Metformin-induced oxidative stress inhibits LNCaP prostate cancer cell survival.

BACKGROUND: Preclinical and clinical studies over the past several decades have indicated the potential value of metformin, a widely utilized treatment for Type 2 diabetes, in prostate cancer therapy. Notably, these studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways, such as androgen signaling, cell cycle, and cellular bioenergetics. In this study we investigated the role of metformin in regulating intracellular redox status and cell survival in LNCaP prostate cancer cells. METHODS AND RESULTS: The cytotoxic effects of metformin with or without the presence of SBI0206965 (AMPK inhibitor) on LNCaP cells were determined using MTT and trypan blue exclusion assays. Seahorse XP extracellular analysis, Liquid Chromatography/ Mass Spectrophotometry (LC/MS), and 2,7- and Dichlorofluoresin diacetate (DCFDA) assay were used to assess the effects of metformin on cellular bioenergetics, redox status, and redox-related metabolites. mRNA expression and protein concentration of redox-related enzymes were measured using Real Time-qPCR and ELISA assay, respectively. Independently of AMP-activated protein kinase, metformin exhibited a dose- and time-dependent inhibition of LNCaP cell survival, a response mitigated by glutathione or N-acetylcysteine (ROS scavengers) treatment. Notably, these findings were concomitant with a decline in ATP levels and the inhibition of oxidative phosphorylation. The results further indicated metformin's induction of reactive oxygen species, which significantly decreased glutathione levels and the ratio of reduced to oxidized glutathione, as well as the transsulfuration metabolite, cystathionine. Consistent with an induction of oxidative stress condition, metformin increased mRNA levels of the master redox transcription factor Nrf-2 (nuclear factor erythroid-derived 2-like), as well as transsulfuration enzymes cystathionine beta-synthase and cystathionase and GSH synthesis enzymes γ-glutamylcysteine synthetase and glutathione synthetase. CONCLUSION: Our findings highlight multiple mechanisms by which metformin-induced formation of reactive oxygen species may contribute to its efficacy in prostate cancer treatment, including promotion of oxidative stress, Nrf2 activation, and modulation of redox-related pathways, leading to its anti-survival action.

An observational cohort study to determine the impact of research capacity building strategies implemented in an Australian metropolitan hospital occupational therapy department.

INTRODUCTION: Research capacity building enhances the abilities of individuals and is critical within health systems for quality patient care and promotes a culture of excellence within the occupational therapy profession. A research capacity building toolkit was proposed identifying strategies to support allied health professionals to undertake research. This study evaluated participant-reported outcomes of research capacity building toolkit implementation in an occupational therapy department. METHODS: An observational pre-post-cohort study at a tertiary hospital with volunteer occupational therapists using the standardised Research Capacity in Context Tool (RCCT) and an author-designed quality improvement (QI) survey was employed. The RCCT measures research capacity and culture at organisation, team and individual levels. Semi-structured interviews were used to elicit reflections regarding participant experience. RESULTS: All levels of the toolkit were implemented successfully. The response rate was 59% (n = 36) at baseline and 49.1% (n = 26) at follow-up. Eighty-five percent of participants held direct clinical roles. Nine clinicians participated in the interviews. There were significant improvements in the estimate mean for the organisation (6.51 [2019] compared with 8.13 [2020], p = <0.001) and the team (5.52 [2019] compared with 7.15 [2020], p = 0.001). The individual level did not significantly change with an estimate mean of 4.20 in 2019 increasing slightly to 4.84 in 2020 (p = 0.128). This was supported by the QI survey where improvements were noted in the department but not at an individual level. The qualitative findings verified the components of the toolkit including 'supporting clinicians in research', 'working together', 'valuing research for excellence' and reflected the importance of 'individual attributes'. CONCLUSION: The toolkit supported the implementation of specific strategies to enhance research capacity and culture. Improvements within the organisation and team were evident; however, these were not seen at an individual level. Further research about the contribution of individual-related factors and processes to the building of research capacity is required.

Alterations in Gene Expression of Renin-Angiotensin System Components and Related Proteins in Colorectal Cancer.

