Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed.

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG(+/+) and UNG(-/-) cells. We report that despite identical growth rates and DSB repair capacities, UNG(-/-) cells accumulated significantly greater uracil and DSBs compared with UNG(+/+) cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG(+/+) cells. Furthermore, DSBs in UNG(+/+) and UNG(-/-) cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG(-/-) cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG(+/+) and UNG(-/-) cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG(-/-) but not UNG(+/+) cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG(-/-) cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.

Canadian integrative oncology research priorities: results of a consensus-building process.

BACKGROUND: In Canada, many diverse models of integrative oncology care have emerged in response to the growing number of cancer patients who combine complementary therapies with their conventional medical treatments. The increasing interest in integrative oncology emphasizes the need to engage stakeholders and to work toward consensus on research priorities and a collaborative research agenda. The Integrative Canadian Oncology Research Initiative initiated a consensus-building process to meet that need and to develop an action plan that will implement a Canadian research agenda. METHODS: A two-day consensus workshop was held after completion of a Delphi survey and stakeholder interviews. RESULTS: FIVE INTERRELATED PRIORITY RESEARCH AREAS WERE IDENTIFIED AS THE FOUNDATION FOR A CANADIAN RESEARCH AGENDA: EffectivenessSafetyResource and health services utilizationKnowledge translationDeveloping integrative oncology models Research is needed within each priority area from a range of different perspectives (for example, patient, practitioner, health system) and in a way that reflects a continuum of integration from the addition of a single complementary intervention within conventional cancer care to systemic change. Strategies to implement a Canadian integrative oncology research agenda were identified, and working groups are actively developing projects in line with those strategic areas. Of note is the intention to develop a national network for integrative oncology research and knowledge translation. CONCLUSIONS: The identified research priorities reflect the needs and perspectives of a spectrum of integrative oncology stakeholders. Ongoing stakeholder consultation, including engagement from new stakeholders, is needed to ensure appropriate uptake and implementation of a Canadian research agenda.

Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair.

Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG(+/+)/UDG(-/-)). UDG(-/-) cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG(+/+) cells were >10 times more resistant to pemetrexed due to the rapid removal of uracil from DNA by UDG and subsequent repair of the resultant AP sites (abasic sites) via the base excision repair (BER). The resistance to pemetrexed in UDG(+/+) cells could be reversed by the addition of methoxyamine (MX), which binds to AP sites and interrupts BER pathway. Furthermore, MX-bound AP sites induced cell death was related to their cytotoxic effect of dual inactivation of UDG and topoisomerase IIα, two genes that are highly expressed in lung cancer cells in comparison with normal cells. Thus, targeting BER-based therapy exhibits more selective cytotoxicity on cancer cells through a synthetic lethal mechanism.

A systematic review of integrative oncology programs.

OBJECTIVE: This systematic review set out to summarize the research literature describing integrative oncology programs. METHODS: Searches were conducted of 9 electronic databases, relevant journals (hand searched), and conference abstracts, and experts were contacted. Two investigators independently screened titles and abstracts for reports describing examples of programs that combine complementary and conventional cancer care. English-, French-, and German-language articles were included, with no date restriction. From the articles located, descriptive data were extracted according to 6 concepts: description of article, description of clinic, components of care, administrative structure, process of care, and measurable outcomes used. RESULTS: Of the 29 programs included, most were situated in the United States (n = 12, 41%) and England (n = 10, 34%). More than half (n = 16, 55%) operate within a hospital, and 7 (24%) are community-based. Clients come through patient self-referral (n = 15, 52%) and by referral from conventional health care providers (n = 9, 31%) and from cancer agencies (n = 7, 24%). In 12 programs (41%), conventional care is provided onsite; 7 programs (24%) collaborate with conventional centres to provide integrative care. Programs are supported financially through donations (n = 10, 34%), cancer agencies or hospitals (n = 7, 24%), private foundations (n = 6, 21%), and public funds (n = 3, 10%). Nearly two thirds of the programs maintain a research (n = 18, 62%) or evaluation (n = 15, 52%) program. CONCLUSIONS: The research literature documents a growing number of integrative oncology programs. These programs share a common vision to provide whole-person, patient-centred care, but each program is unique in terms of its structure and operational model.

