Understanding radiation response and cell cycle variation in brain tumour cells using Raman spectroscopy.

Radiation therapy is currently utilised in the treatment of approximately 50% of cancer patients. A move towards patient tailored radiation therapy would help to improve the treatment outcome for patients as the inter-patient and intra-patient heterogeneity of cancer leads to large differences in treatment responses. In radiation therapy, a typical treatment outcome is cell cycle arrest which leads to cell cycle synchronisation. As treatment is typically given over multiple fractions it is important to understand how variation in the cell cycle can affect treatment response. Raman spectroscopy has previously been assessed as a method for monitoring radiation response in cancer cells and has shown promise in detecting the subtle biochemical changes following radiation exposure. This study evaluated Raman spectroscopy as a potential tool for monitoring cellular response to radiation in synchronised versus unsynchronised UVW human glioma cells in vitro. Specifically, it was hypothesised that the UVW cells would demonstrate a greater radiation resistance if the cell cycle phase of the cells was synchronised to the G1/S boundary prior to radiation exposure. Here we evaluated whether Raman spectroscopy, combined with cell cycle analysis and DNA damage and repair analysis (γ-H2AX assay), could discriminate the subtle cellular changes associated with radiation response. Raman spectroscopy combined with principal component analysis (PCA) was able to show the changes in radiation response over 24 hours following radiation exposure. Spectral changes were assigned to variations in protein, specifically changes in protein signals from amides as well as changes in lipid expression. A different response was observed between cells synchronised in the cell cycle and unsynchronised cells. After 24 hours following irradiation, the unsynchronised cells showed greater spectral changes compared to the synchronised cells demonstrating that the cell cycle plays an important role in the radiation resistance or sensitivity of the UVW cells, and that radiation resistance could be induced by controlling the cell cycle. One of the main aims of cancer treatment is to stop the proliferation of cells by controlling or halting progression through the cell cycle, thereby highlighting the importance of controlling the cell cycle when studying the effects of cancer treatments such as radiation therapy. Raman spectroscopy has been shown to be a useful tool for evaluating the changes in radiation response when the cell cycle phase is controlled and therefore highlighting its potential for assessing radiation response and resistance.

Effectiveness of early diagnosis of cerebral palsy guideline implementation: a systematic review.

INTRODUCTION: Tailored implementation interventions are required to overcome the diagnostic research-practice gap for cerebral palsy (CP). Evaluating the impact of interventions on patient outcomes is a priority. This review aimed to summarize the established evidence for the effectiveness of guideline implementations in lowering the age of CP diagnosis. EVIDENCE ACQUISITION: A systematic review was conducted according to PRISMA. CINAHL, Embase, PubMed and MEDLINE were searched (2017-October 2022). Inclusion criteria were studies that evaluated effect of CP guideline interventions on health professional behaviour or patient outcomes. GRADE was used to determine quality. Studies were coded for use of theory (Theory Coding Scheme). Meta-analysis was performed and a standardized metric used to summarize statistics of intervention effect estimates. EVIDENCE SYNTHESIS: Of (N.=249) records screened, (N.=7) studies met inclusion, comprising interventions following infants less than 2 years of age with CP risk factors (N.=6280). Guideline feasibility in clinical practice was established through health professional adherence and patient satisfaction. Efficacy of patient outcome of CP diagnosis by 12 months of age was established in all studies. Weighted averages were: (1) high-risk of CP (N.=2) 4.2 months and (2) CP diagnosis (N.=5) at 11.6 months. Meta-analysis of (N.=2) studies found a large, pooled effect size Z = 3.00 (P=0.003) favoring implementation interventions lowering age of diagnosis by 7.50 months, however study heterogeneity was high. A paucity of theoretical frameworks were identified in this review. CONCLUSIONS: Multifaceted interventions to implement the early diagnosis of CP guideline are effective in improving patient outcomes by lowering the age of CP diagnosis in high-risk infant follow-up clinics. Further targeted health professional interventions including low-risk infant populations are warranted.

Towards a fair and transparent research participant compensation and reimbursement framework in Vietnam.