MATERIALS AND METHODS: Quantitative expression of the RNA of these 17 genes in normal and cancerous tissues obtained using chip arrays from the public functional genomics data repository, Gene Expression Omnibus (GEO) application, was compared statistically. RESULTS: Expression of four genes, AGT (angiotensinogen), ENPEP (aminopeptidase A) MME (neprilysin), and PREP (prolyl endopeptidase), was significantly upregulated in CRC specimens. Expression of REN (renin), THOP (thimet oligopeptidase), NLN (neurolysin), PRCP (prolyl carboxypeptidase), ANPEP (aminopeptidase N), and MAS1 (Mas receptor) was downregulated in CRC specimens. CONCLUSIONS: Presuming gene expression parallel protein expression, these results suggest that increased production of the angiotensinogen precursor of angiotensin (ANG) peptides, with the reduction of the enzymes that metabolize it to ANG II, can lead to accumulation of angiotensinogen in CRC tissues. Downregulation of THOP, NLN, PRCP, and MAS1 gene expression, whose proteins contribute to the ACE2/ANG 1-7/Mas axis, suggests that reduced activity of this RAS branch could be permissive for oncogenicity. Components of the RAS may be potential therapeutic targets for treatment of CRC.

A Systematic Literature Review of Efficiency Measurement in Nursing Homes.

Background: As our population ages at an increasing rate, the demand for nursing homes is rising. The challenge will be for nursing homes to maintain efficiency with limited resources while not compromising quality. This study aimed to review the nursing home efficiency literature to survey the application of efficiency methods and the measurements of inputs, outputs, facility characteristics and operational environment, with a special focus on quality measurement. Methods: We systematically searched three databases for eligible studies published in English between January 1995 and December 2018, supplemented by an exhaustive search of reference lists of included studies. The studies included were available in full text, their units of analysis were nursing homes, and the analytical methods and efficiency scores were clearly reported. Results: We identified 39 studies meeting the inclusion criteria, of which 31 accounted for quality measures. Standard efficiency measurement techniques, data envelopment analysis and stochastic frontier method, and their specifications (orientation, returns to scale, functional forms and error term assumptions) were adequately applied. Measurements of inputs, outputs and control variables were relatively homogenous while quality measures varied. Notably, most studies did not include all three quality dimensions (structure, process and outcome). One study claimed to include quality of life; however, it was not a well-validated and widely used measure. The impacts of quality on efficiency estimates were mixed. The effect of quality on the ranking of nursing home efficiency was rarely reported. Conclusions: When measuring nursing home efficiency, it is crucial to adjust for quality of care and resident's quality of life because the ultimate output of nursing homes is quality-adjusted days living in the facility. Quality measures should reflect their multidimensionality and not be limited to quality of throughput (health-related events). More reliable estimation of nursing home efficiencies will require better routine data collection within the facility, where well-validated quality measures become an essential part of the minimum data requirement. It is also recommended that different efficiency methods and assumptions, and alternative measures of inputs, outputs and quality, are used for sensitivity analyses to ensure the robustness and validity of findings.

Drug-Resistance and Population Structure of Plasmodium falciparum Across the Democratic Republic of Congo Using High-Throughput Molecular Inversion Probes.

A better understanding of the drivers of the spread of malaria parasites and drug resistance across space and time is needed. These drivers can be elucidated using genetic tools. Here, a novel molecular inversion probe (MIP) panel targeting all major drug-resistance mutations and a set of microsatellites was used to genotype Plasmodium falciparum infections of 552 children from the 2013-2014 Demographic and Health Survey conducted in the Democratic Republic of the Congo (DRC). Microsatellite-based analysis of population structure suggests that parasites within the DRC form a homogeneous population. In contrast, sulfadoxine-resistance markers in dihydropteroate synthase show marked spatial structure with ongoing spread of double and triple mutants compared with 2007. These findings suggest that parasites in the DRC remain panmictic despite rapidly spreading antimalarial-resistance mutations. Moreover, highly multiplexed targeted sequencing using MIPs emerges as a cost-effective method for elucidating pathogen genetics in complex infections in large cohorts.

A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse.

Human babesiosis caused by Babesia microti is an emerging tick-borne zoonosis of increasing importance due to its rising incidence and expanding geographic range(1). Infection with this organism, an intraerythrocytic parasite of the phylum Apicomplexa, causes a febrile syndrome similar to malaria(2). Relapsing disease is common among immunocompromised and asplenic individuals(3,4) and drug resistance has recently been reported(5). To investigate the origin and genetic diversity of this parasite, we sequenced the complete genomes of 42 B. microti samples from around the world, including deep coverage of clinical infections at endemic sites in the continental USA. Samples from the continental USA segregate into a Northeast lineage and a Midwest lineage, with subsequent divergence of subpopulations along geographic lines. We identify parasite variants that associate with relapsing disease, including amino acid substitutions in the atovaquone-binding regions of cytochrome b (cytb) and the azithromycin-binding region of ribosomal protein subunit L4 (rpl4). Our results shed light on the origin, diversity and evolution of B. microti, suggest possible mechanisms for clinical relapse, and create the foundation for further research on this emerging pathogen.

