Formal evaluation of PYNK: Breast Cancer Program for Young Women-the patient perspective.

PURPOSE: The aim of the present study was to assess patient satisfaction with pynk: Breast Cancer Program for Young Women so as to determine how the program might be improved and to provide feedback to donors. METHODS: All pynk patients who had consented to have their information entered in our database and who supplied us with their e-mail address were invited to complete a 58-item online questionnaire consisting of multiple choice and open-ended questions. Domains included demographics, provision of written and spoken information, support, infertility risk, research awareness, attitudes toward discharge, and general feedback. RESULTS: Of 120 pynk patients approached, 61 (51%) participated. More than 90% were satisfied or very satisfied with the timing, usefulness, and clarity of spoken and written information given, and 69% found the service and support provided by the nurse navigator to be the most helpful component of the program. Of those who had received systemic therapy, 93% recalled a health care provider initiating a discussion of the risk of treatment-related infertility, and 67% were referred to a fertility clinic. On the negative side, 11%-27% were unaware of various services provided by pynk, and 11% were unaware of pynk's ongoing research. One third of patients were unhappy or ambivalent about the prospect of discharge from the program. CONCLUSIONS: Patient satisfaction with this novel program for young women with breast cancer is high. This study highlights the critical role that the nurse navigator plays in patient support and dissemination of information. In contrast to other reported surveys of young cancer patients, pynk patients are routinely given the opportunity to undergo fertility preservation.

Greater need but reduced access: a population study of planned and elective surgery rates in adult mental health service users.

AIMS: Timely access to surgery is an essential part of healthcare. People living with mental health (MH) conditions may have higher rates of chronic illness requiring surgical care but also face barriers to care. There is limited evidence about whether unequal surgical access contributes to health inequalities in this group. METHODS: We examined 1.22 million surgical procedures in public and private hospitals in New South Wales (NSW), Australia, in 2019. In a cross-sectional study of 76,320 MH service users aged 18 and over, surgical procedure rates per 1,000 population were compared to rates for 6.23 million other NSW residents after direct standardisation for age, sex and socio-economic disadvantage. Rates were calculated for planned and emergency surgery, for major specialty groups, for the top 10 procedure blocks in each specialty group and for 13 access-sensitive procedures. Subgroup analyses were conducted for hospital and insurance type and for people with severe or persistent MH conditions. RESULTS: MH service users had higher rates of surgical procedures (adjusted incidence rate ratio [aIRR]: 1.53, 95% CI: 1.51-1.56), due to slightly higher planned procedure rates (aIRR: 1.22, 95% CI: 1.19-1.24) and substantially higher emergency procedure rates (aIRR: 3.60, 95% CI: 3.51-3.70). Emergency procedure rates were increased in all block groups with sufficient numbers for standardisation. MH service users had very high rates (aIRR > 4.5) of emergency cardiovascular, skin and plastics and respiratory procedures, higher rates of planned coronary artery bypass grafting, coronary angiography and cholecystectomy but lower rates of planned ophthalmic surgery, cataract repair, shoulder reconstruction, knee replacement and some plastic surgery procedures. CONCLUSIONS: Higher rates of surgery in MH service users may reflect a higher prevalence of conditions requiring surgical care, including cardiac, metabolic, alcohol-related or smoking-related conditions. The striking increase in emergency surgery rates suggests that this need may not be being met, particularly for chronic and disabling conditions which are often treated by planned surgery in private hospital settings in the Australian health system. A higher proportion of emergency surgery may have serious personal and health system consequences.

A mechanism for surface attachment in spores of a plant pathogenic fungus.

Rice blast disease is caused by a fungus that attacks all above-ground parts of the rice plant. In a study of the means by which the fungus attaches to the hydrophobic rice leaf surface, it was found that spores(conidia) of the rice blast fungus Magnaporthe grisea have a mechanism for immediate and persistent attachment to various surfaces, including Teflon. This attachment occurs at the spore apex and is blocked by the addition of the lectin concanavalin A. Microscopy of hydrated conidia shows that a spore tip mucilage that binds concanavalin A is expelled specifically from the conidial apex before germ tube emergence. Ultrastructural analysis of dry conidia shows a large periplasmic deposit, presumably spore tip mucilage, at the apex. The results indicate a novel mechanism for the attachment of phytopathogenic fungal spores to a plant surface.

Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea.

