Apoptosis-modulatory miR-361-3p as a novel treatment target in endocrine-responsive and endocrine-resistant breast cancer.

Breast cancer (BC) is the most diagnosed cancer in women worldwide. In estrogen receptor (ER)-positive disease, anti-estrogens and aromatase inhibitors (AI) improve patient survival; however, many patients develop resistance. Dysregulation of apoptosis is a common resistance mechanism; thus, agents that can reinstate the activity of apoptotic pathways represent promising therapeutics for advanced drug-resistant disease. Emerging targets in this scenario include microRNAs (miRs). To identify miRs modulating apoptosis in drug-responsive and -resistant BC, a high-throughput miR inhibitor screen was performed, followed by high-content screening microscopy for apoptotic markers. Validation demonstrated that miR-361-3p inhibitor significantly increases early apoptosis and reduces proliferation of drug-responsive (MCF7), plus AI-/antiestrogen-resistant derivatives (LTED, TamR, FulvR), and ER- cells (MDA-MB-231). Importantly, proliferation-inhibitory effects were observed in vivo in a xenograft model, indicating the potential clinical application of miR-361-3p inhibition. RNA-seq of tumour xenografts identified FANCA as a direct miR-361-3p target, and validation suggested miR-361-3p inhibitor effects might be mediated in part through FANCA modulation. Moreover, miR-361-3p inhibition resulted in p53-mediated G1 cell cycle arrest through activation of p21 and reduced BC invasion. Analysis of publicly available datasets showed miR-361-3p expression is significantly higher in primary breast tumours vspaired normal tissue and is associated with decreased overall survival. In addition, miR-361-3p inhibitor treatment of BC patient explants decreased levels of miR-361-3p and proliferation marker, Ki67. Finally, miR-361-3p inhibitor showed synergistic effects on BC growth when combined with PARP inhibitor, Olaparib. Together, these studies identify miR-361-3p inhibitor as a potential new treatment for drug-responsive and -resistant advanced BC.

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer.

BACKGROUND: miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent. METHODS: RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq). RESULTS: miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy. CONCLUSIONS: A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

The antiandrogen enzalutamide downregulates TMPRSS2 and reduces cellular entry of SARS-CoV-2 in human lung cells.

SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably prostate. We show here that treatment with the antiandrogen enzalutamide-a well-tolerated drug widely used in advanced prostate cancer-reduces TMPRSS2 levels in human lung cells and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

Martius labial fat pad graft (use in rectovaginal fistula repair).

INTRODUCTION AND HYPOTHESIS: The objective is to demonstrate the utility of the Martius labial fat pad graft in pelvic fistula repair. METHODS: An incision is made over the labium majus from the level of the clitoral hood superiorly and extending inferiorly to the level of the labiocrural fold. The fibrofatty graft is then mobilized from the adjacent labium majus. The flap can be divided either at its anterior or at its posterior pedicle. A subepithelial defect is created through which the flap will be passed. The flap is sewn into place by attaching it to the underlying rectovaginal fascia without associated tensioning. Next, the posterior vaginal wall is closed over the graft with a series of everting subepithelial mattress sutures followed by a reinforcing layer of interrupted #1 Vicryl through the vaginal epithelium. CONCLUSIONS: The advantages of the Martius flap in fistula repair include low morbidity, lack of a cosmetic defect, and the need for only a single surgical field. Its prominent fibrous component makes it a stronger graft than adipose tissue from other areas and its abundant blood supply promotes rapid neovascularization and lends itself well to many surgical modifications that can be utilized in even the most difficult of fistula repairs.

Postoperative Opioid Prescribing Following Gynecologic Surgery for Pelvic Organ Prolapse.

OBJECTIVES: The aim of this study was to evaluate postoperative pain scores, quantity of prescribed opioids at hospital discharge, and need for additional opioid prescriptions among women undergoing surgical treatment of pelvic organ prolapse. METHODS: Institutional billing data were used to identify all patients undergoing pelvic reconstructive surgery between January 1, 2012, and May 30, 2017. Inpatient records were utilized to obtain prescription data (reported in oral morphine equivalents for standardization) and pain scores. The cohort was organized by surgical approach (open, endoscopic, vaginal), number of concomitant procedures, and patient age stratified by decade. These factors were then matched to postoperative pain scores, amount of opioid prescribed at discharge, and number of subsequent opioid refills. Pain scores and opioid use were also compared for correlation. RESULTS: One thousand eight hundred thirty patients underwent surgical treatment of pelvic organ prolapse and met criteria for study participation. A significant decrease in pain scores, mean oral morphine equivalents prescribed, and opioid refill rates was seen with increasing patient age by decade regardless of surgical approach. Pain scores were significantly different only between patients undergoing vaginal surgery with 0 concomitant procedures versus 1 or more concomitant procedures. Finally, pain scores were directly correlated to the amount of opioid prescribed. CONCLUSIONS: Pain scores, opioid prescription amounts, and refills varied by patient age and surgical approach but were unaffected by concomitant procedures. Further work in correlating pain scores to opioid utilization is needed to ensure appropriate prescribing patterns and reduce risks of opioid dependence and diversion.

Implementation of a clinical prediction tool for pulmonary embolism diagnosis in a tertiary teaching hospital reduces the number of computed tomography pulmonary angiograms performed.

