Fasciolosis in the Mediterranean island of Corsica (France): Insights from epidemiological and malacological investigations.

Fasciolosis is a re-emergent parasitic disease of worldwide significance with a major global impact on livestock health and production. In the French Mediterranean island of Corsica, fasciolosis has been recognized for a long time but little is known about its dynamic as the main investigations are outdated. Three compartments - definitive domestic hosts, intermediate hosts and environment - involved in fasciolosis transmission were studied by applying an integrative and extensive approach: (1) farm and abattoir surveys, (2) snail sampling, identification and infection prospection, and (3) snail habitat analysis; and (4) a questionnaire-based survey to inquire about husbandry practices and environmental risks. Our results indicate a significant circulation of the liver flukes in Corsican livestock, with 90% (252/279) of the sampled farms testing positive for anti-F. hepatica antibodies. At the abattoir, 46% (67/149) of cattle were positive for F. hepatica antibodies and eggs were present in the bile of 19% (26/139) bovines. In addition, high prevalence of Dicrocoelium dendriticum (69%) was observed in slaughtered cattle. Malacological surveys registered the occurrence of several lymnaeid species in a variety of habitats throughout the island. In particular, we report for the first time the presence of the invasive lymnaeid snail Pseudosuccinea columella in Corsica, a potential intermediate host for F. hepatica. We also found that the presence of Galba truncatula and, to a lesser extent, that of Peregriana peregra, is associated with altitude. Fasciola hepatica DNA was detected in the latter species occurring at two different sites. Finally, a questionnaire-based study revealed risky management practices among Corsican farmers, low perception of transmission and a suboptimal use of flukicide treatments as main control strategy. Our results show that animal fasciolosis in Corsica is characterised by a significant circulation and a favourable epidemiological scenario for transmission to occur.

Resistance of Cabbage Loopers to Bacillus thuringiensis (Bt) Toxin Cry1F and to Dual-Bt Toxin WideStrike Cotton Plants.

The Cry proteins from Bacillus thuringiensis (Bt) are major insecticidal toxins in formulated Bt sprays and are expressed in genetically engineered Bt crops for insect pest control. However, the widespread application of Bt toxins in the field imposes strong selection pressure on target insects, leading to the evolution of insect resistance to the Bt toxins. Identification and understanding of mechanisms of insect resistance to Bt toxins are an important approach for dissecting the modes of action of Bt toxins and providing knowledge necessary for the development of resistance management technologies. In this study, cabbage looper (Trichoplusia ni) strains resistant to the transgenic dual-Bt toxin WideStrike cotton plants, which express Bt toxins Cry1Ac and Cry1F, were selected from T. ni strains resistant to the Bt formulation Bt-DiPel. The WideStrike-resistant T. ni larvae were confirmed to be resistant to both Bt toxins Cry1Ac and Cry1F. From the WideStrike-resistant T. ni, the Cry1F resistance trait was further isolated to establish a T. ni strain resistant to Cry1F only. The levels of Cry1F resistance in the WideStrike-resistant and the Cry1F-resistant strains were determined, and the inheritance of the Cry1F-resistant trait in the two strains was characterized. Genetic association analysis of the Cry1F resistance trait indicated that the Cry1F resistance in T. ni isolated in this study is not shared with the Cry1Ac resistance mechanism nor is it associated with a mutation in the ABCC2 gene, as has so far been reported in Cry1F-resistant insects. IMPORTANCE Insecticidal toxins from Bacillus thuringiensis (Bt) are highly effective for insect control in agriculture. However, the widespread application of Bt toxins exerts strong selection for Bt resistance in insect populations. The continuing success of Bt biotechnology for pest control requires the identification of resistance and understanding of the mechanisms of resistance to Bt toxins. Cry1F is an important Bt toxin used in transgenic cotton, maize, and soybean varieties adopted widely for insect control. To understand the mode of action of Cry1F and mechanisms of Cry1F resistance in insects, it is important to identify Cry1F-specific resistance and the resistance mechanisms. In this study, Trichoplusia ni strains resistant to commercial "WideStrike" cotton plants that express Bt toxins Cry1Ac and Cry1F were selected, and a Cry1F-specific resistant strain was isolated. The isolation of the novel Cry1F-specific resistance in the T. ni provided an invaluable biological system to discover a Cry1F-specific novel resistance mechanism.