Effects of nitrocompounds on uric acid-utilizing microorganisms, nitrogen retention, and microbial community in laying hen manure.

A study was conducted to evaluate effects of nitrocompounds on growth of uric acid-utilizing microorganisms, nitrogen retention, and microbial community in laying hen manure. There were three treatments: control, 100 mM nitropropanol (NPL), and 100 mM nitropropionic acid (NPC). The mixed laying hen manure was divided into 3 groups and incubated at 23 degrees C for 7 days. On Days 0, 3, and 7, samples were collected to measure the quantity of uric acid-utilizing microorganisms, total nitrogen retention, and microbial community changes. Both nitrocompounds significantly reduced growth of the uric acid-utilizing microorganisms on Day 3 (P < 0.05). Inhibitory effects of both nitrocompounds remained until Day 7 when the experiment was terminated. NPL treatment retained significantly more manure nitrogen compared to the control on both Days 3 and 7. Manure nitrogen levels of NPC treatment were also significantly higher than the control on Day 7. We further investigated the effects of NPL and NPC on microbial community changes during a 7-day incubation. NPC treatment and control on Day 7 exhibited 94% community similarity. NPC on Day 3 and NPL on Day 7 also showed high community similarity (approximately 94%). Control on Day 0 and Day 7 yielded less than 80% community similarity. Control and NPL treatment groups on Day 3 gave the lowest community similarity (approximately 64%) compared to the other groups. This result indicated that incubation time and treatment moderately influenced microbial community changes. In summary, these results indicate that both nitrocompounds increased manure nitrogen retention by inhibiting the growth of uric acid-utilizing microorganisms, and that NPL and NPC could be used as manure treatments in order to reduce ammonia volatilization and nitrogen retention in poultry manure. Moreover, nitrocompounds may have potential as feed additives to reduce ammonia volatilization.

Principles to guide integrative oncology and the development of an evidence base.

BACKGROUND: Integrative oncology uses both conventional and complementary medicine to meet the needs of individual patients and to focus on the whole person. The core principles of integrative oncology include individualization, holism, dynamism, synergism, and collaboration, but the nature of the evidence to guide the development of integrative oncology has been given little attention. OBJECTIVES: To discuss the need for evidence to support the integration of complementary therapies for integrative oncology care. To emphasize that the evidence base must be valid and respect the underlying principles of individual complementary therapies and integrative oncology practice. To suggest ways to begin developing the evidence base. REVIEW AND DISCUSSION: Although the evidence for safety and efficacy seems paramount for supporting the integration of an individual complementary therapy into mainstream cancer care, the need for evidence to support the overall practice of integrative oncology has to be considered as well. We argue that developing an evidence base for integrative oncology requires a contextual and comprehensive research approach that assesses a range of outcomes over a suitable period of time that the patient and the patient's family, in addition to the health care providers, deem important. CONCLUSION: A whole-systems framework to the development of the evidence base for integrative oncology can guide the development of evidence that respects the complex nature of many complementary and integrative practices and their underlying principles of care delivery.

Patient decision-making about complementary and alternative medicine in cancer management: context and process.

OBJECTIVE: In this paper, we set out to describe the personal and social contexts of treatment decisions made by cancer patients concerning complementary and alternative medicine (CAM) and also the process through which cancer patients reach cam decisions throughout the cancer trajectory. METHODS: We selected and reviewed a variety of CAM decision-making models published in the past 10 years within the Canadian health literature. RESULTS: The cam decision-making process is influenced by a variety of sociodemographic, disease-related, psychological, and social factors. We reviewed four main phases of the cam decision-making process: Taking stock of treatment options. Gathering and evaluating CAM information. Making CAM decisions. Revisiting the cam decision. Immediately following diagnosis, cancer patients become interested in taking stock of the full spectrum of conventional and CAM treatment options that may enhance the effectiveness of their treatment and mediate potential side effects. Information about CAM is then gathered from numerous information sources that vary in terms of credibility and scientific legitimacy, and is evaluated. When making a decision regarding CAM options, patients attempt to make sense of the diverse information obtained, while acknowledging their beliefs and values. The CAM decision is often revisited at key milestones, such as the end of conventional treatment and when additional information about disease, prognosis, and treatment is obtained. CONCLUSIONS: The CAM decision-making process is a dynamic and iterative process that is influenced by a complex array of personal and social factors. Oncology health professionals need to be prepared to offer decision support related to CAM throughout the cancer trajectory.

