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.

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.

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.

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.

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.

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.

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.

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.

Impaired nucleotide excision repair in UV-irradiated human oral keratinocytes immortalized with type 16 human papillomavirus genome.

We previously reported that 'high risk' human papillomaviruses (HPV) induce genetic instability in human oral keratinocytes. To understand the mechanisms of HPV-induced genetic instability, we determined the nucleotide excision repair (NER) capacity of normal (NHOK) and human papillomavirus type-16 immortalized oral keratinocytes (HOK-16B) by strand-specific removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from a 16 Kb fragment of the p53 gene. In NHOK the NER activity was initiated in both DNA strands immediately, although the process in the non-transcribed strand was notably slower than that of the transcribed strand. In HOK-16B cells the initiation of CPDs removal was delayed for at least 8 h in both DNA strands, and the process was significantly slower than that in NHOK. UV-irradiation enhanced the p53 protein level more than 30-fold in NHOK, but it did not significantly alter the protein level in the HOK-16B cells. UV-irradiation also increased the p21WAF1/CIP1 protein level only in NHOK. These data indicate that 'high risk' HPV induces genetic instability by impairing NER capacity of cells. Impaired NER activity of HOK-16B cells may be implicated with their inability to enhance active p53 when challenged by genotoxic stress.

Differential gene expression in neoplastic and human papillomavirus-immortalized oral keratinocytes.

We have previously demonstrated that normal human oral keratinocytes immortalized by transfection with human papillomavirus type-16 Dna became tumorigenic after exposure to a chemical carcinogen. In an effort to detect differentially regulated genes associated with this transition from the immortal to the malignant phenotype, we employed representational differences analysis (a PCR-coupled subtractive hybridization technique). After analysing 50 colonies, 12 putative messages were identified. Northern analysis comparison using the identified cDNAs as probes was made between normal human oral keratinocyte, papillomavirus-immortalized human oral keratinocytes (HOK-16B), a neoplastic cell line derived from HOK-16B (HOK-16B-BaP-T) and the human oral cancer cell lines Hep-2, SCC-9 and Tu-177. We found that mRNAs encoding for cyclophilin A, c-myc binding protein 1, the heat shock protein 90alpha and one unknown transcript were up-regulated in the oral cancer cell lines analysed as well as in HOK-16B cells. We also detected a downregulation of the mRNAs encoding the skin-derived antileukoproteinase SKALP/elafin, the translationally regulated p23 protein and one unknown transcript. Whether these messages are associated to the neoplastic conversion of human keratinocytes remains to be determined.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Molecular structure and early events in the replication of Tacaribe arenavirus S RNA.

Tacaribe arenavirus S RNA was cloned and analysis of its nucleotide sequence revealed two open reading frames of significant size, one in the virus-sense strand, the other in the virus-complementary strand. The predicted amino acid sequences of the two reading frames were compared with the predicted primary structures of the nucleoprotein (N) and glycoprotein precursor (GPC) of LCM, Pichinde and Lassa viruses. The results indicated a high degree of homology between the proteins of similar properties. It was also found that in Tacaribe virus-infected cells a subgenomic viral-sense GPC RNA and a subgenomic viral-complementary N RNA are synthesized in addition to the full length viral (v) RNA and viral complementary (vc) RNAs. These results support the conclusion that in Tacaribe virus--as in Pichinde and lymphocytic choriomeningitis arenavirus-S RNA encodes the viral N and GPC proteins and has an 'ambisense' coding strategy. Analysis of the S-derived RNA species at early times post-infection in cells incubated with or without inhibitors of protein synthesis indicated that for primary transcription of the N mRNA, protein synthesis is not required; whereas synthesis of the vc RNA, GPC mRNA and v RNA does require protein synthesis to take place.

Quantitative analysis of the endogenous reverse transcriptase reactions of HIV type 1 variants with decreased susceptibility to azidothymidine and nevirapine.

A large number of nucleoside analog and nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) have been developed for clinical use. Data confirm that resistant variants of HIV-1 rapidly emerge in response to the selective pressure of treatment with these agents. Detection of drug resistance generally involves detection of specific mutations in the viral genome or demonstrating a failure of the drug to suppress virus replication in culture. We have developed a PCR-based method to quantitatively examine HIV-1 DNA synthesis in vitro in endogenous reverse transcription reactions and tested it as a method to detect resistance to RT inhibitors. Under certain conditions, we were able to distinguish HIV strains with high-level resistance to azidothymidine triphosphate inhibition from sensitive strains. This method was quite useful as an assay to detect resistance to nevirapine, a nonnucleoside RT inhibitor; in reconstruction experiments, nevirapine-resistant virus was detectable when it represented 10 to 25% of the total amount of virus present in reaction mixtures. These data are examined in the light of current models of the mechanisms of action of nucleoside nonnucleoside RT inhibitors. This assay may be useful for detecting the emergence of drug-resistant HIV-1 variants during therapy.

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.

[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.

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.

Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins.

We investigated the properties of ts51, an influenza virus (A/WSN/33) temperature-sensitive RNA segment 7 mutant. Nucleotide sequence analysis revealed that ts51 possesses a single nucleotide mutation, T-261----C, in RNA segment 7, resulting in a single amino acid change. Phenylalanine (position 79) in the wild-type M1 protein was substituted by serine in ts51. This mutation was phenotypically characterized by dramatic nuclear accumulation of the M1 protein and interfered with some steps at the late stage of virus replication, possibly affecting the assembly and/or budding of viral particles. However, although M1 protein was retained within the nucleus, export of the newly synthesized viral ribonucleoprotein containing the minus-strand RNA into the cytoplasm was essentially the same at both permissive and nonpermissive temperatures. The roles of M1 in the export of viral ribonucleoproteins from the nucleus into the cytoplasm and in the virus particle assembly process are discussed.

HIV-1 Gag protein associates with F-actin present in microfilaments.

Several studies have provided evidence that the cellular cytoskeleton may be involved in the assembly and budding of retroviruses. In fractionation studies of HIV-1-infected CEM cells, the majority of the unprocessed Gag polyprotein cofractionated with the cellular cytoskeleton. In vivo and in vitro analyses of this interaction indicated that the unprocessed Gag polyprotein is capable of association with polymerized actin (F-actin). Binding of Gag to F-actin may be involved in the assembly or budding of HIV-1.

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.