Prevalence and risk factors associated with urogenital schistosomiasis among primary school pupils in Nigeria.

Urogenital schistosomiasis is a neglected tropical disease that is endemic to Nigeria and one which continues to pose a public health problem especially among school-age children in rural communities. This study was carried out in remote areas where most people depend on natural water bodies and rainwater for their daily water needs. The present research investigates the prevalence of urogenital schistosomiasis and the significant risk factors associated with the infection among primary school children in Nigeria. From August 2019 to December 2019, a total of 5514 primary school-age children from twelve sites were diagnosed with the presence of Schistosoma haematobium eggs in their urine. Socio-demographic, sociocultural, and socioeconomic indices and data on behaviors (e.g contact frequency with freshwater bodies) were also collected for each diagnosed individual through the use of a questionnaire. Associations between each of these variables and disease infection were tested using a multivariate logistic regression. A total of 392 of the 5514-urine samples were positive for the infection, the overall prevalence reached 7.1% and ranged from 4.6% (East Nigeria) to 15,9% (West Nigeria). Multivariate logistic regression analyses showed that the significant risk factors associated with S. haematobium infection are frequent contact with freshwater bodies (rivers/steams), with an adjusted odds ratio (AOR) of 4.92; 3.34-7.24, washing/swimming, AOR: 46.49; 27.64-78.19, and fishing, AOR: 11.57; 8.74-15.32. For socioeconomic factors, primary education of fathers which resulted in an AOR of 1.63; 1.01-2.45 was significantly associated with the infection. The socio-demographic factor for the 12-14 year age group had an AOR of 1.68; 1.21-2.33, and was also significantly associated with the disease. Nigeria remains endemic for urogenital schistosomiasis as indicated by the data obtained from all the studied sites, and it is clear that efforts need to be intensified in order to control and eradicate the disease throughout the country.

Bt Cry1Ac resistance in Trichoplusia ni is conferred by multi-gene mutations.

The three-domain Cry toxin Cry1Ac from Bacillus thuringiensis （Bt） is an important insecticidal toxin in Bt sprays and has been used in transgenic Bt-crops to confer insect resistance. The cabbage looper, Trichoplusia ni, has developed resistance to Bt sprays in commercial greenhouses, and the resistance to Cry1Ac has been previously identified to be associated with altered expression of the APN1 and APN6 genes and be genetically linked to a locus on chromosome 15. In this study, the Cry1Ac resistance locus in T. ni was further finely mapped, and the specific Cry1Ac resistance-conferring mutation in the resistance locus was identified to be a 4 bp frameshift insertion in the ABCC2 gene by whole genome resequencing, midgut transcriptome analysis, candidate gene cDNA sequencing and mutation site genomic DNA sequencing. By CRISPR/Cas9 mutagenesis, a series of ABCC2 and ABCC3 mutant T. ni strains were generated, and the role of ABCC2 in the toxicity of Cry1Ac in T. ni was confirmed. The results from this study also showed that knockout of ABCC2 in T. ni conferred resistance to Cry1Ac at a level lower than that in the greenhouse-derived resistant T. ni strain and that the Cry1Ac resistance-associated alteration of APN1 and APN6 expression was independent of ABCC2 gene mutations, indicating that the altered expression of APN1 and APN6 was controlled by another gene mutation in Cry1Ac resistant T. ni. Furthermore, T. ni larval bioassays showed that the level of Cry1Ac resistance in F1 families from reciprocal crosses of the Cry1Ac resistant strain with an ABCC2 knockout CRISPR strain was significantly higher than that in ABCC2 knockout strain, indicating the presence of additional Cry1Ac resistance-conferring mutation(s) in the Cry1Ac resistant strain. Therefore, the resistance to Cry1Ac in T. ni is conferred by a mutation in ABCC2 and an additional mutation (or mutations) which leads to altered expression of APN1 and APN6. The additional Cry1Ac resistance mutation or mutations remain to be identified.