MPG1, a pathogenicity gene of the rice blast fungus Magnaporthe grisea, is expressed during pathogenesis and in axenic culture during nitrogen or glucose limitation. We initiated a search for regulatory mutations that would impair nitrogen metabolism, MPG1 gene expression, and pathogenicity. First, we developed a pair of laboratory strains that were highly fertile and pathogenic toward barley. Using a combinatorial genetic screen, we identified mutants that failed to utilize a wide range of nitrogen sources (e.g., nitrate or amino acids) and then tested the effect of these mutations on pathogenicity. We identified five mutants and designated them Nr- (for nitrogen regulation defective). We show that two of these mutations define two genes, designated NPR1 and NPR2 (for nitrogen pathogenicity regulation), that are essential for pathogenicity and the utilization of many nitrogen sources. These genes are nonallelic to the major nitrogen regulatory gene in M. grisea and are required for expression of the pathogenicity gene MPG1. We propose that NPR1 and NPR2 are major regulators of pathogenicity in M. grisea and may be novel regulators of nitrogen metabolism in fungi.

DNA Fingerprinting with a Dispersed Repeated Sequence Resolves Pathotype Diversity in the Rice Blast Fungus.

The poor definition of pathotype variation in the rice blast fungus has historically handicapped strategies for reducing blast disease damage to the world's rice crop. We have employed a probe for a dispersed repeated DNA sequence called MGR [Hamer et al. (1989). Proc. Natl. Acad. Sci. USA 86, 9981-9985] to construct genotype-specific, EcoRl restriction fragment length profiles (MGR-DNA fingerprints) from United States field isolates of this fungus. By using a blind-test design, we demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups. These results resolved a lingering controversy regarding rice blast pathotype stability and illustrated new opportunities for tracking the population dynamics and evolution of this important crop pathogen.

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea.

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.

Functional organization of the Aspergillus nidulans trpC promoter.

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

Infection-related development in the rice blast fungus Magnaporthe grisea.

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.

Karyotope and survival in human acute leukemia.

Of 111 patients presenting with acute forms of leukemia, 44% had a chromosomally abnormal cell line in the bone marrow at diagnosis. In each of the acute leukemia forms (myeloid, lymphatic, stem-cell), patients with karyotypic abnormalities showed mean and median survival times like those with normal chromosomes. Both groups showed a wide range of survival times. The mean and median survival times of patients with mixed populations of chromosomally normal and abnormal cells did not differ from those of patients with exclusively abnormal cells in the bone marrow. The karyotypic abnormalities associated with acute leukemia, as well as having no etiologic significance, probably do not determine the subsequent course of the leukemia.

[Estimation of the jugular venous pressure]. / Het beoordelen van de centraalveneuze druk.

Estimation of jugular venous pressure (JVP) is valuable for the differentiation between dyspnoea of cardiac or pulmonary origin, and for determining the cause of oedema. JVP assessments are useful for evaluation of treatment of right ventricular failure. The correlation between non-invasive JVP and invasive measurement of the central venous pressure (CVP) is remarkably better than previously reported. Correlation between JVP - determined via the external jugular vein - and CVP is excellent when the outcomes are categorised into low, normal and elevated pressure. Optimal measurement configurations include: extended expiration (without Valsalva manoeuvre), and during ventricular diastole. In the literature, these measurement configurations concerning the respiratory cycle and cardiac cycle have not been applied uniformly. To investigate in detail the correlation between JVP and CVP, the methods need to be standardized, and tests performed simultaneously and correctly.

The ebb and flow of a fungal genome.

Transposable DNA elements have only recently been described in a few species of filamentous fungi, but may be more abundant than previously believed. Several different elements have been isolated from the rice blast pathogen Magnaporthe grisea. The distribution and amplification of these elements suggest a potential role in the evolution of the fungal genome.

Recent advances in large-scale transposon mutagenesis.

Transposons were identified as mobile genetic elements over fifty years ago and subsequently became powerful tools for molecular-genetic studies. Recently, transposon-mutagenesis strategies have been developed to identify essential and pathogenicity-related genes in pathogenic microorganisms. Also, a number of in vitro transposition systems have been used to facilitate genome sequence analysis. Finally, transposon mutagenesis of yeast and complex eukaryotes has provided valuable functional genomic information to complement genome-sequencing projects.

hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.

Mutations at the smo genetic locus affect the shape of diverse cell types in the rice blast fungus.