BACKGROUND: Providing compensation for participants in clinical research is well established and while international guidelines exist, defining a context-specific and fair compensation for participants in low-resource settings is challenging due to ethical concerns and the lack of practical, national compensation and reimbursement frameworks. METHODS: We reviewed Oxford University Clinical Research Unit (OUCRU) internal reimbursement documentation over a 10-y period and conducted a scoping literature review to expand our knowledge of compensation and reimbursement practices including ethical concerns. We developed a preliminary reimbursement framework that was presented to community advisory boards (CAB) and clinical investigators to assess its applicability, fairness and transparency. RESULTS: The main topics discussed at the workshops centered on fairness and whether the reimbursements could be perceived as financial incentives. Other decisive factors in the decision-making process were altruism and the loss of caregivers' earnings. Investigators raised the issue of additional burdens, whereas the CAB members were focused on non-monetary elements such as the healthcare quality the patients would receive. All elements discussed were reviewed and, where possible, incorporated into the final framework. CONCLUSION: Our new reimbursement framework provides a consistent, fair and transparent decision-making process and will be implemented across all future OUCRU clinical research in Vietnam.

Survival and Prognostic Factors in C9orf72 Repeat Expansion Carriers: A Systematic Review and Meta-analysis.

Importance: The c9orf72 repeat expansion (c9 or c9orf72RE) confers a survival disadvantage in amyotrophic lateral sclerosis (ALS); its effect on prognosis in frontotemporal dementia (FTD) remains uncertain. Data on prognostic factors in c9orf72RE disorders could inform patient care, genetic counseling, and trial design. Objective: To examine prognostic factors in c9ALS, c9FTD, c9ALS-FTD, and atypical phenotypes. Data Sources: The MEDLINE, Embase, Amed, ProQuest, PsychINFO, CINAHL, and LILACS databases were searched between January 2011 and January 2019. Keywords used were c9orf72 and chromosome 9 open reading frame 72. Reference lists, citations of eligible studies, and review articles were also searched by hand. Study Selection: Studies reporting disease duration for patients with a confirmed c9orf72RE and a neurological and/or psychiatric disorder were included. A second author independently reviewed studies classified as irrelevant by the first author. Analysis began in January 2019. Data Extraction and Synthesis: Data were extracted by 1 author; a further author independently extracted 10% of data. Data were synthesized in univariate and multivariable Cox regression and are displayed as hazard ratios (HR) and 95% confidence intervals. Main Outcomes and Measures: Survival after symptom onset. Results: Overall, 206 studies reporting on 1060 patients were included from 2878 publications identified (c9ALS: n = 455; c9FTD: n = 296; c9ALS-FTD: n = 198; atypical phenotypes: n = 111); 197 duplicate cases were excluded. The median (95% CI) survival (in years) differed significantly between patients with c9ALS (2.8 [2.67-3.00]), c9FTD (9.0 [8.09-9.91]), and c9ALS-FTD (3.0 [2.73-3.27]); survival in atypical phenotypes varied substantially. Older age at onset was associated with shorter survival in c9ALS (HR, 1.03; 95% CI, 1.02-1.04; P < .001), c9FTD (HR, 1.04; 95% CI, 1.02-1.06; P < .001), and c9ALS-FTD (HR, 1.02; 95% CI, 1.004-1.04; P = .016). Bulbar onset was associated with shorter survival in c9ALS (HR, 1.64; 95% CI, 1.27-2.08; P < .001). Age at onset and bulbar onset ALS remained significant in multivariable regression including variables indicating potential diagnostic ascertainment bias, selection bias, and reporting bias. Family history, sex, study continent, FTD subtype, or the presence of additional pathogenic sequence variants were not significantly associated with survival. Clinical phenotypes in patients with neuropathologically confirmed frontotemporal lobar degeneration-TDP-43, motor neuron disease-TDP-43 and frontotemporal lobar degeneration-motor neuron disease-TDP-43 were heterogenous and impacted on survival. Conclusions and Relevance: Several factors associated with survival in c9orf72RE disorders were identified. The inherent limitations of our methodological approach must be considered; nonetheless, the reported prognostic factors were not significantly associated with the bias indicators examined.

