bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.

Iron and copper on Botrytis cinerea: new inputs in the cellular characterization of their inhibitory effect.

Certain metals play key roles in infection by the gray mold fungus, Botrytis cinerea. Among them, copper and iron are necessary for redox and catalytic activity of enzymes and metalloproteins, but at high concentrations they are toxic. Understanding the mechanism requires more cell characterization studies for developing new, targeted metal-based fungicides to control fungal diseases on food crops. This study aims to characterize the inhibitory effect of copper and iron on B. cinerea by evaluating mycelial growth, sensitivity to cell wall perturbing agents (congo red and calcofluor white), membrane integrity, adhesion, conidial germination, and virulence. Tests of copper over the range of 2 to 8 mM and iron at 2 to 20 mM revealed that the concentration capable of reducing mycelial growth by 50% (IC50) was 2.87 mM and 9.08 mM for copper and iron, respectively. When mixed at equimolar amounts there was a significant inhibitory effect mostly attributable to copper. The effect of Cu50, Fe50, and Cu50-Fe50 was also studied on the mycelial growth of three wild B. cinerea strains, which were more sensitive to metallic inhibitors. A significant inhibition of conidial germination was correlated with adhesion capacity, indicating potential usefulness in controlling disease at early stages of crop growth. Comparisons of the effects of disruptive agents on the cell wall showed that Cu, Fe, and Cu-Fe did not exert their antifungal effect on the cell wall of B. cinerea. However, a relevant effect was observed on plasma membrane integrity. The pathogenicity test confirmed that virulence was correlated with the individual presence of Cu and Fe. Our results represent an important contribution that could be used to formulate and test metal-based fungicides targeted at early prevention or control of B. cinerea.

Impaired stem cell differentiation and somatic cell reprogramming in DIDO3 mutants with altered RNA processing and increased R-loop levels.

Embryonic stem cell (ESC) differentiation and somatic cell reprogramming are biological processes governed by antagonistic expression or repression of a largely common set of genes. Accurate regulation of gene expression is thus essential for both processes, and alterations in RNA processing are predicted to negatively affect both. We show that truncation of the DIDO gene alters RNA splicing and transcription termination in ESC and mouse embryo fibroblasts (MEF), which affects genes involved in both differentiation and reprogramming. We combined transcriptomic, protein interaction, and cellular studies to identify the underlying molecular mechanism. We found that DIDO3 interacts with the helicase DHX9, which is involved in R-loop processing and transcription termination, and that DIDO3-exon16 deletion increases nuclear R-loop content and causes DNA replication stress. Overall, these defects result in failure of ESC to differentiate and of MEF to be reprogrammed. MEF immortalization restored impaired reprogramming capacity. We conclude that DIDO3 has essential functions in ESC differentiation and somatic cell reprogramming by supporting accurate RNA metabolism, with its exon16-encoded domain playing the main role.

Molecular identification and characterization of Botrytis cinerea associated to the endemic flora of semi-desert climate in Chile.

Botrytis cinerea is a phytopathogenic fungus that infects over 200 plant species and can cause significant crop losses in local and worldwide agricultural industries. However, its presence in the endemic flora in the Coquimbo Region and its impact on local flora have not been studied yet. In order to determine whether Botrytis spp is present in the native plant in the Coquimbo Region, fifty-two field-samples were analysed. A total of 30 putative Botrytis spp were isolated and phenotypic and genetically characterized. The internal transcribed spacer (ITS) analysis of these isolates revealed that it corresponded to genus Botrytis. For further confirmation, nuclear protein-coding genes (G3PDH, HSP60, and RPB2) were sequenced and showed 100% identity against B. cinerea. Complementary to this, Botrytis can also be clustered in two different groups, group I (B. pseudocinerea) and group II (B. cinerea), based on DNA polymorphism, the Botrytis isolates were identified as member of group II. On the order hand, we investigated the presence and frequency distribution of the transposable elements boty and flipper in the isolates obtained. The results indicate that 83.3% of the isolates presented both transposable elements, boty and flipper, indicating that the most prevalent genotype was transpose. In addition, 16.6% of the isolates showed substantially reduced virulence in apple fruit in comparison to B05.10 strain. According to fungicide resistance studies, the results indicate that resistance to Fenhexamid or Boscalid was observed in the 22.6% of isolates. The results show for the first time that B. cinerea has not been described before in fourteen new host plants and contributes to our fundamental understanding of the presence of B. cinerea in the native plant in the Coquimbo Region and the possible ecological impact of this disease on native and endemic plants.