Near-Complete Genome Sequence of a SARS-CoV-2 VOC 202012/01 Strain in Peru.

A near-complete genome sequence was obtained for a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) 202012/01 strain obtained from an oropharyngeal swab sample from a Peruvian patient with coronavirus syndrome who had contact with an individual who had recently returned from England.

Population genetics of African Schistosoma species.

Blood flukes within the genus Schistosoma (schistosomes) are responsible for the major disease, schistosomiasis, in tropical and sub-tropical areas. This disease is predominantly present on the African continent with more than 85% of the human cases. Schistosomes are also parasites of veterinary importance infecting livestock and wildlife. Schistosoma population genetic structure and diversity are important characteristics that may reflect variations in selection pressures such as those induced by host (mammalian and snail) environments, habitat change, migration and also treatment/control interventions, all of which also shape speciation and evolution of the whole Schistosoma genus. Investigations into schistosome population genetic structure, diversity and evolution has been an area of important debate and research. Supported by advances in molecular techniques with capabilities for multi-locus genetic analyses for single larvae schistosome genetic investigations have greatly progressed in the last decade. This paper aims to review the genetic studies of both animal and human infecting schistosome. Population genetic structures are reviewed at different spatial scales: local, regional or continental (i.e. phylogeography). Within species genetic diversities are discussed compared and the compounding factors discussed, including the effect of mass drug administration. Finally, the ability for intra-species hybridisation questions species integrities and poses many questions in relation to the natural epidemiology of co-endemic species. Here we review molecularly confirmed hybridisation events (in relation to human disease) and discuss the possible impact for ongoing and future control and elimination.

Mutation of ABC transporter ABCA2 confers resistance to Bt toxin Cry2Ab in Trichoplusia ni.

Insecticidal proteins from Bacillus thuringiensis (Bt) are the primary recombinant proteins expressed in transgenic crops (Bt-crops) to confer insect resistance. Development of resistance to Bt toxins in insect populations threatens the sustainable application of Bt-crops in agriculture. The Bt toxin Cry2Ab is a major insecticidal protein used in current Bt-crops, and resistance to Cry2Ab has been selected in several insects, including the cabbage looper, Trichoplusia ni. In this study, the Cry2Ab resistance gene in T. ni was mapped to Chromosome 17 by genetic linkage analyses using a whole genome resequencing approach, and was then finely mapped using RNA-seq-based bulked segregant analysis (BSA) and amplicon sequencing (AmpSeq)-based fine linkage mapping to a locus containing two genes, ABCA1 and ABCA2. Mutations in ABCA1 and ABCA2 in Cry2Ab resistant T. ni were identified by both genomic DNA and cDNA sequencing. Analysis of the expression of ABCA1 and ABCA2 in T. ni larvae indicated that ABCA2 is abundantly expressed in the larval midgut, but ABCA1 is not a midgut-expressed gene. The mutation in ABCA2 in Cry2Ab resistant T. ni was identified to be an insertion of a transposon Tntransib in ABCA2. For confirmation of ABCA2 as the Cry2Ab-resistance gene, T. ni mutants with frameshift mutations in ABCA1 and ABCA2 were generated by CRISPR/Cas9 mutagenesis. Bioassays of the T. ni mutants with Cry2Ab verified that the mutations of ABCA1 did not change larval susceptibility to Cry2Ab, but the ABCA2 mutants were highly resistant to Cry2Ab. Genetic complementation test of the ABCA2 allele in Cry2Ab resistant T. ni with an ABCA2 mutant generated by CRISPR/Cas9 confirmed that the ABCA2 mutation in the Cry2Ab resistant strain confers the resistance. The results from this study confirmed that ABCA2 is essential for the toxicity of Cry2Ab in T. ni and mutation of ABCA2 confers the resistance to Cry2Ab in the resistant T. ni strain derived from a Bt resistant greenhouse population.

Evolutionary history of the little fire ant Wasmannia auropunctata before global invasion: inferring dispersal patterns, niche requirements and past and present distribution within its native range.