Teflon film surfaces are highly conducive to the formation of infection structures (appressoria) in the plant pathogenic fungus, Magnaporthe grisea. We have utilized Teflon films to screen and select for mutants of M. grisea that are defective in appressorium formation. This approach and several others yielded a group of 14 mutants with a similar phenotype. All the mutant strains make abnormally shaped conidia and appressoria. When two mutant strains are crossed, abnormally shaped asci are formed. Ascus shape is normal when a mutant strain is crossed with a wild-type strain. Despite dramatic alterations in cell shape these strains otherwise grow, form conidia, undergo meiosis, and infect plants normally. This mutant phenotype, which we have termed Smo(-), for abnormal spore morphology, segregates in simple Mendelian fashion in crosses with wild-type strains. Some ascospore lethality is associated with smo mutations. In genetic crosses between mutants, smo mutations fail to recombine and do not demonstrate complementation of the abnormal ascus shape phenotype. We conclude that the smo mutations are alleles of a single genetic locus and are recessive with regard to the the ascus shape defect. Mutations at the SMO locus also permit germinating M. grisea conidia to differentiate appressoria on surfaces that are not normally conducive to infection structure formation. A number of spontaneous smo mutations have been recovered. The frequent occurrence of this mutation suggests that the SMO locus may be highly mutable.

Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis.

Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokines mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis.

Hypomorphic bimA(APC3) alleles cause errors in chromosome metabolism that activate the DNA damage checkpoint blocking cytokinesis in Aspergillus nidulans.

The Aspergillus nidulans sepI(+) gene has been implicated in the coordination of septation with nuclear division and cell growth. We find that the temperature-sensitive (ts) sepI1 mutation represents a novel allele of bimA(APC3), which encodes a conserved component of the anaphase-promoting complex/cyclosome (APC/C). We have characterized the septation, nuclear division, cell-cycle checkpoint defects, and DNA sequence alterations of sepI1 (renamed bimA10) and two other ts lethal bimA(APC3) alleles, bimA1 and bimA9. Our observations that bimA9 and bimA10 strains had morphologically abnormal nuclei, chromosome segregation defects, synthetic phenotypes with mutations in the DNA damage checkpoint genes uvsB(MEC1/rad3) or uvsD(+), and enhanced sensitivity to hydroxyurea strongly suggest that these strains accumulate errors in DNA metabolism. We found that the aseptate phenotype of bimA9 and bimA10 strains was substantially relieved by mutations in uvsB(MEC1/rad3) or uvsD(+), suggesting that the presence of a functional DNA damage checkpoint inhibits septation in these bimA(APC3) strains. Our results demonstrate that mutations in bimA(APC3) lead to errors in DNA metabolism that indirectly block septation.

Lipitoids--novel cationic lipids for cellular delivery of plasmid DNA in vitro.

BACKGROUND: Although synthetic nonviral vectors hold promise for the delivery of plasmid DNA, their gene-transfer efficiencies are far from matching those of viruses. To systematically investigate the structure-activity relationship of cationic lipids, a small library of cationic lipid-peptoid conjugates (lipitoids) was synthesized. The compounds were evaluated for their ability to form complexes with plasmid DNA and to mediate DNA transfer in vitro. RESULTS: Lipid-peptoid conjugates were conveniently prepared in high yield using solid-phase synthesis. Several lipitoids condensed plasmid DNA into 100 nm spherical particles and protected the DNA and DNase digestion. A subset of lipitoids with a repeated (aminoethyl, neutral, neutral) sidechain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA transfer with high efficiency. CONCLUSIONS: Automated solid-phase synthesis of cationic lipids allowed the rapid synthesis of a diverse set of transfection reagents. The most active compound DMPE-(Nae-Nmpe-Nmpe)3 (Nae, N-aminoethyl glycine; Nmpe, N-p-methoxyphenethyl-glycine) is more efficient than lipofectin or DMRIE-C (two commercial cationic lipid transfection reagents) and is active in the presence and absence of serum. The activity in the presence of serum suggests potential for applications in vivo.

Chemotherapy immediately following autologous stem-cell transplantation in patients with advanced breast cancer.