Breakpoint: Cell Wall and Glycoproteins and their Crucial Role in the Phytopathogenic Fungi Infection.

The cell wall that surrounds fungal cells is essential for their survival, provides protection against physical and chemical stresses, and plays relevant roles during infection. In general, the fungal cell wall is composed of an outer layer of glycoprotein and an inner skeletal layer of ß-glucans or α- glucans and chitin. Chitin synthase genes have been shown to be important for septum formation, cell division and virulence. In the same way, chitin can act as a potent elicitor to activate defense response in several plant species; however, the fungi can convert chitin to chitosan during plant infection to evade plant defense mechanisms. Moreover, α-1,3-Glucan, a non-degradable polysaccharide in plants, represents a key feature in fungal cell walls formed in plants and plays a protective role for this fungus against plant lytic enzymes. A similar case is with ß-1,3- and ß-1,6-glucan which are essential for infection, structure rigidity and pathogenicity during fungal infection. Cell wall glycoproteins are also vital to fungi. They have been associated with conidial separation, the increase of chitin in conidial cell walls, germination, appressorium formation, as well as osmotic and cell wall stress and virulence; however, the specific roles of glycoproteins in filamentous fungi remain unknown. Fungi that can respond to environmental stimuli distinguish these signals and relay them through intracellular signaling pathways to change the cell wall composition. They play a crucial role in appressorium formation and penetration, and release cell wall degrading enzymes, which determine the outcome of the interaction with the host. In this review, we highlight the interaction of phypatophogen cell wall and signaling pathways with its host and their contribution to fungal pathogenesis.

HPV and cervical cancer testing and prevention: knowledge, beliefs, and attitudes among Hispanic women.

Cervical cancer is a preventable disease resulting from infection with high-risk types of sexually transmitted human papillomaviruses (HPVs). Public knowledge of HPVs and their link to cervical cancer is limited. Participation in cervical cancer prevention programs, including Pap and HPV screening and HPV vaccine acceptance, is crucial for limiting the incidence of cervical cancer. Hispanic women suffer the highest cervical cancer incidence rates in the United States. In this study, we conducted community-based focus groups with Hispanic women to explore knowledge and attitudes relating to cervical cancer, HPV, HPV testing, and HPV vaccination. Study findings suggest a need to increase public health literacy in relation to HPV, the link between HPV and cervical cancer, and HPV primary and secondary prevention options. Health care providers should be prepared to share information with patients that supports and promotes informed decision making about HPV testing and vaccines and their complementary roles in cervical cancer screening and prevention.

Recent Advances in the Study of the Plant Pathogenic Fungus Botrytis cinerea and its Interaction with the Environment.

The primary contact between the fungal phytopathogen Botrytis cinerea and its host takes place at the cell surface of both organisms. The fungal cell wall is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. Some of these glycoproteins have structural or enzymatic functions, or are involved in conidial adhesion. After landing on the host surface and sensing appropriate signals, B. cinerea conidia produce a germ tube and secrete phytotoxic fungal metabolites and cell wall-degrading enzymes (CWDEs), facilitating host penetration. In fact, 118 genes encoding putative Carbohydrate-Active Enzymes (CAZymes) have been identified in the B. cinerea genome. This large enzymatic repertoire could explain, at least in part, the ability of B. cinerea to infect a vast number of plant species. In recent years, several genes and signaling factors have been identified as playing key roles in pathogenesis, particularly in appressorium formation and penetration. These include the NOX Complex, MAPK cascades, heterotrimeric G proteins, histidine kinases and cAMP signaling pathways. Some of these pathways could also be responsible for controlling the expression and secretion of CWDEs and/or secondary metabolites during infection. Herein, putative virulence factors that are linked to the cell wall, as well as recentlydescribed genes and components that allow the sensing of environmental cues, are highlighted.