The evolutionary history of invasive species within their native range may involve key processes that allow them to colonize new habitats. Therefore, phylogeographic studies of invasive species within their native ranges are useful to understand invasion biology in an evolutionary context. Here we integrated classical and Bayesian phylogeographic methods using mitochondrial and nuclear DNA markers with a palaeodistribution modelling approach, to infer the phylogeographic history of the invasive ant Wasmannia auropunctata across its native distribution in South America. We discuss our results in the context of the recent establishment of this mostly tropical species in the Mediterranean region. Our Bayesian phylogeographic analysis suggests that the common ancestor of the two main clades of W. auropunctata occurred in central Brazil during the Pliocene. Clade A would have differentiated northward and clade B southward, followed by a secondary contact beginning about 380,000 years ago in central South America. There were differences in the most suitable habitats among clades when considering three distinct climatic periods, suggesting that genetic differentiation was accompanied by changes in niche requirements, clade A being a tropical lineage and clade B a subtropical and temperate lineage. Only clade B reached more southern latitudes, with a colder climate than that of northern South America. This is concordant with the adaptation of this originally tropical ant species to temperate climates prior to its successful establishment in the Mediterranean region. This study highlights the usefulness of exploring the evolutionary history of invasive species within their native ranges to better understand biological invasions.

Thelytokous parthenogenesis, male clonality and genetic caste determination in the little fire ant: new evidence and insights from the lab.

Previous studies indicate that some populations of the little fire ant, Wasmannia auropunctata, display an unusual reproduction system polymorphism. Although some populations have a classical haplodiploid reproduction system, in other populations queens are produced by thelytokous parthenogenesis, males are produced by a male clonality system and workers are produced sexually. An atypical genetic caste determination system was also suggested. However, these conclusions were indirectly inferred from genetic studies on field population samples. Here we set up experimental laboratory nests that allow the control of the parental relationships between individuals. The queens heading those nests originated from either putatively clonal or sexual populations. We characterized the male, queen and worker offspring they produced at 12 microsatellite loci. Our results unambiguously confirm the unique reproduction system polymorphism mentioned above and that male clonality is strictly associated with thelytokous parthenogenesis. We also observed direct evidence of the rare production of sexual gynes and arrhenotokous males in clonal populations. Finally, we obtained evidence of a genetic basis for caste determination. The evolutionary significance of the reproduction system polymorphism and genetic caste determination as well as future research opportunities are discussed.

La Ecografia de Alta Resolucion y Doppler Color en la Patologia Nodular Tiroidea. Su Correlacion con la Anatomia Patologica / The Ultrasonography of high resolution and color Doppler in the Nodular Pathology Thyroid-correlato pathological anatomo

