LOGAN-CV: A Prospective Study of a Multifaceted Intervention Targeting United States Clinicians to Improve Guideline-Based Management of Lipid-Lowering Therapy.

INTRODUCTION: The 2018 American Heart Association (AHA)/American College of Cardiology (ACC)/Multisociety blood cholesterol guidelines recommend clinicians consider adding non-statin therapy for patients with very high-risk (VHR) atherosclerotic cardiovascular disease (ASCVD) and low-density lipoprotein cholesterol (LDL-C) ≥ 70 mg/dl while receiving maximally tolerated statins. However, according to a recent study, only 17.1% of patients with established ASCVD received appropriate lipid-lowering therapy (LLT) intensification. Here, we describe the design of a prospective, 12-month study (LOGAN-CV) evaluating a multifaceted site-level intervention to enhance clinicians' adherence to guidelines to improve LDL-C levels for patients with VHR ASCVD. METHODS: Clinicians from up to ten research sites are eligible if they care for adult patients with ASCVD. Interventions include educational modules, a cloud-based performance platform providing clinicians a tailored summary of their LDL-C management performance, newsletters, periodic peer-to-peer calls, and pre- and post-intervention surveys evaluating knowledge, attitudes, and beliefs around LDL-C management, with additional interventions for clinicians demonstrating a lower readiness to make treatment decisions based on guideline recommendations. Patients with VHR ASCVD, defined as having recent myocardial infarction and LDL-C ≥ 70 mg/dl despite statin treatment, will be included in the study. Patient data will be collected from electronic medical records from baseline (clinician enrollment) through the 12-month intervention. The study started in October 2022, with anticipated completion in March 2024. PLANNED OUTCOMES: The change in proportion of patients with LDL-C < 70 mg/dl achieved at any time during the 12-month intervention (primary); LLT intensification, changes in guideline-aligned LDL-C testing and LLT titration over 12 months, and change in overall clinicians' knowledge, attitudes, and beliefs are key outcomes of interest. The LOGAN-CV study addresses a critical unmet need in LDL-C control in patients with VHR ASCVD and evaluates the effect of a multifaceted intervention targeting clinicians to improve their adherence to guidelines and consequently improve clinical outcomes for patients.

Elevated incidence of fractures in women with invasive breast cancer.

UNLABELLED: This study evaluates the incidence of bone fractures in women with BC.We found that women with invasive breast cancer are at an increased risk for bone fractures, with fractures most commonly occurring at lower extremity and vertebral sites. The risk is further increased in women undergoing cancer therapy. INTRODUCTION: Bone loss and fractures in breast cancer have generally been attributed to aromatase inhibitor use. This study assessed the incidence of fractures after invasive breast cancer diagnosis and evaluated bone density and FRAX risk calculation at time of fracture occurrence. METHODS: Retrospective cohort study of women with invasive breast cancer [June 2003-December 2011] who participated in an academic hospital based genetic biobank. Demographic and clinical characteristics were abstracted from the electronic medical record (EMR). RESULTS: A total of 422 women with invasive breast cancer were assessed; 79 (28 %) sustained fractures during the observation period; fractures occurred at multiple skeletal sites in 27 cases (116 fractures). The incidence of fractures was 40 per 1000 person-years. Women who sustained fractures were mostly white and had a family history of osteoporosis (36.9 %, p = 0.03) or history of a prior fracture (6/79, p = 0.004). Fractures occurred 4.0 years (range 0-12 years) after cancer diagnosis. Fracture cases had femoral neck bone mineral density (BMD) of 0.72 + 0.12 g/cm(2), T-score of -1.2, that is, within the low bone mass range. Fractures most commonly occurred in lower extremities, vertebral, and wrist sites. Hip fractures accounted for 11 % of fractures, occurring at a median age of 61 years. CONCLUSIONS: Fractures occur shortly after commencing cancer therapy. Rapid bone loss associated with cancer therapy may precipitate fractures. Fractures occur at relatively higher BMD in BC. Occurrence of fractures in invasive breast cancer raises the possibility of cancer-induced impairment in bone quality.