Scanning tunnelling microscopy imaging of [3 x 3] Mn nonanuclear grids.

The scanning tunnelling microscopy imaging of [3 x 3] Mn(II) nonanuclear grids on gold substrates is described. Self-assembled behaviour is observed at both high and low coverage, with submolecular resolution of individual molecules displayed at low deposition concentrations. The importance of proper image processing techniques is demonstrated in resolving the layer structure at high coverage.

Past goals/future challenges.

At the 2001 MHA Annual Corporate Membership Meeting in June, the Michigan Association of Hospital Auxiliaries (MAHA) saw one president step down and a new one begin a one-year appointment. The following are the thoughts of each woman as they assume their respective positions as counselor and president.

Remodeling of the vessel wall after copper-induced injury is highly attenuated in mice with a total deficiency of plasminogen activator inhibitor-1.

Clinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1(-/-)) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1(-/-) mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1(-/-) mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1(-/-) arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation.

Utility of flow cytometry immunophenotyping for the diagnosis and classification of lymphoma in community hospital clinical needle aspiration/biopsies.

CONTEXT: Flow cytometry immunophenotyping (FC) of needle aspiration/biopsy (NAB) samples has been reported to be useful for the diagnosis and classification of lymphoma in university and cancer center-based settings. Nevertheless, there is no agreement on the utility of these methods. OBJECTIVE: To further define the utility of adjunctive FC of clinical NAB for the diagnosis and classification of lymphoma, and to determine if this approach is practicable in a routine clinical practice setting. SETTING: A community-based hospital. METHODS: Clinical NABs were submitted for adjunctive FC between June 1996 and September 1999 if initial smears were suspicious for lymphoma. Smears and cell block or needle core tissues were routinely processed and paraffin-section immunostains were performed if indicated. The final diagnosis was determined by correlating clinical and pathologic data, and the revised European-American classification criteria were used to subtype lymphomas. RESULTS: Needle aspiration/biopsies from 60 different patients were submitted for FC. Final diagnoses were lymphoma (n = 38), other neoplasm (n = 15), benign (n = 6), or insufficient (n = 1). For 38 lymphomas (20 primary, 18 recurrent), patients ranged in age from 32 to 86 years (mean, 62 years); samples were obtained from the retroperitoneum (n = 11), lymph node (n = 9), abdomen (n = 8), mediastinum (n = 6), or other site (n = 4); and lymphoma subtypes were indolent B-cell (n = 20; 2 small lymphocytic, 14 follicle center, 4 not subtyped), aggressive B-cell (n = 14; 3 mantle cell, 10 large cell, 1 not subtyped), B-cell not further specified (n = 2), or Hodgkin disease (n = 2). For the diagnosis of these lymphomas, FC was necessary in 20 cases, useful in 14 cases, not useful in 2 cases, and misleading in 2 cases. Thirty-two of 36 lymphoma patients with follow-up data received antitumor therapy based on the results of NAB plus FC. CONCLUSIONS: Adjunctive FC of NABs is potentially practicable in a community hospital, is necessary or useful for the diagnosis and subtyping of most B-cell lymphomas, and can help direct lymphoma therapy. Repeated NAB or surgical biopsy is necessary for diagnosis or treatment in some cases.

Germline mutations in the vhl gene in patients presenting with phaeochromocytomas.

