Underexpression of tumour suppressor LKB1 in clear cell renal cell carcinoma is common and confers growth advantage in vitro and in vivo.

BACKGROUND: Evidence suggests that dysregulation of energy-sensing pathways closely associates with renal cell carcinoma (RCC) development. The metabolic regulation is largely controlled by 5'-AMP activated protein kinase (AMPK) which is activated through phosphorylation by LKB1. METHODS: The expression of LKB1 was determined by reverse transcription-PCR using 10 clinical clear cell RCC (ccRCC) samples and their adjacent normal renal parenchyma, and by immunohistochemical staining of two tissue microarrays containing 201 ccRCC and 26 normal kidney samples. Expression of LKB1 was knocked down in human ccRCC 786-O cells (shLKB1) and compared with cells expressing scrambled control shRNA (shControl). AMPK signalling, proliferation, invasion, and VEGF secretion was measured. The cells were subcutaneously injected into mice to determine tumour growth in vivo. RESULTS: At the protein and transcript levels, a significant reduction in LKB1 expression in tumour compared with normal tissue was found. In vitro, knockdown of LKB1 resulted in reduced AMPK signalling and increased cellular proliferation, invasion, and VEGF secretion compared with shControl cells. In vivo, growth of shLKB1 ccRCC xenografts in nude mice was significantly increased compared with shControl xenografts. CONCLUSION: Collectively, our results suggest that LKB1 acts as a tumour suppressor in most sporadic cases of ccRCC and that underexpression of LKB1 is a common event in the disease.

Why people choose to not use complementary therapies during cancer treatment: a focus group study.

While 50% of cancer patients use complementary therapies (CT) during treatment, few studies have examined why individuals choose not to use CT. This study aimed to address this gap in knowledge using a focus group methodology, where 36 participants took part in one of eight groups; participants were recruited until saturation of themes was achieved. Three categories of participants were investigated: patients/recent survivors (n= 14); volunteers/advocates (n= 16), the majority of whom were also long-term survivors; and health professionals (n= 6). Focus groups were digitally audio-recorded, transcribed, and coded thematically using NVivo software. Reasons for non-use fell into four broad themes: (1) Resource barriers, particularly the cost and lack of time; (2) fear and distrust, including the potential for drug interactions; (3) lack of evidence, including the unproven nature of many CT practices; and (4) satisfaction with conventional treatment. Two further themes related to the benefits of non-use and reasons for discontinuation. A sub-analysis indicated that reasons for non-use differed by CT category, with non-use being mentioned more frequently for biologically based and body-based therapies. Differences in understanding CT non-use emerged between patients, volunteers and health professionals. Findings have clinical implications regarding tailoring information for patients during and after cancer.

The impact of COVID-19 on cancer patients, their carers and oncology health professionals: A qualitative study.

OBJECTIVE: Cancer patients, carers and oncology health professionals have been impacted by the COVID-19 pandemic in many ways, but their experiences and psychosocial responses to the pandemic are still being explored. This study aimed to document the experience of Australians living with cancer, family carers, and Oncology health professionals (HPs) when COVID-19 first emerged. METHODS: In this qualitative study, participants (cancer patients currently receiving treatment, family carers and HPs) completed a semi-structured interview exploring their experiences of COVID-19 and the impact it had on cancer care. Participants also completed the Hospital Anxiety and Depression Scale (patients) and the Depression, Anxiety and Stress Scale (carers and HPs) to assess emotional morbidity. Thematic analysis was undertaken on qualitative data. RESULTS: 32 patients, 16 carers and 29 HPs participated. Qualitative analysis yielded three shared themes: fear and death anxiety, isolation, and uncertainty. For HPs, uncertainty incorporated the potential for moral distress and work-stress. Patients and carers scoring high on anxiety/depression measures were more likely to have advanced disease, expressed greater death anxiety, talked about taking more extreme precautionary measures, and felt more impacted by isolation. CONCLUSION: Cancer and COVID-19 can have compounding psychological impacts on all those receiving or giving care. PRACTICE IMPLICATIONS: Screening for distress in patients, and burnout in HPs, is recommended. Increased compassionate access and provision of creative alternatives to face-to-face support are warrented.

The mechanism of loading of the FLP recombinase onto its DNA target sequence.

The FLP recombinase interacts with its target sequence with the formation of three distinct DNA-protein complexes. The first complex leaves neither a DNase footprint nor is the DNA protected from methylation by dimethyl sulfate. We have found, however, that the FLP protein is bound predominantly to only one of the three 13 base-pair (bp) symmetry elements. This asymmetric loading of the FLP site seems to require the presence of an adjacent directly repeated 13 bp element. We speculate that this asymmetric filling of the target site may be accompanied by the unique order of cleavage and exchange of DNA strands.

Synaptic intermediates promoted by the FLP recombinase.