Advancing pharmacovigilance through academic-legal collaboration: the case of gadolinium-based contrast agents and nephrogenic systemic fibrosis-a Research on Adverse Drug Events and Reports (RADAR) report.

OBJECTIVE: To compare and contrast three databases, that is, The International Centre for Nephrogenic Systemic Fibrosis Registry (ICNSFR), the Food and Drug Administration Adverse Event Reporting System (FAERS) and a legal data set, through pharmacovigilance and to evaluate international nephrogenic systemic fibrosis (NSF) safety efforts. METHODS: The Research on Adverse Drug events And Reports methodology was used for assessment-the FAERS (through June 2009), ICNSFR and the legal data set (January 2002 to December 2010). Safety information was obtained from the European Medicines Agency, the Danish Medicine Agency and the Food and Drug Administration. RESULTS: The FAERS encompassed the largest number (n = 1395) of NSF reports. The ICNSFR contained the most complete (n = 335, 100%) histopathological data. A total of 382 individual biopsy-proven, product-specific NSF cases were analysed from the legal data set. 76.2% (291/382) identified exposure to gadodiamide, of which 67.7% (197/291) were unconfounded. Additionally, 40.1% (153/382) of cases involved gadopentetate dimeglumine, of which 48.4% (74/153) were unconfounded, while gadoversetamide was identified in 7.3% (28/382) of which 28.6% (8/28) were unconfounded. Some cases involved gadobenate dimeglumine or gadoteridol, 5.8% (22/382), all of which were confounded. The mean number of exposures to gadolinium-based contrast agents (GBCAs) was gadodiamide (3), gadopentetate dimeglumine (5) and gadoversetamide (2). Of the 279 unconfounded cases, all involved a linear-structured GBCA. 205 (73.5%) were a non-ionic GBCA while 74 (26.5%) were an ionic GBCA. CONCLUSION: Clinical and legal databases exhibit unique characteristics that prove complementary in safety evaluations. Use of the legal data set allowed the identification of the most commonly implicated GBCA. ADVANCES IN KNOWLEDGE: This article is the first to demonstrate explicitly the utility of a legal data set to pharmacovigilance research.

Reporting of serious adverse events during cancer clinical trials to the institutional review board: an evaluation by the research on adverse drug events and reports (RADAR) project.

Global introspection is considered an unreliable method for attribution of causality of serious adverse events (SAEs), yet remains widely used for cancer drug clinical trials. Here, we compare structured case abstraction (SCA) to the routine method for detecting, evaluating, and reporting ADEs during cancer drug clinical trials to an Institutional Review Board (IRB). We obtained all SAE reports (2001-2008) received by one IRB for six clinical trials involving bevacizumab or oxaliplatin for treatment of gastrointestinal cancers. We compared the routine IRB SAE method to SCA for adverse event detection and causality attribution. Of 205 adverse events, 182 events (75%) were not reported; of these, 6 (20%) of 30 SAEs requiring an IRB report were unreported. For the 10 item Naranjo score, the amount of information useful for causality attribution was higher with SCA than the routine method (6.0 vs. 2.4 items, P < .0001). One-fifth of SAEs requiring an IRB report were unreported to the IRB via the routine method. SCA provided more useful information as to whether an SAE was caused by a cancer drug exposure. Our results suggest that SCA may improve SAE detection and the accuracy of attribution of causality during cancer drug clinical trials.

Progestogens for preterm birth prevention: a systematic review and meta-analysis by drug route.