Sustainability and power in health promotion: community-based participatory research in a reproductive health policy case study in New Mexico.

Health promotion programs are commonly viewed as value-free initiatives which seek to improve health, often through behavior change. An opposing view has begun to emerge that health promotion efforts, especially ones seeking to impact health policy and social determinants of health, are vulnerable to political contexts and may depend on who is in power at the time. This community-based participatory research study attempts to understand these interactions by applying a conceptual model focused on the power context, diverse stakeholder roles within this context, and the relationship of political levers and other change strategies to the sustainability of health promotion interventions aimed at health policy change. We present a case study of a health promotion coalition, New Mexico for Responsible Sex Education (NMRSE), as an example of power dynamics and change processes. Formed in 2005 in response to federal policies mandating abstinence-only education, NMRSE includes community activists, health promotion staff from the New Mexico Department of Health, and policy-maker allies. Applying an adapted Mayer's 'power analysis' instrument, we conducted semi-structured stakeholder interviews and triangulated political-context analyses from the perspective of the stakeholders.We identified multiple understandings of sustainability and health promotion policy change, including: the importance of diverse stakeholders working together in coalition and social networks; their distinct positions of power within their political contexts; the role of science versus advocacy in change processes; the particular challenges for public sector health promotion professionals; and other facilitators versus barriers to action. One problem that emerged consisted of the challenges for state employees to engage in health promotion advocacy due to limitations imposed on their activities by state and federal policies. This investigation's results include a refined conceptual model, a power-analysis instrument, and new understandings of the intersection of power and stakeholder strategies in the sustainability of health promotion and health in all policies.

Experimentally Induced Articular Cartilage Injury: The Combined Use of Misoprostol and Diclofenac as Therapeutic Agents.

Injection of chymopapain (CP) into the rabbit knee causes rapid depletion of proteoglycans (PGs) in the articular cartilage. Successful repair of the articular cartilage follows injection of only 0.2 mg CP whereas in animals injected with 0.2 mg CP, the repair phase fails leading to cartilage destruction within 21 days. Previous work by the authors indicated that daily oral diclofenac reduced the severity and incidence of CP-induced cartilage injury after 21 days. The present study was designed to determine if administration of diclofenac promoted articular cartilage repair after CP injury over a longer period of time and if coadministration with misoprostol could reduce the concentration of diclofenac needed to promote repair as well as confer protection to the gastric mucosa. Adolescent male New Zealand white rabbits received CP-induced cartilage injury (2.0 mg) followed by daily oral diclofenac, diclofenac/misoprostol or misoprostol. Control animals received diclofenac, diclofenac/misoprostol, or misoprostol or no drug treatment with no cartilage injury. All animals were sacrificed after 2 months. Examination of the stomachs from animals receiving no drug or misoprostol only were completely normal. Most animals receiving diclofenac only had abnormal gastric mucosa. In contrast, coadministration of diclofenac/misoprostol revealed significantly fewer abnormalities. Analysis of the composite histologic score of patellae and measurements of cartilage PG content revealed no significant differences in groups which received CP injury with and without drug treatment. Histology scores and cartilage PG content from animals in groups which did not receive CP injury, but were treated with diclofenac/misoprostol, diclofenac or misoprostol were within normal limits and thus did not differ significantly from untreated control cartilage. The results of this study indicate that daily administration of diclofenac, diclofenac/misoprostol or misoprostol has no protective effect on CP-induced cartilage injury after 2 months. Administration of diclofenac only results in severe gastric abnormalities which are significantly reduced if diclofenac is coadministered with misoprostol. Administration of diclofenac, diclofenac/misoprostol or misoprostol has no effect on PG content of cartilage from otherwise normal joints.