We have devised a novel assay to trap nucleoprotein synaptic intermediates of the FLP recombination reaction. DNase I footprinting analysis of these intermediates indicates that synapsis is mediated by protein-protein interactions between FLP molecules bound to each FLP recombination target (FRT) site. Under certain conditions we have observed a synaptic structure in which the FRT sites have come together in an aberrant arrangement. Although our analysis shows that homology between the core sequences of the sites is not a prerequisite for synapsis, the data suggest that homology between cores dictates the directionality of the reaction. Many of the intermediates contain a Holliday junction indicating that the FLP protein has catalysed strand exchanges between the FRT sites. The general scheme of the assay should prove useful to analyse nucleoprotein intermediates in other site-specific recombination systems, and to investigate protein-protein and protein-DNA interactions in intermediates important for DNA replication and transcription.

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.

Isolation of intermediates in the binding of the FLP recombinase of the yeast plasmid 2-micron circle to its target sequence.

We describe a method for isolating and characterizing intermediates in the binding of the FLP recombinase, encoded by the yeast plasmid 2-micron circle to its target sequence. On a wild-type substrate, three specific complexes are formed. Footprinting analysis of the gel-purified complexes shows that each complex is the result of a unique FLP-DNA association. On the basis of the behavior of various FLP target sequences in the gel-binding assay, we propose a model describing the steps that lead to the formation of a stable FLP-DNA complex.

Cross-national interrater agreement on the clinical diagnostic criteria for dementia. WHO-PRA Age-Associated Dementia Working Group, WHO-Program for Research on Aging, Health of Elderly Program.

We assessed the interobserver agreement on the clinical diagnosis of dementia syndrome and dementia subtypes as part of a cross-national project on the prevalence of dementia. Fourteen clinicians from the participating countries (Canada, Chile, Malta, Nigeria, Spain, and the United States) independently assessed the diagnosis of 51 patients whose clinical information was in standard records written in English. We used the DSM-III-R and ICD-10 criteria for dementia syndrome, the NINCDS-ADRDA criteria for Alzheimer's disease (AD), and the ICD-10 criteria for other dementing diseases, and measured interobserver agreement. We found comparable levels of agreement on the diagnosis of dementia using the DSM-III-R (kappa = 0.67) as well as the ICD-10 criteria (kappa = 0.69). Cognitive impairment without dementia was a major source of disagreement (kappa = 0.10). The kappa values were 0.58 for probable AD, 0.12 for possible AD, and rose to 0.72 when the two categories were merged. The interrater reproducibility of the diagnosis of vascular dementia was 0.66 in terms of kappa index; the diagnoses of other dementing disorders as a whole reached a kappa value of 0.40. This study suggests that clinicians from different cultures and medical traditions can use the DSM-III-R and the ICD-10 criteria for dementia effectively and thus reliably identify dementia cases in cross-national research. The interrater agreement on the diagnosis of dementia might be improved if clear-cut guidelines in the definition of cognitive impairment are provided. To improve the reliability of AD diagnosis in epidemiologic studies, we suggest that the NINCDS-ADRDA "probable" and "possible" categories be merged.

Prophylaxis of heart transplant rejection with either antithymocyte globulin-, Minnesota antilymphocyte globulin-, or an OKT3-based protocol.

We compared an equine antithymocyte globulin (ATGAM)-based protocol with a Minnesota antilymphocyte globulin (MALG)-based protocol and a murine monoclonal CD-3 (OKT-)-based protocol in 3 groups of heart transplant (HT) recipients. Thirty-four recipients received a four-day course of ATGAM. Thirty HT recipients received a 14-day course of OKT3. Fifteen HT recipients received MALG for an average of 10 days. The ATGAM group received cyclosporine beginning preoperatively, while the OKT3 and MALG groups received CyA beginning on post-transplant day 4. All three groups received identical azathioprine and similar steroid therapy. The 3 groups were similar in age, donor/recipient HLA mismatches, and donor/recipient gender mismatches. The MALG and OKT3 groups had 20% and 17% females, respectively, while the ATG group had 41% (p < 0.05). Average follow-up exceeded 14 months for each group. The ATGAM group received a higher dose of CyA during "induction" therapy than the OKT3 and MALG groups, and experienced a greater rise in post-transplant serum creatinine levels. We found no difference between the 3 groups in: preoperative creatinine levels, one-year post-transplant creatinine levels, number of patients who could be successfully "weaned" from steroids, or one-year survival. Other data are tabulated as episodes/patient: [table: see text] We conclude that ATG plus preoperative CyA is superior for rejection prophylaxis following heart transplantation when compared with either MALG plus postoperative CyA or OKT3 plus postoperative CyA.

Attitudes of Iowa dental hygienists regarding self-regulation and practice.

PURPOSE: This study investigated Iowa dental hygienists' perceptions about education, practice, and licensure regarding issues related to self-regulation. Furthermore, it was the purpose of the study to determine if these perceptions were statistically related to membership in the American Dental Hygienists' Association (ADHA). METHODS: In October 1992, 150 Iowa dental hygienists were randomly selected to participate in a mail survey. The questionnaire included items on self-regulation, licensure, practice, supervision, delivery systems, and independent practice. Responses were analyzed by frequency distributions and the chi-square statistic. RESULTS: Ninety responses were received, representing 80% of the 112 subjects with valid Iowa addresses, but only 60% of the selected sample. Of the 36 variables analyzed, only two were statistically significantly different by ADHA membership. Thus, data were combined and reported for all respondents. The majority of Iowa dental hygienists (71.6%) believed dental hygiene was pursuing self-regulation to improve quality of care. They wanted more dental hygiene representation on licensure boards and almost 50% wanted separate boards. The majority of respondents favored control of dental hygiene practice and 70% wanted alternatives to the current dental hygiene care delivery system. Most respondents stated general supervision and independent practice would increase access to oral healthcare without increasing patients' risk of health problems. CONCLUSIONS: The majority of Iowa dental hygienists support the dental hygiene profession's goal of self-regulation. ADHA members and nonmembers have similar attitudes and opinions about dental hygiene self-regulation, licensure, delivery systems, and practice.