PURPOSE: Progestogen has been investigated as a preventive intervention among women with increased preterm birth risk. Our objective was to systematically review the effectiveness of intramuscular (IM), vaginal, and oral progestogens for preterm birth and neonatal death prevention. METHODS: We included articles published from January 1966 to January 2013 and found 27 randomized trials with data for Bayesian meta-analysis. RESULTS: Across all studies, only vaginal and oral routes were effective at reducing preterm births (IM risk ratio [RR] 0.95, 95 % Bayesian credible interval [BCI]: 0.88-1.03; vaginal RR 0.87, 95 % BCI: 0.80-0.94; oral RR 0.64, 95 % BCI: 0.49-0.85). However, when analyses were limited to only single births all routes were effective at reducing preterm birth (IM RR 0.77, 95 % BCI: 0.69-0.87; vaginal RR 0.80, 95 % BCI: 0.69-0.91; oral RR 0.66, 95 % BCI: 0.47-0.84). Only IM progestogen was effective at reducing neonatal deaths (IM RR 0.78, 95 % BCI: 0.56-0.99; vaginal RR 0.75, 95 % BCI: 0.45-1.09; oral RR 0.72, 95 % BCI: 0.09-1.74). Vaginal progestogen was effective in reducing neonatal deaths when limited to singletons births. CONCLUSIONS: All progestogen routes reduce preterm births but not neonatal deaths. Future studies are needed that directly compare progestogen delivery routes.

Progestogens for preterm birth prevention: a systematic review and meta-analysis.

OBJECTIVE: We systematically reviewed the effectiveness of progestogens for prevention of preterm birth among women with prior spontaneous preterm birth, multiple gestations, preterm labor, short cervix, or other indications. DATA SOURCES: We searched MEDLINE and EMBASE databases for English language articles published from January 1966 to October 2011. METHODS OF STUDY SELECTION: We excluded publications that were not randomized controlled trials or had fewer than 20 participants, identifying 34 publications, of which 19 contained data for Bayesian meta-analysis. TABULATION, INTEGRATION, AND RESULTS: Two reviewers independently extracted data and assigned overall quality ratings based on predetermined criteria. Among women with prior preterm birth and a singleton pregnancy (five randomized controlled trials), progestogen treatment decreased the median risk of preterm birth by 22% (relative risk [RR] 0.78, 95% Bayesian credible interval 0.68-0.88) and neonatal death by 42% (RR 0.58, 95% Bayesian credible interval 0.27-0.98). The evidence suggests progestogen treatment does not prevent prematurity (RR 1.02, 95% Bayesian credible interval 0.87-1.17) or neonatal death (RR 1.44, 95% Bayesian credible interval 0.46-3.18) in multiple gestations. Limited evidence suggests progestogen treatment may prevent prematurity in women with preterm labor (RR 0.62, 95% Bayesian credible interval 0.47-0.79) and short cervix (RR 0.52, 95% Bayesian credible interval 0.36-0.70). Across indications, evidence about maternal, fetal, or neonatal health outcomes, other than reducing preterm birth and neonatal mortality, is inconsistent, insufficient, or absent. CONCLUSION: Progestogens prevent preterm birth when used in singleton pregnancies for women with a prior preterm birth. In contrast, evidence suggests lack of effectiveness for multiple gestations. Evidence supporting all other uses is insufficient to guide clinical care. Overall, clinicians and patients lack longer-term information to understand whether intervention has the ultimately desired outcome of preventing morbidity and promoting normal childhood development.

Evidence to inform decisions about maternal-fetal surgery: technical brief.

OBJECTIVE: To summarize the state of research in maternal-fetal surgery regarding the surgical repair of abnormalities in fetuses in the womb. DATA SOURCES: We searched MEDLINE from 1980 to 2010 for studies of maternal-fetal surgery for the following conditions: twin-twin transfusion syndrome, obstructive uropathy, congenital diaphragmatic hernia, myelomeningocele, thoracic lesions, cardiac malformations, and sacrococcygeal teratoma. METHODS OF STUDY SELECTION: We used pilot-tested data collection forms to screen publications for inclusion and to extract data. We compiled information about how fetal diagnoses were defined, maternal inclusion criteria, type of surgery, study design, country, setting, comparators used, length of follow-up, outcomes measured, and adverse events. TABULATION, INTEGRATION, AND RESULTS: Two reviewers independently extracted data and discordance was resolved by a third party. Of 1,341 articles located, we retained 258 (comprising 166 unique study populations). Three studies were randomized controlled trials; the majority of the evidence was observational (116 case series [70%], 36 retrospective [22%], and 11 prospective [7%] cohorts). Twin-twin transfusion is the most studied condition, with 84 studies including 2,532 pregnancies. Fewer than 500 pregnancies are represented in the literature for each of the other conditions except congenital diaphragmatic hernia (n=503). Inclusion criteria were poorly specified. Outcomes typically measured were survival to birth, preterm birth, and neonatal death. Longer-term outcomes were sparse but included pulmonary, renal, and neurologic status and developmental milestones. Maternal outcome data were rare. CONCLUSION: Although developing rapidly, maternal-fetal surgery research has yet to achieve the typical quality of studies and aggregate strength of evidence needed to optimally inform care.