Altered immune parameters associated with Koala Retrovirus (KoRV) and Chlamydial infection in free ranging Victorian koalas (Phascolarctos cinereus).

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas (Phascolarctos cinereus) and to underlie the koala's susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A (p`0.023) and IFNγ (p = 0.044) gene expression along with a decreased CD4:CD8 gene expression ratio (p = 0.025) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

Altered Immune Cytokine Expression Associated with KoRV B Infection and Season in Captive Koalas.

Koala (Phascolarctos cinereus) populations are increasingly vulnerable and one of the main threats is chlamydial infection. Koala retrovirus (KoRV) has been proposed as an underlying cause of the koala's susceptibility to infection with Chlamydia and high rates of lymphoid neoplasia; however, the regionally ubiquitous, endogenous nature of this virus suggests that KoRV A infection is not sufficient for immune suppression to occur. A recently discovered exogenous variant of KoRV, KoRV B, has several structural elements that cause increased pathogenicity in related retroviruses and was associated with lymphoid neoplasia in one study. The present study assesses whether KoRV B infection is associated with alterations in immune function. Cytokine gene expression by mitogen stimulated lymphocytes of KoRV B positive (n = 5-6) and negative (n = 6-7) captive koalas was evaluated by qPCR four times (April 2014-February 2015) to control for seasonal variation. Key immune genes in the Th1 pathway (IFNγ, TNFα), Th2 pathway (IL 10, IL4, IL6) and Th17 pathway (IL17A), along with CD4:CD8 ratio, were assessed. KoRV B positive koalas showed significantly increased up-regulation of IL17A and IL10 in three out of four sampling periods and IFNγ, IL6, IL4 and TNFα in two out of four. IL17A is an immune marker for chlamydial pathogenesis in the koala; increased expression of IL17A in KoRV B positive koalas, and concurrent immune dysregulation, may explain the differences in susceptibility to chlamydial infection and severity of disease seen between individuals and populations. There was also marked seasonal variation in up-regulation for most of the cytokines and the CD4:CD8 ratio. The up-regulation in both Th1 and Th2 cytokines mirrors changes associated with immune dysregulation in humans and felids as a result of retroviral infections. This is the first report of altered immune expression in koalas infected by an exogenous variant of KoRV and also the first report of seasonal variation in cytokine up-regulation and CD4:CD8 ratio in marsupials.

Expression profiles of the immune genes CD4, CD8ß, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus) by qRT-PCR.

Investigation of the immune response of the koala (Phascolarctos cinereus) is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV), which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala's susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1) and Th2 lymphocyte responses are important to an individual's susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala's adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNγ) along with CD4 and CD8ß. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A). Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not consistently up-regulated by any of the protocols. Expression of CD4 and CD8ß was down-regulated by mitogen stimulation. We found that the reference genes GAPDH and 28s are valid for normalising cytokine expression by koala lymphocytes after mitogen stimulation.

Claustrophobia: a proxy for psychological distress in patients with back pain.