Objetivos:Los nodulos tienen una alta prevalencia en la poblacion general, especialmente en las mujeres y se incrementan con la edad. Para evaluar si la ecografia de alta resolucion y el Doppler color permiten diferenciar nodulos benignos de malignos, y dado que la conducta ante un nodulo menor de 1cm o no palpable, es motivo de controversias, relacionamos la ecografia de alta resolucion con la Anatomia Patologica (AP) de los pacientes operados. Metodologia:Se analizaron las ecografias de 83 pacientes operados por nodulo de tiroides:72 mujeres(m) y 11 varones(v), de ente 10 y 78 años de edad.Las mismas fueron realizadas con Ecografo ATL HDI 3500con Doppler color, Angiopower y Trnasductador LIneal 12/5 38 MHZ, tomandose 5 criterios sospechosos de malignidad:Hipoecogenicidad, Flujo Intranodular, Microcalcificaciones, Contornos Irregulares y Ausencia de Halo de Seguridad. Se las correlaciono con la AP, clasificandolas en tres grupos:Grupo I:Carcinoma (Ca), GrupoII:Adenoma Folicular(AF), y Grupo III:Bocio Multinodular(BMN)-Nodulo Hiperplasico (NH). Resultados: Grupo I: 37 pacientes: 44,5 por ciento ((33 Ca papilar: 9 v y 24 m; 4 de ellos variante folicular; 2 m Ca folicular; 1 m Ca medular y 1 m Ca indiferenciado). El 94,6 por ciento tenia entre 3 a 5 cirterios de malignidad : 94,6 por ciento Hipoecogenicos, 89,2 por ciento Hipervascularizados, 54 por ciento Calcificaciones finas, 62,1 por ciento Bordes irregulares y 67,5 por ciento Ausencia de Halo de Seguridad. Se detectaron 7 pac. con microcarcinoma papilar (ndulo <1cm:18,9 por ciento) 4 con MTS ganglionar y tenian 3 a 5 patrones de alarma.; 17 multinodulares (MN); 8 Ca multicentricos y 4 en nodulo no dominante(teniendo estos mayor grado de sospecha ecografica): 2 pac. se encontraban hipotiroideos y 6 con anticuerpo antiperoxidasa tiroidea (ATPO) (-). GrupoIIII:22 pac. (20m y 2v): 26,5 por ciento. El 27,2 por ciento con 3 a 5 criterios; 59 por ciento Hipoecogenicos, 54,5 por ciento Hipervascularizados, 13,6 por ciento Calcificaciones finas, 31,8 por ciento Bordes irregulares y 9 por ciento Ausencia de Halo; 14 MN, 2 pac hipotiroideos y 4 ATPO(+). Conclusiones: No existe ningun patron ecografico patognomonico de enfermedad nodular maligna, pero si el nodulo reune 3 o mas patrones de sospecha, la incidencia de malignidad aumenta de manera significativa. Aconsejamos...

La Ecografia de Alta Resolucion y Doppler Color en la Patologia Nodular Tiroidea. Su Correlacion con la Anatomia Patologica / The Ultrasonography of high resolution and color Doppler in the Nodular Pathology Thyroid-correlato pathological anatomo

Objetivos:Los nodulos tienen una alta prevalencia en la poblacion general, especialmente en las mujeres y se incrementan con la edad. Para evaluar si la ecografia de alta resolucion y el Doppler color permiten diferenciar nodulos benignos de malignos, y dado que la conducta ante un nodulo menor de 1cm o no palpable, es motivo de controversias, relacionamos la ecografia de alta resolucion con la Anatomia Patologica (AP) de los pacientes operados. Metodologia:Se analizaron las ecografias de 83 pacientes operados por nodulo de tiroides:72 mujeres(m) y 11 varones(v), de ente 10 y 78 años de edad.Las mismas fueron realizadas con Ecografo ATL HDI 3500con Doppler color, Angiopower y Trnasductador LIneal 12/5 38 MHZ, tomandose 5 criterios sospechosos de malignidad:Hipoecogenicidad, Flujo Intranodular, Microcalcificaciones, Contornos Irregulares y Ausencia de Halo de Seguridad. Se las correlaciono con la AP, clasificandolas en tres grupos:Grupo I:Carcinoma (Ca), GrupoII:Adenoma Folicular(AF), y Grupo III:Bocio Multinodular(BMN)-Nodulo Hiperplasico (NH). Resultados: Grupo I: 37 pacientes: 44,5 por ciento ((33 Ca papilar: 9 v y 24 m; 4 de ellos variante folicular; 2 m Ca folicular; 1 m Ca medular y 1 m Ca indiferenciado). El 94,6 por ciento tenia entre 3 a 5 cirterios de malignidad : 94,6 por ciento Hipoecogenicos, 89,2 por ciento Hipervascularizados, 54 por ciento Calcificaciones finas, 62,1 por ciento Bordes irregulares y 67,5 por ciento Ausencia de Halo de Seguridad. Se detectaron 7 pac. con microcarcinoma papilar (ndulo <1cm:18,9 por ciento) 4 con MTS ganglionar y tenian 3 a 5 patrones de alarma.; 17 multinodulares (MN); 8 Ca multicentricos y 4 en nodulo no dominante(teniendo estos mayor grado de sospecha ecografica): 2 pac. se encontraban hipotiroideos y 6 con anticuerpo antiperoxidasa tiroidea (ATPO) (-). GrupoIIII:22 pac. (20m y 2v): 26,5 por ciento. El 27,2 por ciento con 3 a 5 criterios; 59 por ciento Hipoecogenicos, 54,5 por ciento Hipervascularizados, 13,6 por ciento Calcificaciones finas, 31,8 por ciento Bordes irregulares y 9 por ciento Ausencia de Halo; 14 MN, 2 pac hipotiroideos y 4 ATPO(+). Conclusiones: No existe ningun patron ecografico patognomonico de enfermedad nodular maligna, pero si el nodulo reune 3 o mas patrones de sospecha, la incidencia de malignidad aumenta de manera significativa. Aconsejamos...(AU)