Treatment of overactive bladder in women.

OBJECTIVES: The Vanderbilt Evidence-based Practice Center systematically reviewed evidence on treatment of overactive bladder (OAB), urge urinary incontinence, and related symptoms. We focused on prevalence and incidence, treatment outcomes, comparisons of treatments, modifiers of outcomes, and costs. DATA: We searched PubMed, MEDLINE, EMBASE, and CINAHL. REVIEW METHODS: We included studies published in English from January 1966 to October 2008. We excluded studies with fewer than 50 participants, fewer than 75 percent women, or lack of relevance to OAB. Of 232 included publications, 20 were good quality, 145 were fair, and 67 poor. We calculated weighted averages of outcome effects and conducted a mixed-effects meta-analysis to investigate outcomes of pharmacologic treatments across studies. RESULTS: OAB affects more than 10 to 15 percent of adult women, with 5 to 10 percent experiencing urge urinary incontinence (UUI) monthly or more often. Six available medications are effective in short term studies: estimates from meta-analysis models suggest extended release forms (taken once a day) reduce UUI by 1.78 (95 percent confidence interval (CI): 1.61, 1.94) episodes per day, and voids by 2.24 (95 percent CI: 2.03, 2.46) per day. Immediate release forms (taken twice or more a day) reduce UUI by 1.46 (95 percent CI: 1.28, 1.64), and voids by 2.17 (95 percent CI: 1.81, 2.54). As context, placebo reduces UUI episodes by 1.08 (95 percent CI: 0.86, 1.30), and voids by 1.48 (95 percent CI: 1.19, 1.71) per day. No one drug was definitively superior to others, including comparison of newer more selective agents to older antimuscarinics. Current evidence is insufficient to guide choice of other therapies including sacral neuromodulation, instillation of oxybutynin, and injections of botulinum toxin. Acupuncture was the sole complementary and alternative medicine treatment, among reflexology and hypnosis, with early evidence of benefit. The strength of the evidence is insufficient to fully inform choice of these treatments. Select behavioral interventions were associated with symptom improvements comparable to medications. Limited evidence suggests no clear benefit from adding behavioral interventions at the time of initiation of pharmacologic treatment. CONCLUSIONS: OAB and associated symptoms are common. Treatment effects are modest. Quality of life and treatment satisfaction measures suggest such improvements can be important to women. The amount of high quality literature available is meager for helping guide women's choices. Gaps include weak or absent data about long-term followup, poorly characterized and potentially concerning harms, information about best choices to minimize side effects, and study of how combinations of approaches may best be used. This is problematic since the condition is chronic and a single treatment modality is unlikely to fully resolve symptoms for most women.

Reflections on the use of electronic health record data for clinical research.

The adoption of electronic health records (EHRs) offers the potential to improve the delivery, quality, and continuity of clinical care, but widespread use has not yet occurred. In this article, we describe our use of clinical (production) data that were derived from outpatient and inpatient visits at a university teaching hospital for clinical research, a use for which the data and their structure were not originally designed. Similar data exist at many outpatient and inpatient clinical facilities, and we believe that our insights are relevant to electronically captured medical data regardless of their origin. We describe the approaches taken to ensure compliance with the Health Insurance Portability and Accountability Act (HIPAA) and to leverage the vast stores of structured and unstructured data that are currently underused. We conclude by reflecting on what we would have done differently and by making recommendations to streamline the process.