STUDY DESIGN: Case-control study. OBJECTIVE: The aim of this study was to assess the levels of psychological distress in patients with back pain who expressed claustrophobia at the time of their magnetic resonance imaging (MRI) scan, compared with sex and age-matched normal controls who did not exhibit claustrophobia. The secondary aim was to document the level of disability and intervention rates in this group. SUMMARY OF BACKGROUND DATA: Psychosocial factors influence the outcomes of low back pain treatment with psychological distress being associated with poorer surgical outcomes in patients with low back pain. Up to 14% of patients experience claustrophobia during MRI scans requiring sedation to complete the scan. The effect of claustrophobia on back pain disability and outcomes has not been previously reported. METHODS: Twenty females and 13 males all requiring MRI scan under sedation for claustrophobia (group 1) were compared with an age and sex-matched cohort that had MRI scan without sedation (group 2). Both groups were drawn from a chronic back clinic. Average age in both groups was 54 years (range, 27 to 79 y). Both groups had standard conservative therapy, together with psychometric evaluation: Zung Depression Index and Modified Somatic Perception Questionnaire. Disability was measured by Oswestry Disability Index. Primary outcome measures were intervention rates (surgery, injections, and physiotherapy sessions) and prevalence of psychological distress. RESULTS: Mean Zung Depression Index in group 1 was significantly higher than in group 2 (59.5 vs. 28.9, P<0.05) as was the mean Modified Somatic Perception Questionnaire score (13.3 vs. 9.2, P<0.05.) Prevalence of psychological distress was higher in group 1 (75.8% vs. 18.2%, P<0.05). Oswestry Disability Index was the same in both groups (50% vs. 48%). Group 1 had 13 interventions (13 patients=39.4%) compared with 26 in group 2 (26 patients=78.8%) (P< 0.05). Twenty-two patients (66.7%) in group 1 were discharged after their MRI scan with no intervention compared with 7 patients (21.2%) in group 2 (P<0.05). CONCLUSIONS: Claustrophobic patients with back pain showed higher levels of depression than nonclaustrophobic patients, with a higher rate of psychological distress. Disability, however, was not higher. The majority of claustrophobic patients did not require surgical intervention. The reasons for this are unclear and require further investigation. Claustrophobia requiring sedation for MRI scans may be a proxy for psychological distress in these patients and psychometric testing is advised during assessment to help with surgical decision making.

Polymerase chain reaction survey of feline haemoplasma infections in Greece.

The aim of this study was to use real-time polymerase chain reaction assays to determine the prevalence of three haemoplasma species in cats from Greece and to evaluate possible associations between haemoplasma infection and age, gender, feline immunodeficiency virus/feline leukaemia virus (FIV/FeLV) status and packed cell volume (PCV). Ninety-seven cats (24 ill anaemic, 55 ill non-anaemic, 18 healthy non-anaemic) were included in the study. Twenty cats (20.6%) were haemoplasma positive; seven cats were infected only with Mycoplasma haemofelis, 10 were infected only with 'Candidatus Mycoplasma haemominutum' and three were co-infected with M haemofelis and 'Candidatus M haemominutum'. 'Candidatus Mycoplasma turicensis' was not detected. Haemoplasma infection was associated with older age (P=0.019). M haemofelis infection tended to be more common in anaemic cats (P=0.058). No association between gender and haemoplasma infection, or haemoplasma relative copy number and PCV, was detected. Retroviral infection rates were very low with only one FeLV proviral positive cat found.

External quality control of Mycobacterium tuberculosis drug susceptibility testing: results of two rounds in endemic countries.

SETTING: Quality assurance for the World Health Organization (WHO)/International Union Against Tuberculosis and Lung Disease (The Union) global tuberculosis (TB) drug resistance surveillance programme. OBJECTIVE: To monitor the quality of drug susceptibility testing (DST) in different countries. METHODS: In 2002-2003 and 2005-2006, 20 Mycobacterium tuberculosis strains were sent by the WHO/Union Supranational Reference Laboratory of Rome to TB reference laboratories in Albania, Bahrain, Kosovo, Mozambique, Oman, Qatar and Turkey for external quality control (EQC). RESULTS: In 2002-2003, the specificity, sensitivity, efficiency, reproducibility and predictive values for resistance/susceptibility were >or=90% for streptomycin (SM), isoniazid (INH) and ethambutol (EMB). In 2005-2006, all statistical values were >or=96% for SM, INH, rifampicin and EMB. CONCLUSION: EQC improved the quality of M. tuberculosis DST in the participating countries.

Simple interventions improve re-attendance when treating the sleep apnoea syndrome.