HIV type 1 Gag and nucleocapsid proteins: cytoskeletal localization and effects on cell motility.

Cell motility is likely to play a pivotal role in HIV infection by promoting the dissemination of infected cells. On the basis of observations indicating an interaction between HIV-1 Gag and target cell filamentous actin, we hypothesized that these interactions would promote cell motility of HIV-infected cells. Indeed, we have found that HIV-1 infection enhances the chemotactic response of macrophages. To specifically investigate the significance of the interactions between Gag and cellular actin, we transfected NIH 3T3 fibroblasts and HeLa cells with a construct that permits the expression of HIV-1 Gag in the absence of any other viral protein. Fractionation experiments showed that Gag was present in cytoskeletal fraction containing long actin filaments and in a high-speed postcytoskeletal fraction with short actin filaments. We have also localized HIV-1 Gag to the lamellipodia of chemoattractant-stimulated cells. Significantly, the motility of Gag-expressing cells was enhanced in chemotaxis assays. In vitro mutagenesis experiments showed that HIV-1 Gag binds filamentous actin through the nucleocapsid domain (NC). An NC-green fluorescent protein fusion had the same cellular distribution as the complete protein, and its expression increased cell motility. These data suggest that interactions between HIV-1 Gag and actin in infected cells enhance cell motility. Ultimately this enhanced motility of infected cells could promote the dissemination of virus into the brain and other tissues.

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation.

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.

Protein kinase D interacts with Golgi via its cysteine-rich domain.

Protein kinase D (PKD)/protein kinase Cmu is a serine/threonine protein kinase that has been localized in the cytosol and in several intracellular compartments including Golgi, mitochondria and plasma membrane. Using real time imaging of fluorescent protein (GFP)-tagged PKD, we have found that the accumulation of PKD in the Golgi compartment, following a temperature shift from 37 to 20 degrees C, was mediated by the cysteine-rich domain (CRD) of PKD. The CRD of PKD also mediates its interaction with the plasma membrane, further supporting the conclusion that the CRD of PKD may act as a subcellular localization signal.

Smoking in Colombian medical schools: the hidden curriculum.

BACKGROUND: Tobacco companies are focusing their interest in less developed countries. In the absence of governmental opposition, physicians are expected to lead tobacco control efforts. We studied Colombian medical students' smoking prevalence and tobacco attitudes. METHODS: First- and fifth-year students from 11 medical schools in seven Colombian cities answered anonymous, self-administered, 38-item questionnaires. Additionally, smokers answered the Fagerström Test for Nicotine Dependence (FTND). RESULTS: Two thousand twenty-one students (males 50.6%; age 15-44, median 19) completed the survey; average response rate was 89.9%. Globally 25.9% of students were current smokers (males 27.9%, females 24.0%). Living at higher altitude and attending private universities were associated with higher prevalence (P < 0.001). Males had a higher chance of having given up smoking (P < 0.05); 91.3% of current smokers would like to quit; 67.3% of all smokers and 44.8% of daily smokers scored 0 in the FTND. Prevalence was similar among first- and fifth-years, but fifth-year students were more complacent with smoking in health centers and showed a lesser desire to quit. CONCLUSIONS: Medical students' smoking prevalence is similar to that of the general population. Tobacco control strategies need to be included in the curriculum. Nicotine addiction does not seem to be the main perpetuating factor.

Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) mu) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.

In vitro replication and differentiation of normal human oral keratinocytes.