Legal, financial, and public health consequences of transfusion-transmitted hepatitis C virus in persons with haemophilia.

BACKGROUND: Since the first cases of acquired immunodeficiency syndrome in persons with haemophilia were reported in 1982, much has been written about the consequences of human immunodeficiency virus (HIV) contamination of the blood supply. Relatively little attention has been paid to similar hepatitis C virus (HCV) concerns since the first cases of HCV-infected persons with haemophilia were identified in 1989. METHODS: We review the history, public health, policy, and financial consequences of blood supply policy decisions made for persons with haemophilia who received HCV-contaminated blood products in eight countries that were severely impacted by viral contamination of the blood supply during the 1980s, contrasting these findings with those reported previously for HIV contamination of the blood supply during the same time-period. A Medline search and a hand search of retrieved bibliographies of English-language articles on HCV concerns in haemophilia patients published from 1989 to 2006 were performed. RESULTS: Our review identified that two- to eightfold more persons with haemophilia in the eight countries contracted HCV vs. HIV from contaminated blood products during the 1980s. Opportunistic infections and immunosuppression-related complications among persons with haemophilia developed shortly after these patients received HIV-infected blood products whereas hepatic complications among HCV-infected persons with haemophilia are just now being diagnosed two decades after these individuals received HCV-contaminated blood products. Policy makers in four countries conducted official public inquiries into blood safety decisions related to HIV- and/or HCV-contamination of the blood supply. More than 20 countries allocated compensation funds for HIV-infected persons with haemophilia (mean award ranging from $37 000 to 400 000) whereas only the UK, Canada, and Ireland allocated compensation funds for HCV-infected persons with haemophilia (mean award ranging from $37 000 to 50 000). CONCLUSION: While the clinical impact among persons with haemophilia of HCV contamination of the blood supply in the 1980s was larger than the impact of HIV contamination of the blood supply during this time-period, the policy response was smaller. Consideration should be given to adopting support programmes for HCV-infected persons with haemophilia in countries that do not have these programs.

Granulocyte-colony stimulating factor administration to healthy individuals and persons with chronic neutropenia or cancer: an overview of safety considerations from the Research on Adverse Drug Events and Reports project.

Granulocyte-colony stimulating factor (G-CSF) is widely administered to donors who provide peripheral blood stem cells (PBSC) for individuals who undergo hematopoietic stem cell transplants. Questions have been raised about the safety of G-CSF in this setting. Herein, the Research on Adverse Drug Events and Reports (RADAR) project investigators reviewed the literature on G-CSF-associated adverse events in healthy individuals or persons with chronic neutropenia or cancer. Toxicities identified included bone pain and rare instances of splenic rupture, allergic reactions, flares of underlying autoimmune disorders, lung injury and vascular events. Among healthy individuals, four patients developed splenic rupture shortly after G-CSF administration and three patients developed acute myeloid leukemia 1 to 5 years after G-CSF administration. Registry studies identified no increased risks of malignancy among healthy individuals who received G-CSF before PBSC harvesting. However, more than 2000 donors would have to be followed for 10 years to detect a 10-fold increase in leukemia risk. Our review identifies bone pain as the most common toxicity of G-CSF administration. There are questions about a causal relationship between G-CSF administration and acute leukemia, but more long-term safety data from database registries are needed to adequately evaluate such a relationship.

Breakthrough zygomycosis after voriconazole administration among patients with hematologic malignancies who receive hematopoietic stem-cell transplants or intensive chemotherapy.