OPA1 mutation and late-onset cardiomyopathy: mitochondrial dysfunction and mtDNA instability.

BACKGROUND: Mitochondrial fusion protein mutations are a cause of inherited neuropathies such as Charcot-Marie-Tooth disease and dominant optic atrophy. Previously we reported that the fusion protein optic atrophy 1 (OPA1) is decreased in heart failure. METHODS AND RESULTS: We investigated cardiac function, mitochondrial function, and mtDNA stability in a mouse model of the disease with OPA1 mutation. The homozygous mutation is embryonic lethal. Heterozygous OPA(+/-) mice exhibit reduced mtDNA copy number and decreased expression of nuclear antioxidant genes at 3 to 4 months. Although initial cardiac function was normal, at 12 months the OPA1(+/-) mouse hearts had decreased fractional shortening, cardiac output, and myocyte contraction. This coincided with the onset of blindness. In addition to small fragmented mitochondria, aged OPA1(+/-) mice had impaired cardiac mitochondrial function compared with wild-type littermates. CONCLUSIONS: OPA1 mutation leads to deficiency in antioxidant transcripts, increased reactive oxygen species, mitochondrial dysfunction, and late-onset cardiomyopathy.

The heat shock paradox and cardiac myocytes: role of heat shock factor.

The induction of the heat shock (HS) response is accepted to be a protective response, reducing injury and improving cell survival. However, when inflammation precedes HS, there is an unexpected increase in injury, known as the HS paradox, which is hypothesized to be a mechanism underlying multiorgan dysfunction. We hypothesized that the HS paradox would occur in adult cardiac myocytes and that HS factor (HSF) 1 would contribute to injury. Heat shock at 42°C and TNF (10 ng/mL) were used as the HS and the inflammatory insult, respectively. The combination of TNF followed by HS (TNF/HS) caused the greatest amount of apoptosis in adult rat cardiac myocytes. TNF/HS resulted in an increase in HS protein (HSP) 60, compared with untreated cells, those receiving HS/TNF, or TNF alone. There was no increase in heme oxygenase 1 in any of the groups. Heat shock protein 72 increased in all the groups, with the greatest levels with TNF/HS. Nuclear factor κB activation was greatest with TNF/HS. Pretreatment with a DNA-binding decoy for HSF-1 prevented the increase in HSPs and decreased apoptosis in all groups. However, the increase in iNOS, seen in all treatment groups, was unaffected by the HSF-1-binding decoy. We conclude that the HS paradox occurs in adult cardiac myocytes, that HSP60 is increased as part of the HS paradox, and that HSF-1 activation contributes to injury.

Effects of hindbrain melanin-concentrating hormone and neuropeptide Y administration on licking for water, saccharin, and sucrose solutions.

Melanin-concentrating hormone (MCH) and neuropeptide Y (NPY) are orexigenic peptides found in hypothalamic neurons that project throughout the forebrain and hindbrain. The effects of fourth ventricle (4V) infusions of NPY (5 microg) and MCH (5 microg) on licking for water, 4 mM saccharin, and sucrose (0.1 and 1.0 M) solutions were compared to identify the contributions of each peptide to hindbrain-stimulated feeding. NPY increased mean meal size only for the sucrose solutions, suggesting that caloric feedback or taste quality is pertinent to the orexigenic effect; MCH infusions under identical testing conditions failed to produce increases for any tastant. A second experiment also observed no intake or licking effects after MCH doses up to 15 microg, supporting the conclusion that MCH-induced orexigenic responses require forebrain stimulation. A third experiment compared the 4V NPY results with those obtained after NPY infusions (5 microg) into the third ventricle (3V). In contrast to the effects observed after the 3V NPY injections and previously reported forebrain intracerebroventricular (ICV) NPY infusion studies, 4V NPY failed to increase meal frequency for any taste solution or ingestion rate in the early phases of the sucrose meals. Overall, 4V NPY responses were limited to intrameal behavioral processes, whereas forebrain ICV NPY stimulation elicited both consummatory and appetitive responses. The dissociation between MCH and NPY effects observed for 4V injections is consistent with reports that forebrain ICV injections of MCH and NPY produced nearly dichotomous effects on the pattern of licking microstructure, and, collectively, the results indicate that the two peptides have separate sites of feeding action in the brain.