The replication kinetics and cytological changes of normal human oral keratinocytes (NHOK) isolated from the basal surface of oral epithelial sheet and cultured as dispersed cells in low (0.15 mM) Ca(2+) medium without serum were analyzed. Replicating NHOK were quantitated by cell count and identified by [(3)H]thymidine uptake. Cell morphology was analyzed by phase contrast and transmission electron microscopy, and by cytochemical staining for endogenous beta-galactosidase (beta-gal) activity, involucrin, and cytokeratin types 1 and 10 (K1/K10). Primary NHOK obtained from 15 different donors whose ages ranged from 21 to 62 years consistently showed three distinct phases of replication, i.e., exponential, senescing, and senescent, which were independent of the donors' age. Initially, the cells replicated exponentially for a period of 20 days with a doubling time of 26.6 +/- 3.5 h. They then gradually entered replication arrest over a period of 18 days. The cells underwent a maximum of 22.1 +/- 2.8 population doublings. The onset of gradual replication arrest coincided with an increase in the fraction of cells, which stopped DNA synthesis within a maximum of 48 h and which stained for beta-gal. The fraction of terminally differentiated cells stained for K1/K10 did not increase until nearly all the cells had stopped replicating (senescent phase) and maximal beta-gal staining had been reached. Subsequently, the percentage of beta-gal stained cells actually decreased while the percentage of those stained for K1/K10 increased to a maximum of 80-90% within 2-3 weeks. Exposure of exponentially replicating NHOK to 5-aza-2'-deoxycytidine (5-aza CdR) inhibited DNA replication within 18-48 h and induced terminal differentiation 6 days later. In contrast, exposure of these cells to 1.5 mM Ca(2+) induced expression of involucrin and K1/K10 within 48 h without inhibiting DNA synthesis. Thus, replication arrest preceded differentiation in NHOK serially subcultured in vitro; however, differentiation could be induced without replication arrest.

Human papillomavirus type 16 E7 oncoprotein represses transcription of human fibronectin.

The E7 oncoprotein encoded by human papillomavirus (HPV) type 16 repressed the transcription of fibronectin, a key component of the extracellular matrix. This repression, detected in several HPV-positive nontumorigenic and tumorigenic cell lines, was abolished when the Cys-X-X-Cys repeats in E7 were disrupted.

[The Mini-Mental State Examination as a selected diagnostic test for dementia: a Colombian population study. GENECO]. / El examen mental abreviado (Mini-Mental State Examination) como prueba de selección para el diagnóstico de demencia: estudio poblacional colombiano. GENECO.

INTRODUCTION: Folstein's Mini Mental State Examination (MMSE) is widely used as screening test for cognitive impairment. OBJECTIVE: To test a Spanish version of the MMSE in a population of high illiteracy rate. MATERIAL AND METHODS: Population-based survey of a stratified random sample of urban and rural residents of five regions of Colombia, followed by neurological and neuropsychological evaluation of suspect cases (phase 2). Dementia was diagnosed using DSM-IV criteria. RESULTS: 1,611 subjects age 50 or older filled out both the WHO Protocol for Epidemiologic Studies of Neurological Disorders and a Spanish version of the MMSE; 55.2% of them had three or less years of schooling; 536 individuals with scores below cutoff points were sent to phase 2. Of the population with satisfactory scores in MMSE 366 (34.0%) were evaluated by neurologists to exclude other neurological conditions. Twelve cases of dementia were diagnosed among individuals with scores below cutoff point and one among subjects with high scores. Age-adjusted prevalence was 8.1 per thousand subjects age 50 or over (95% CI: 3.7-12.5); and 34.2 per thousand for ages 75 or over (95% CI: 12.2-56.2). Sensitivity and specificity were 92.3 and 53.7%; 16 of the 19 questions show significant differences (p < 0.001) according to educational level. A gender gap is significant in low educational levels (p < 0.001) but not in subjects with more than five years of schooling. CONCLUSIONS: MMSE scores correlated closely with level of education. Low specificity leads to many non-demented subjects with low educational status requiring further investigation.