It has been shown that an appreciable percentage of patients presenting with primary, apparently sporadic phaeochromocytomas may in fact have von-Hippel-Lindau (VHL) disease. In order to investigate this, we retrospectively screened 68 patients, who had been operated on for phaeochromocytomas, for the presence of germline mutations in the vhl gene. DNA was isolated from peripheral-blood leukocytes and used to screen the entire coding sequence and the intron-exon boundaries of the vhl gene for mutations, using a PCR-based SSCP strategy. When an abnormal pattern was found in the SSCP analysis, sequence analysis was carried out. We found SSC variants in the vhl gene in 8 of the 68 patients. Of 6 patients, 2 turned out to be related (an uncle and his nephew), and they carried the same mis-sense mutation: R64P. In 4 other patients, mis-sense mutations, P25L, L63P, G144Q and I147T, were also identified. None of these mutations has been described, and 3 of them (P25L, L63P and R64P) are located closer to the N terminus of the vhl protein than any reported vhl mutation. In the remaining 2 cases, the mutations were localized not in the coding sequence but in the intronic sequence (but not within splice-sites), adjacent to the exon, so they were probably not related to the disease. Our results show that a relatively high proportion (6/68, or 8.8%), though not as high as the 20% reported earlier, of patients with apparently sporadic phaeochromocytomas may carry germline mutations in the vhl gene.

Common C-to-T substitution at position -480 of the hepatic lipase promoter associated with a lowered lipase activity in coronary artery disease patients.

We studied the molecular basis of low hepatic lipase (HL) activity in normolipidemic male patients with angiographically documented coronary artery disease (CAD). In 18 subjects with a lowered HL activity (< 225 mU/mL), all nine exons of the HL gene and part of the promoter region (nucleotides -524 to +7) were sequenced. No structural mutations in the coding part of the HL gene were found, but 50% of the subjects showed a C-to-T substitution at nucleotide -480. Screening for the base substitution in 782 patients yielded an allele frequency of 0.213 (297 heterozygotes, 18 homozygotes). In a group of 316 nonsymptomatic control subjects, the allele frequency was 0.189, which is significantly less than in the CAD patients (P = .035). In the CAD patients, the C-to-T substitution was associated with a lowered lipase activity (heterozygotes -15%, homozygotes -20%). The patients were divided into quartiles on the basis of HL activity. Sixty percent (allele frequency 0.32) of the patients in the lowest quartile (HL activity < 306 mU/mL) had the gene variant against 27% (allele frequency 0.14) in the highest quartile (HL activity > 466 mU/mL). In the noncarriers, but not in the carriers, HL activity was related with plasma insulin, being increased at higher insulin concentration. Homozygous carriers had a significantly higher HDL cholesterol level-than noncarriers (1.13 +/- 0.28 mmol/L versus 0.92 +/- 0.22 mmol/L, P < .02). Our results show that a C-to-T substitution at -480 of the HL promoter is associated with a lowered HL activity. The base substitution, or a closely linked gene variation, may contribute to the variation in HL activity and affect plasma lipoprotein metabolism.

Graduate nursing education. What are the benefits and costs to hospitals?

In 1989, Hermann Hospital and University of Texas-Houston Health Science Center School of Nursing applied for and received approval to implement a 5-year demonstration project funded by the Health Care Finance Administration using Medicaid pass-through funds to educate nurses at the graduate level. The project aimed to evaluate the impact of advanced practice nurses and researchers on the practice of professional nursing at a 655-bed acute-care hospital in a large metropolitan area. The authors describe the factors that served to galvanize the two institutions to apply for such a grant, discuss the barriers encountered along the course of the project, and list the outcomes achieved by 1994 or project end. Predictions regarding the second 5-year segment (phase 2) are summarized as well. The article includes suggestions to other institutions interested in replicating such a project.

Diminished activity of ceftizoxime in comparison to cefotaxime and ceftriaxone against Streptococcus pneumoniae.