BACKGROUND AND PURPOSE: Interventions to improve treatment outcomes in sleep apnoea-hypopnoea syndrome (SAHS) have had mixed success. Most have concentrated on following the use of a continuous positive airway pressure (CPAP) machines; poorer users may not return for machine readings, so any compliance study must take into account rates of attendance rates. We hypothesised that a series of additional, early support measures would improve re-attendance over a sustained period. PATIENTS AND METHODS: Prospective, single-blinded interventional study. Seventy-two consecutive patients starting CPAP for SAHS were randomised to receive standard follow-up or extra early support. Attendance rates, CPAP use, Epworth scores, side-effects scores and number of changes to equipment were compared, by intent to treat, in both groups at 1 and 12 months. RESULTS: Re-attendance rates were higher in the intervention group at 1 month (P=0.04), 6 months (P=0.07) and 12 months (P=0.12). Those who defaulted tended previously to be poor users of the CPAP machine. For those who re-attended there was no difference in machine use or other outcomes. CONCLUSIONS: Simple interventions while commencing CPAP improve re-attendance with maximal benefit early on. This could provide more opportunities for solving problems early or considering alternative treatments. By confirming that poorer CPAP users eventually have higher default rates we recommend that future studies on CPAP compliance should first account for re-attendance rates.

Does one size fit all? Drug resistance and standard treatments: results of six tuberculosis programmes in former Soviet countries.

SETTING: After the collapse of the Soviet Union, countries in the region faced a dramatic increase in tuberculosis cases and the emergence of drug resistance. OBJECTIVE: To discuss the relevance of the DOTS strategy in settings with a high prevalence of drug resistance. DESIGN: Retrospective analysis of one-year treatment outcomes of short-course chemotherapy (SCC) and results of drug susceptibility testing (DST) surveys of six programmes located in the former Soviet Union: Kemerovo prison, Russia; Abkhasia, Georgia; Nagorno-Karabagh, Azerbaijan; Karakalpakstan, Uzbekistan; Dashoguz Velayat, Turkmenistan; and South Kazakhstan Oblast, Kazakhstan. Results are reported for new and previously treated smear-positive patients. RESULTS: Treatment outcomes of 3090 patients and DST results of 1383 patients were collected. Treatment success rates ranged between 87% and 61%, in Nagorno-Karabagh and Kemerovo, respectively, and failure rates between 7% and 23%. Any drug resistance ranged between 66% and 31% in the same programmes. MDR rates ranged between 28% in Karakalpakstan and Kemerovo prison and 4% in Nagorno-Karabagh. CONCLUSION: These results show the limits of SCC in settings with a high prevalence of drug resistance. They demonstrate that adapting treatment according to resistance patterns, access to reliable culture, DST and good quality second-line drugs are necessary.

Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells.

BACKGROUND: Tracheary element (TE) differentiation in single cells in culture isolated from Zinnia elegans leaves involves programmed cell death (PCD) co-ordinated with key morphological developments. We have used flow cytometry to analyze physiological and nuclear changes in the differentiating cells. Flow cytometry allows the identification of subpopulations, thereby removing the obscuring effect of population heterogeneity that occurs with the use of other techniques. METHODS: Cell viability, plasma membrane integrity, oxidative activity, intracellular calcium and pH, cell wall thickening, the possible role of microtubule rearrangement, chromatin condensation, and DNA breakdown were followed by flow cytometry from the first stages of TE induction. RESULTS: TE differentiation could be enhanced and made more synchronous by a centrifugation step at 72 h after cell isolation. Size and shape changes were the first changes identified in differentiating cells, and these properties could be used to isolate differentiating populations by back-gating. Chromatin condensation and nDNA breakdown followed patterns characteristic of programmed cell death. CONCLUSIONS: We have used flow cytometry to characterize the morphological and physiological changes that occur during TE differentiation, and our findings indicate that this process is a form of autophagic PCD in which microtubule rearrangement appears to play a role.

Spontaneous rupture of the popliteus tendon in a 74-year-old woman and review of the literature.

