysian Borneo

The cestode fauna of the shortfin devilray, Mobula kuhlii (Müller & Henle, 1841) was examined for the first time. The work resulted in the discovery of a new genus and species of rhinebothriidean tapeworm. Crassuseptum pietrafacei, n. gen. n. sp. is erected here on the basis of its unique scolex and proglottid morphology. Histological sections and examination by light and scanning electron microscopy demonstrated that this new genus differs from all other rhinebothriidean genera in its possession of bothridia in which the proximal and distal sides are confluent, i.e., not separated by a rim of tissue, and in its possession of testes that extend to the posterior margin of the ovary. This new species is characterized in part by its possession of stalked, elongate bothridia lacking lateral constrictions, with 13-15 prominent transverse bothridial septa and 4 reduced transverse septa, craspedote proglottids, each with 2-3 layers of testes in cross section, and a vas deferens that joins the cirrus sac at its anterior margin. Histological and optical sections through bothridial septa revealed that the transverse septa are formed by septal muscles separate from bothridial radial musculature, extending from the anterior side to the posterior side of each septum. This is only the second species rhinebothriidean cestode reported from mobulids. This study adds to the number of new species and genera of elasmobranch cestodes discovered off of the island of Borneo.     

A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter.     

The Proteocephalus species-aggregate (Cestoda) in cyprinoids,pike, eel,smelt and cavefish of the Nearctic region (North America): diversity,host associations and distribution

Systematic Parasitology - Tapeworms (Cestoda) of the Proteocephalus-species aggregate from cyprinoids, pike, eel, smelt and cavefish in the Nearctic region (North America) are reviewed, based on a...     

Intestinal helminths of freshwater stingrays in southeastern Peru, and a new genus and two new species of cestode

Helminths of the spiral intestine of neotropical freshwater stingrays (Potamotrygonidae) were examined in Peru for the first time. The stingrays examined for helminths included Paratrygon aiereba, Potamotrygon motoro, and Potamotrygon cf. castexi. Present in P. aiereba were the cestodes Nandocestus guariticus (Marques, Brooks, and Lasso, 2001) n. gen. n. comb., Rhinebothrium copianullum n. sp., Rhinebothrium sp. 1, Rhinebothroides sp., Potamotrygonocestus cf. fitzgeraldae, and 1 species each of Cucullanus and Rhabdochona. Nandocestus n. gen. is erected to house N. guariticus, which is formally transferred from Anindobothrium Marques, Brooks, and Lasso, 2001. The new genus is unique among phyllobothriids in its possession of circummedullary vitelline follicles and a submarginal genital pore, in combination with bothridia with a single apical sucker and marginal bothridial loculi. The helminths of P. motoro included the cestodes Paraoncomegas araya, Acanthobothrium peruviense n. sp., Acanthobothrium cf. ramiroi, Rhinebothrium sp. 1, Rhinebothroides sp. 1, Potamotrygonocestus sp., the nematode Brevimulticaecum regoi, a species of Cucullanus, and a species of the digenean superfamily Diplostomoidea. The helminths of P. cf. castexi included the cestodes P. araya, N. guariticus n. gen. n. comb., Acanthobothrium cf. peruviense, Potamotrygonocestus sp., Rhinebothrium sp. 1, Rhinebothroides sp. 2, the nematode species Echinocephalus daileyi and B. regoi, 1 species each of Cucullanus, Rhabdochona, and Procamallanus, and a species of the digenean superfamily Hemiuroidea. All taxa were examined via light microscopy; the cestode taxa were also examined using scanning electron microscopy. Each helminth species recorded in this study is a first report from Peru. The study suggests that the diversity and host specificity of the cestodes in potamotrygonid stingrays may be greater than previously thought. The known numbers of genera and species of tetraphyllidean cestodes parasitizing neotropical freshwater stingrays are now 6 and 22, respectively.     

A comparison of biological performances among a laboratory-isolated population and two wild populations of Moniliformis moniliformis

Divergence of biological performance of a laboratory-reared population of Moniliformis moniliformis (Acanthocephala) was investigated after 31 yr, or approximately 60 generations, of genetic isolation. An isolate of the laboratory-reared population and isolates of 2 wild populations were used to begin 3 independent life cycles that were maintained for 1 generation for interbreeding and life history trait comparison. Both wild population isolates represented populations with open gene flow. One wild population isolate represented a present-day sample of descendants of the parent population of the laboratory isolate. All 3 populations hybridized, and egg production occurred in all mixed-sex pairs of different populations. The 3 populations did not differ significantly in prepatent period, mean daily egg production, or establishment within the definitive host Rattus norvegicus. The 3 populations varied in patent period, but the laboratory-isolated worms differed from the 2 wild population isolates no more than they did from each other. A positive correlation between mean daily egg production and duration of patent period resulted in different cumulative egg productions. A 31-yr period of isolation did not produce greater divergence in a laboratory population of M. moniliformis than occurs between wild populations with respect to the biological parameters measured.