Evaluating the impact of cancer on complementary and alternative medicine use, distress and health related QoL among Australian women: a prospective longitudinal investigation.

OBJECTIVES: While several cross-sectional studies have examined psychological correlates of complementary and alternative medicine (CAM) use and cancer, few prospective longitudinal investigations have been reported. This study examined whether CAM use moderated distress and quality of life (HRQoL) from pre- to post-cancer. DESIGN: A prospective longitudinal national cohort design. SETTING: Participants were 718 mid-aged women from the Australian longitudinal study on women's health who did not have cancer at survey 1, but who subsequently developed cancer. For each participant, three waves of data were extracted: the wave prior to diagnosis ('pre'), at diagnosis ('cancer'), and after cancer ('post'). MAIN OUTCOME MEASURES: CAM use was measured by the question 'in the past 12 months have you consulted an alternative health practitioner'. Distress was measured by perceived stress (PSS) and depression (CES-D 10), HRQoL was measured by physical and mental health functioning (SF-36). RESULTS: CAM use significantly moderated the change over time in stress [F(561)=3.09, p=0.04], depression [F(494)=3.14, p=0.04], but not HRQoL. CAM-users were significantly more stressed than non-users pre-cancer (p<0.05), but there were no significant differences at subsequent surveys. CAM-users were significantly less depressed post-cancer compared to non-users (p<0.05). CONCLUSIONS: Findings indicated that CAM users may be more psychologically vulnerable than non-users with respect to stress, with CAM acting as an effective psychological, but not HRQoL, intervention.

Characteristics of operators of adult psychiatric foster homes.

To discover characteristics of adult foster home operators that might improve future selection and training procedures, the authors studied 39 operators who lived and worked with chronic psychiatric patients from a VA hospital. The findings supported their hypothesis that foster home operators have higher behavioral expectations of the mentally ill than does the normative population, in this case a group of college sophomores. Other findings included the fact that the operators most preferred clients diagnosed as being depressed or anxious and least preferred those who were alcoholic. The reason most gave for being foster home operators was that they wanted to help people.

In vitro recombination of bacteriophage T7 DNA: further characterization of the reaction using plasmid DNA.

We have used a plasmid which contains a cloned fragment of T7 DNA to study the properties of general recombination of phage T7 in vitro. It was shown that T7-infected cell extracts promote recombination by the exchange of double strands of DNA. While both products of these double-strand exchanges were detected, we were unable to show that they were formed during a single recombination event.

The FLP protein contacts both major and minor grooves of its recognition target sequence.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences.

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.

Dimensions of happiness: a qualitative study of family practice residents.

BACKGROUND: Happiness is related to both positive and negative forces. Positive factors, or happiers, that compensate for stressors in residency have only recently been recognized in a pilot study of 14 family practice residents. These happiers are positivism, the pursuit of goals, relationships, a religious belief system, and feedback. The purpose of this qualitative study was to describe happiers of residency in family practice in each postgraduate year and relate the findings to established theories on well-being. METHODS: A qualitative interview with participant observation was used to study 59 residents at four family practice residency sites. Interviews were semistructured and audiotaped. Tapes and field notes were analyzed for trends. The Faces Scale was administered as a happiness visual analog scale. RESULTS: The importance of a well-balanced lifestyle, as well as the pursuit of goals, relationships, and accentuation of the positive, were found to be happiers for all residents interviewed. Religious commitment, feedback, and a sense of control or autonomy were important to the level of happiness of more than 60 percent of residents interviewed. Feedback was more important to 1st-year residents, and a sense of control was more important to 2nd-year and 3rd-year residents. CONCLUSION: The seven happiers described in this study were similar to happiness factors reported in the social science literature. Our findings could contribute to theories of resident well-being and further the research on well-being in residency training.

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene.

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.

Site-specific recombination of yeast 2-micron DNA in vitro.

Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two 599-base-pair precise inverted repeats across which a site-specific inversion event occurs in vivo. This inversion is promoted by a plasmid-encoded function called "FLP." We have cloned the FLP gene of 2-micron DNA under control of a strong yeast promoter and transformed yeast cells with a plasmid containing the cloned FLP gene. Cell-free extracts from such a transformant promote highly efficient inversion of 2-micron DNA in vitro. The reaction requires a cation and works efficiently on supercoiled, relaxed circular, or linear DNA. The FLP activity bears certain similarities to the cre protein, a site-specific recombinase encoded by bacteriophage P1.