Spontaneous rupture of the popliteus tendon has not previously been reported. We report the case of a 74-year-old patient, discuss its diagnosis and management, and review the current literature on isolated popliteus lesions. A multi-database electronic literature review suggests that the injury is invariably traumatic and has essentially been reported only in young, active individuals. It should be suspected in a patient with acute lateral knee pain who, on examination, has a hemarthrosis and a stable, possibly locked knee. The diagnosis is readily made using arthroscopy. While both conservative treatment with physiotherapy and open repair have been advocated, we describe a successful outcome of arthroscopic resection in an elderly patient and propose that popliteus injury is not confined to a younger age group. The balance of opinion in the current literature is that acute traumatic rupture in the young patient is managed by primary surgical repair, but successful outcome is also reported with nonoperative treatment.

Salicylic acid-mediated potentiation of Hsp70 induction correlates with reduced apoptosis in tobacco protoplasts.

BACKGROUND: Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. METHODS: Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 microM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. RESULTS AND CONCLUSIONS: SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection.

Early predictors of CPAP use for the treatment of obstructive sleep apnea.

STUDY OBJECTIVES: To identify factors before a trial of nasal continuous positive airway pressure (CPAP) treatment that are associated with lower compliance. DESIGN: A prospective cohort study. Initial Hospital Anxiety and Depression Scale scores and other demographic data were noted. Machine use was recorded by clock timer after a 1-month trial of treatment. SETTING: District General Hospital sleep-disordered breathing clinic. PATIENTS OR PARTICIPANTS: Eighty consecutive patients with symptoms of sleep apnea-hypopnea syndrome and a 4% Sao2 desaturation index greater than 10 events per hour. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Those reporting 'initial problems' with CPAP went on to have an average of 2.4 hours of on time per night, while those not reporting initial problems had an on time of 5.0 hours per night (P < .001). Those living alone had a machine on time of 3.2 hours compared with 4.5 hours for those with partners (P = .04). Pearson's correlations between hours on time were -0.08 (P = .48) for initial Anxiety score and 0.10 (P = .37) for initial Depression score. CONCLUSIONS: There was no association between baseline anxiety and depression scores, as measured by the Hospital Anxiety and Depression Scale, and subsequent machine use. Other factors observable prior to commencing treatment, in particular, 'initial problems' (reported at autotitration), 'recent life-events' and 'living alone' were associated with lower machine use. Regarding all initial variables, reporting problems after the first night of nCPAP seems the most important predictor of ensuing machine use. A single screening question immediately after autotitration is useful in identifying those at high risk of treatment failure.

[A review of methods for measuring plant programmed cell death (PCD)].

Programmed cell death (PCD) is an active way for plant cells marching to death, which plays an important role in plant development and stress responses. Cytological, biochemical, molecular and physiological methods for measuring plant PCD were reviewed. Application of flow cytometer to plant PCD research was also covered.

Oxidative stress is generated via the mitochondrial respiratory chain during plant cell apoptosis.

BACKGROUND: We present evidence that in plant cells DNA damage induced by the topoisomerase trapping drug camptothecin induces oxidative stress via the mitochondrial respiratory chain. METHODS: Flow cytometry was used to analyse mitochondrial respiratory chain activity by simultaneous measurement of mitochondrial generation of reactive oxygen intermediates (ROI) and mitochondrial membrane potential (DeltaPsim), in live functioning sugarbeet protoplasts. The mitochondrial respiratory chain function was assessed by the addition of substrate and inhibitors to the digitonin-permeabilised protoplasts. Mitochondrial activity was also measured in protoplasts at different stages of induced apoptosis by camptothecin. RESULTS: The addition of camptothecin to sugarbeet protoplasts resulted in increased mitochondrial generation of ROI, occurring concurrently with the hyperpolarisation of the mitochondrial inner membrane. Subsequently, there was a decrease in mitochondrial-generated ROI, in association with a decrease in DeltaPsim. These camptothecin-treated digitonin-permeabilised protoplasts could be re-energised and ROI decreased by the addition of malic acid in the early stages of apoptosis induction, but not in the latter stages. There was a depolarisation of the mitochondrial inner membrane in the final stages of apoptosis. CONCLUSIONS: These results are consistent with impaired adenine nucleotide exchange across the mitochondrial membrane, suggesting that similar mechanisms regulate plant apoptosis as those described for mammalian apoptosis.