Most patients relapse after high-dose chemotherapy (HDCT) with autologous stem-cell transplantation (ASCT) for metastatic breast cancer. Further chemotherapy immediately after hematopoietic recovery from ASCT is not given for fear of irreversibly damaging the newly engrafted stem cells. In a pilot chemoprotection trial, autologous CD34+ cells from patients with metastatic breast cancer were exposed to a replication-incompetent retroviral vector carrying MDR-1 cDNA and then reinfused after HDCT. Immediately on recovery, patients received multiple courses of escalating dose paclitaxel. All of the 10 patients tolerated reinfusion of modified cells without any toxicity and had myeloid engraftment within 12 days (range, 11-14). The bone marrow cells of three patients contained vector MDR-1-positive cells only at the time of the first course of posttransplant paclitaxel, indicating that the MDR-1 vector-modified cells had only short-term engrafting potential. A total of 83 courses of paclitaxel were administered starting at a median of 30 (range, 21-32) days from ASCT. The median dose of paclitaxel was 225 mg/m2 and the median interval between paclitaxel cycles of therapy was 21 (range, 20-41) days. Five of the six CR patients were able to receive all of the 12 courses of paclitaxel. Three patients who had achieved less than a complete response to the HDCT (2 patients) and partial response (1 patient) were converted to complete clinical responses during the 12 cycles of paclitaxel. No delayed toxicity or bone marrow failure was noted in these patients with a median follow-up of 2 years from ASCT. This is the first study of chemotherapy immediately after transplantation with autologous CD34+ cells. These data indicate that paclitaxel can be safely administered immediately after ASCT without any delayed toxicities. Paclitaxel given immediately after ASCT can further improve the response to pretransplant chemotherapy in patients with advanced breast cancer.

Observations on the biphasic nature of digitalis electrophysiological actions in the human right atrium.

Cardiac electrophysiological effects of a digitalis glycoside have been investigated by right atrial intracardiac stimulation and recording in 12 patients with paroxysmal supraventricular tachyarrhythmias. Measurements were made of atrial effective refractoriness by pacing together with programmed premature extrastimulation. Simultaneous recordings of atrial action potential duration from a site close to the sinoatrial node and from a more distal atrial site were made using an endocardial contact-injury potential technique. All subjects received methyldigoxin 10.0 micrograms X kg-1 intravenously, while half were also pretreated with atropine. A biphasic response to methyldigoxin was observed, with initial action potential prolongation, maximal at 20 min post-infusion, followed by significant action potential shortening which persisted to the end of the study period at 40 min. The initial phase, that of prolongation, was associated with smaller increases in atrial effective refractoriness and increased vulnerability to atrial tachyarrhythmia initiation. During the subsequent phase of action potential shortening, the gap between the termination of effective refractoriness and completion of action potential repolarisation was narrowed, coinciding with diminished vulnerability to tachyarrhythmias. Slight but significant atrioventricular conduction delay was apparent 30 to 40 min after glycoside infusion, indicating enhanced vagal activity during the phase of action potential shortening. Prior atropinisation reduced the magnitude of both early and late components of the biphasic action potential response to digitalis, supporting the proposition that both components are mediated via cardiac muscarinic receptors. Since vagal effects on the atrioventricular junction appeared during the later phase, it is suggested that initial action potential prolongation by digitalis may have been effected via local acetylcholine release, while subsequent action potential shortening may have been caused by a combination of vagally and locally mediated activity.

Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea.

The fungal phytopathogen Magnaporthe grisea parasitizes a wide variety of gramineous hosts. In the course of investigating the genetic relationship between pathogen genotype and host specificity we identified a retroelement that is present in some strains of M. grisea that infect finger millet and goosegrass (members of the plant genus Eleusine). The element, designated grasshopper (grh), is present in multiple copies and dispersed throughout the genome. DNA sequence analysis showed that grasshopper contains 198 base pair direct, long terminal repeats (LTRs) with features characteristic of retroviral and retrotransposon LTRs. Within the element we identified an open reading frame with sequences homologous to the reverse transcriptase, RNaseH, and integrase domains of retroelement pol genes. Comparison of the open reading frame with sequences from other retroelements showed that grh is related to the gypsy family of retrotransposons. Comparisons of the distribution of the grasshopper element with other dispersed repeated DNA sequences in M. grisea indicated that grasshopper was present in a broadly dispersed subgroup of Eleusine pathogens, suggesting that the element was acquired subsequent to the evolution of this host-specific form. We present arguments that the amplification of different retroelements within populations of M. grisea is a consequence of the clonal organization of the fungal populations.