Zygomycosis is increasingly reported as a cause of life-threatening fungal infections. A higher proportion of cases reported over the last decades have been in cancer patients, with or without hematopoietic stem cell transplantation (HSCT). The new anti-fungal agent voriconazole is a recently identified risk factor for developing zygomycosis. We reviewed the clinical characteristics and outcomes of a large cohort of cancer patients who developed zygomycosis after exposure to voriconazole. Health care professionals at 13 large cancer centers provided clinical information on cancer patients with zygomycosis and prior exposure to voriconazole. Criteria for inclusion were 5 days or more of voriconazole use and diagnostic confirmation with tissue or histology. Fifty-eight cases were identified among patients with hematologic malignancies, 62% including patients who underwent a HSCT procedure. Fifty-six patients received voriconazole for primary or secondary prophylaxis against fungal infection. In addition to prior exposure to voriconazole, patients also had several of the previously established risk factors for zygomycosis. Amphotericin B was the most commonly prescribed anti-fungal therapy. Overall mortality was 73%. We conclude that zygomycosis after exposure to voriconazole is a recently described entity that is frequently fatal, despite treatment with currently available anti-fungal agents and surgery.

Pleiotrophic cellular deficiencies conferred by the blm5-1 mutation of Saccharomyces cerevisiae.

Mutational alteration of the BLM5 gene of the model eukaryote, Saccharomyces cerevisiae, confers extreme hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins. Additional properties conferred by the blm5-1 mutation in haploid and diploid strains were investigated for the current report. Only one copy of blm5-1 together with the normal BLM5 allele was sufficient to produce mitotic and meiotic defects in diploids, and greatly increase killing by bleomycin beyond wild type levels. Mitotic growth rates of blm5-1/blm5-1 homozygous mutant strains were slower than wild type or BLM5/blm5-1 heterozygous strains at 30 degrees C, and growth was nearly completely inhibited at 37 degrees C. Meiosis was inhibited at 30 degrees C and 37 degrees C in mutant homozygotes, and at 37 degrees C in BLM5/blm5-1 heterozygotes, while meiosis occurred at equivalent frequencies in wild type strains at both temperatures. Surprisingly, mutant strains were found to associate extremely low quantities of [S-methyl-3H]bleomycin A2, in contrast to normal strains that associated quite high amounts. However, the fractions of the total associated radioactivities that were released from normal and blm5-1 cells were equivalent. These results suggested that the extremely high killing suffered by blm5-1 mutant strains in response to bleomycin treatments results from something other than increased intracellular drug concentrations.

The novel BLM3 gene encodes a protein that protects against lethal effects of oxidative damage.

Mutational alteration of the BLM3 gene in Saccharomyces cerevisiae confers hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins. Bleomycin is used clinically in the treatment of many types of cancers, including Kaposi's sarcoma. The BLM3 gene was cloned from a genomic library by complementing the drug hypersensitivities conferred by the codominant blm3-1 mutation. The nucleotide sequence of BLM3 encodes a predicted integral protein of 1804 amino acids with seven to ten potential transmembrane domains and additional motifs. The blm3 null mutation was created by gene replacement, and found not to be essential for growth in the absence of the bleomycin-phleomycin antibiotics. Sequence analyses suggest the Blm3p could be a potential member of the major facilitator superfamily (MFS) of permeases. Northern dot blot analyses using a human RNA master tissue blot containing RNA from fifty different fetal and adult tissues revealed sequence homology in adult tissues to BLM3, but no sequence homology in fetal tissues. The function of the Blm3p is presently unknown. We propose several functions for the Blm3p in protecting cells against oxidative agents, including roles in detoxification, transport and defending against DNA damage.

DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae.

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase.

The vast majority of AIDS-related deaths are associated with opportunistic infections. For fungal infections, there are few effective antifungals, particularly for systemic use. The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls. This report describes one of the genes cloned by complementation of the blm1-1 mutation of S. cerevisiae using a YCp50-based yeast genomic library. Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases. The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity. Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle. Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design.

A mutant approach and molecular strategy to study fungal cell walls.

