Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits

Functional mapping of hospitals by diagnosis-dominant case-mix analysis

BACKGROUND: Principles and methods for the allocation of healthcare resources among healthcare providers have long been health policy research issues in many countries. Healthcare reforms including the development of a new case-mix system, Diagnosis Procedure Combination (DPC), and the introduction of a DPC-based payment system are currently underway in Japan, and a methodology for adequately assessing the functions of healthcare providers is needed to determine healthcare resource allocations. METHODS: By two-dimensional mapping of the rarity and complexity of diagnoses for patients receiving treatment, we were able to quantitatively demonstrate differences in the functions of different healthcare service provider groups. RESULTS: On average, inpatients had diseases that were 3.6-times rarer than those seen in outpatients, while major teaching hospitals treated inpatients with diseases 3.0-times rarer on average than those seen at small hospitals. CONCLUSION: We created and evaluated a new indicator for DPC, the diagnosis-dominant case-mix system developed in Japan, whereby the system was used to assess the functions of healthcare service providers. The results suggest that it is possible to apply the case-mix system to the integrated evaluation of outpatient and inpatient healthcare services and to the appropriate allocation of healthcare resources among health service providers

EMQN best practice guidelines for the laboratory diagnosis of osteogenesis imperfecta

Osteogenesis imperfecta (OI) comprises a group of inherited disorders characterized by bone fragility and increased susceptibility to fractures. Historically, the laboratory confirmation of the diagnosis OI rested on cultured dermal fibroblasts to identify decreased or abnormal production of abnormal type I (pro)collagen molecules, measured by gel electrophoresis. With the discovery of COL1A1 and COL1A2 gene variants as a cause of OI, sequence analysis of these genes was added to the diagnostic process. Nowadays, OI is known to be genetically heterogeneous. About 90% of individuals with OI are heterozygous for causative variants in the COL1A1 and COL1A2 genes. The majority of remaining affected individuals have recessively inherited forms of OI with the causative variants in the more recently discovered genes CRTAP, FKBP10, LEPRE1,PLOD2, PPIB, SERPINF1, SERPINH1 and SP7, or in other yet undiscovered genes. These advances in the molecular genetic diagnosis of OI prompted us to develop new guidelines for molecular testing and reporting of results in which we take into account that testing is also used to ‘exclude' OI when there is suspicion of non-accidental injury. Diagnostic flow, methods and reporting scenarios were discussed during an international workshop with 17 clinicians and scientists from 11 countries and converged in these best practice guidelines for the laboratory diagnosis of OI

Competition between Replicative and Translesion Polymerases during Homologous Recombination Repair in Drosophila

In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis

Quorum Sensing Regulation of the Two hcp Alleles in Vibrio cholerae O1 Strains

BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS. METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor. CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp

Preventive Effects of Omega-3 and Omega-6 Fatty Acids on Peroxide Mediated Oxidative Stress Responses in Primary Human Trabecular Meshwork Cells

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H2O2, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H2O2 further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H2O2 stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H2O2 mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H2O2 induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease

Amygdala inputs to prefrontal cortex guide behavior amid conflicting cues of reward and punishment

Orchestrating appropriate behavioral responses in the face of competing signals that predict either rewards or threats in the environment is crucial for survival. The basolateral nucleus of the amygdala (BLA) and prelimbic (PL) medial prefrontal cortex have been implicated in reward-seeking and fear-related responses, but how information flows between these reciprocally connected structures to coordinate behavior is unknown. We recorded neuronal activity from the BLA and PL while rats performed a task wherein competing shock- and sucrose-predictive cues were simultaneously presented. The correlated firing primarily displayed a BLA→PL directionality during the shock-associated cue. Furthermore, BLA neurons optogenetically identified as projecting to PL more accurately predicted behavioral responses during competition than unidentified BLA neurons. Finally photostimulation of the BLA→PL projection increased freezing, whereas both chemogenetic and optogenetic inhibition reduced freezing. Therefore, the BLA→PL circuit is critical in governing the selection of behavioral responses in the face of competing signals.National Institutes of Health (U.S.) (Award 1R25-MH092912-01)National Institute of Mental Health (U.S.) (Grant R01- MH102441-01)National Institutes of Health (U.S.) (Award DP2- DK-102256-01