Previous reports documenting the essentially identical spectra of activity of ceftizoxime, cefotaxime, and ceftriaxone prompted our hospital formulary committee to replace the latter two drugs with ceftizoxime on the basis of cost differences. However, we subsequently observed that every one of 60 isolates of Streptococcus pneumoniae tested was less susceptible to ceftizoxime than to either cefotaxime or ceftriaxone. The difference between minimal inhibitory concentrations (MICs) was greatest for strains moderately or fully resistant to penicillin, which at our institution represent approximately 32% of all isolates of S. pneumoniae. Ten isolates with cefotaxime and ceftriaxone MICs of 2.0-6.0 micrograms/mL had ceftizoxime MICs of > or = 256 micrograms/mL. Time-kill kinetic studies assessing bactericidal activity confirmed the diminished activity of ceftizoxime against penicillin-resistant isolates of S. pneumoniae. Ceftizoxime should not be used to treat proven or suspected pneumococcal infection in areas where resistance to penicillin is prevalent.

Regional anaesthesia and subsequent long-term pain.

The incidence of long-term pain (between 1 and 48 weeks and at 2 year follow-up) unrelated to the surgical site following either regional brachial plexus or general anaesthesia was determined. In 834 patients with regional anaesthesia, the incidence (11.1%) was significantly higher than in the 86 patients with general anaesthesia (3.6%; P = 0.03). The incidence of pain was not significantly different among four common techniques of positioning the needle tip in the axillary sheath (9.9 to 11.1%). Parascalene blocks had a slightly but not significantly higher rate (16.3%). A regional re-block was not associated with a higher incidence when compared to those blocked only once. A more distal local re-block was associated with a higher incidence of pain (23%). 2 years post-operatively, 0.5% of patients had pain related to the regional block. A significant proportion of patients developed some long-lasting post-operative pain following regional brachial plexus anaesthesia, although ultimate morbidity was minimal.

Inhibitory and bactericidal activity of selected beta-lactam agents alone and in combination with beta-lactamase inhibitors compared with that of cefoxitin and metronidazole against cefoxitin-susceptible and cefoxitin-resistant isolates of the Bacteroides fragilis group.

The inhibitory activity of five beta-lactam agents, alone and in combination with a beta-lactamase inhibitor, was compared with that of cefoxitin and metronidazole against 300 beta-lactamase producing Bacteroides fragilis group isolates. Each of the beta-lactamase inhibitors significantly potentiated the activity of the respective beta-lactam. In the presence of clavulanate, the MIC90 (minimum inhibitory concentration) values of amoxicillin and ticarcillin were reduced 64-fold and 32-fold, respectively. Similarly, sulbactam enhanced the activity of ampicillin and cefoperazone 16-fold and 8-fold, respectively, whereas tazobactam potentiated the activity of piperacillin 16-fold. Few strains were resistant to the beta-lactam-beta-lactamase inhibitor combinations and were comprised of strains of B. fragilis, B. thetaiotamicron, and B. distasonis. Of the strains, 7% were resistant to cefoxitin, and none to metronidazole. Using time-kill kinetic studies, the bactericidal activity of the various beta-lactam agents, with and without beta-lactamase inhibitors, was determined and compared with that of cefoxitin and metronidazole against cefoxitin-susceptible and cefoxitin-resistant isolates of the B. fragilis group. Overall, metronidazole was the most bactericidal agent with all isolates being killed with less than or equal to 4 micrograms/ml at 24 hr. Ampicillin-sulbactam was the next most bactericidal agent with all isolates being killed with less than or equal to 16/8 micrograms/ml of ampicillin-sulbactam at 24 hr. Amoxicillin-clavulanate and cefoperazone-sulbactam had bactericidal activity similar to that of ampicillin-sulbactam. Piperacillin-tazobactam and ticarcillin-clavulanate were bactericidal at higher concentrations with all isolates killed with 64 micrograms/ml of piperacillin and 128 micrograms/ml of ticarcillin combined with their respective beta-lactamase inhibitors. None of the beta-lactam agents alone was able to kill more than 19 of the 26 isolates. We conclude that beta-lactam agents combined with beta-lactamase inhibitors have both inhibitory and bactericidal activity against cefoxitin-resistant members of the B. fragilis group provided that the concentrations achieved for these combinations are at the upper limits for maximum recommended dosing. Although isolates of the B. fragilis group have been reported to produce unusual beta-lactamases that are refractory to beta-lactamase inhibitors, none of the cefoxitin-resistant isolates tested in this study were resistant to the beta-lactam-beta-lactamase inhibitor combinations.