The current study describes recombinant plasmids which complement the hypersusceptibility to killing bleomycin of blm1-1 mutant cells of Saccharomyces cerevisiae, and a strategy developed and used to recover active clones from a stable yeast genomic library. The resistance of a spontaneous revertant isolated from the original blm1-1 mutant strain and of mutant cells transformed with each of several recombinant plasmids which complemented the recessive blm1-1 mutation was comparable to the resistance of the parental (non-mutant) strain from which the original blm1-1 mutant was derived. The strategy for cloning S. cerevisiae DNA was based on complementation and in situ hybridization. This strategy employed 32P-labelled 6.6-kb BamHI and 3.8-kb BamHI-ClaI probes from a cloned DNA fragment to recover clones which either fully or partially complemented the hypersensitivity of mutant cells to killing by bleomycin. This method considerably reduced the time and effort required to recover biologically active clones from a genomic library.

Bleomycin affects cell wall anchorage of mannoproteins in Saccharomyces cerevisiae.

Bleomycin induces strand breakage in DNA through disruption of glycosidic linkages. We investigated the ability of bleomycin to damage yeast cell walls, which are composed primarily of carbohydrate. Bleomycin treatment of intact yeast cells facilitated enzymatic conversion of yeasts to spheroplasts. Bleomycin treatment also altered anchorage of mannoproteins to the cell wall matrix in intact cells or isolated cell walls. Cell surface mannoproteins were labelled with 125I, and their solubilization was monitored. Seventeen hour treatments with bleomycin released some of the label directly into treatment supernatants and facilitated extraction of mannoproteins by dithiothreitol and lytic enzymes. Bleomycin treatments as short as 10 min caused changes in extraction of mannoproteins from intact cells. Specifically, cell wall anchorage of several mannoproteins was affected by the drug. There were drug-induced changes in extractability of mannoproteins with apparent molecular weights of 96,000, 80,000, 61,000, 41,000, 31,500, and 21,000 (determined after deglycosylation with endo-N-acetylglucosaminidase H). The similarity of results obtained in the presence and absence of cycloheximide, the appearance of cell wall effects after only 10 min of treatment, and the similarity of effects in intact cells and isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanism dependent on expression of bleomycin-damaged genes or other intracellular mediators. The results are consistent with bleomycin-mediated increases in cell wall permeability through disruption of glycosidic cross-linking structures in the cell wall.

Lesions and preferential initial localization of [S-methyl-3H]bleomycin A2 on Saccharomyces cerevisiae cell walls and membranes.

Extensive lesions were produced in cell walls of Saccharomyces cerevisiae by the bleomycin family of anticancer antibiotics (30 min to 4 h). Electron micrographs revealed that the alterations were most frequently large breaks and small interruptions or holes in cell walls, which sometimes extended into cell membranes. Large portions of cell walls were sometimes lost. Cell walls were frequently ruptured in one or more positions. More than 75% of bud scar regions in single-plane sections and all bud scars in serial sections exhibited many interruptions and breaks after 3 or 4 h of treatment. The discovery of extensive damage to cell walls was consistent with the preferential (approximately 70%) association of radiolabeled bleomycin with cell walls and perimeters of bud scar regions after short exposures (30 min). After longer exposures, the distribution of silver grains changed from a predominant association with cell walls (30 min) to an increased association with the cell cytoplasm (1 to 4 h). This correlated with increased ultrastructural damage, since damage to cell walls was generally more frequent and more severe with increasing length of treatment (30 min to 4 h) or dose (25 to 100 micrograms/ml). Although DNA lesions are believed to be the lethal properties of bleomycins, the lesions produced in cell walls are also lethal properties of the antibiotics. The distributions of lesions on cell walls suggested a generalized interaction of the antibiotic with a cell wall component. These results led us to hypothesize a mechanism of effective antifungal action for the bleomycin family of antibiotics.

Disseminated cutaneous and synovial Mycobacterium marinum infection in a patient with systemic lupus erythematosus.

A patient with systemic lupus erythematosus had a protracted skin infection with Mycobacterium marinum after a puffer fish sting. Disseminated cutaneous and synovial disease was associated with clinically active systemic lupus erythematosus two years after the initial infection. The infection was poorly responsive to multiple antituberculous regimens. Hematogenous spread of infection was the likely route of dissemination.