AIM: To evaluate the effect of implementing the Wells score clinical prediction tool (CPT) on rationalising the use of computed tomography pulmonary angiography (CTPA) for diagnosing pulmonary embolism (PE). METHODS: Within a tertiary teaching hospital, a retrospective study was conducted applying Wells score to all CTPA ordered in the first quarter of 2007. Subsequently, an algorithm including Wells score and d-dimer assay was developed to assist clinicians in rationalising their ordering of CTPA. A prospective study was performed from February to August 2009 to assess the impact of this algorithm. CTPA results, d-dimer levels, referral sources and dates were recorded. The number of CTPA performed over a 7-month period following implementation of the algorithm was compared with the same period during the previous year. PE prevalence within each risk category was compared with the published literature. RESULTS: Three hundred and thirty-three patients were investigated with CTPA in the prospective study period. Two hundred and sixty-eight patients (80.4% of cases) had complete data. The prevalence of PE in the present study was 13.8% with 57 (21.2%) patients stratified to low risk, 169 (63.0%) to intermediate risk and 42 (15.6%) to high risk. Subgroup prevalence was 8.8%, 11.8% and 23.8% respectively. Compared with the same period in 2008, 121 (26.6%) less CTPA were performed. CONCLUSION: Institutional implementation of a clinical prediction tool into the decision-making process is feasible and significantly reduces the number of CTPA being performed, with substantial cost savings and patient benefits.

Essential oil composition and antimicrobial activity of wild and cultivated mint timija (Mentha suaveolens subsp. timija (Briq.) Harley), an endemic and threatened medicinal species in Morocco.

The hydro-distilled essential oils obtained from aerial parts of the wild (w) and cultivated (c) mint timija (Mentha suaveolens subsp. timija), an endemic medicinal species of Morocco, have been analyzed by GC-MS and screened for antimicrobial activity. In total, 35 compounds representing more than 98% of the oils were identified. Menthone (39.4(w)-10.8(c)%), pulegone (62.3(c)-34.3(w)%) and isomenthone (9.3(c)-7.8(w)%) were found as the main components for the two oils. The volatiles of the wild and cultivated material differed significantly in both the percentage of the main components and antimicrobial effect. Pulegone was more dominant in cultivated mint timija (62.3%) than in wild one (34.3%), while menthone was more abundant in the wild material (39.4%). In the antimicrobial assays, both oils displayed good to excellent activity against all microorganisms tested with the oil of the cultivated form being more active.

Drug-related death following release from prison: a brief review of the literature with recommendations for practice.

Mortality from drug-related death is a significant contributor to the loss of life of young people in the UK. Despite attention, the high death rate from this cause continues to persist. One of the most frequently cited factors involved in drug-related death (DRD) is release from prison. This review aims to examine the published literature with a view to quantifying the risk associated with recent prison release and identifying risk factors and prevention strategies. Most deaths following release from prison are caused by overdose, usually from opioid use. The risk of death is greatest within the first week of release but, when compared with the general population, continues to be elevated for several weeks. Relative risk estimates suggest that those released from prison are up to 40 times more likely to die than similar individuals from the general population. Other than gender and an association with poor mental health, there is little in the way of robust risk factors for post-release death that could be identified from the literature. In-prison pharmacological maintenance treatment with methadone and buprenorphine has been shown to reduce the rate of heroin use, in the period immediately following release, in a small number of randomised controlled trials. It is widely recognised that continuity of care, of any form, is critical in avoiding DRDs. For problem drug users, packages of education, including information on the associated risks, treatments, and recognition of DRD after release from prison, are seen as a basic minimum requirement of the prison services. However, special protocols may be required for those drug-using prisoners who have a possibility of being released at short notice.

Changes in water quality of the River Frome (UK) from 1965 to 2009: is phosphorus mitigation finally working?

The water quality of the River Frome, Dorset, southern England, was monitored at weekly intervals from 1965 until 2009. Determinands included phosphorus, nitrogen, silicon, potassium, calcium, sodium, magnesium, pH, alkalinity and temperature. Nitrate-N concentrations increased from an annual average of 2.4 mg l⁻¹ in the mid to late 1960s to 6.0 mg l⁻¹ in 2008-2009, but the rate of increase was beginning to slow. Annual soluble reactive phosphorus (SRP) concentrations increased from 101 µg l⁻¹ in the mid 1960s to a maximum of 190 µg l⁻¹ in 1989. In 2002, there was a step reduction in SRP concentration (average=88 µg l⁻¹ in 2002-2005), with further improvement in 2007-2009 (average=49 µg l⁻¹), due to the introduction of phosphorus stripping at sewage treatment works. Phosphorus and nitrate concentrations showed clear annual cycles, related to the timing of inputs from the catchment, and within-stream bioaccumulation and release. Annual depressions in silicon concentration each spring (due to diatom proliferation) reached a maximum between 1980 and 1991, (the period of maximum SRP concentration) indicating that algal biomass had increased within the river. The timing of these silicon depressions was closely related to temperature. Excess carbon dioxide partial pressures (EpCO2) of 60 times atmospheric CO2 were also observed through the winter periods from 1980 to 1992, when phosphorus concentration was greatest, indicating very high respiration rates due to microbial decomposition of this enhanced biomass. Declining phosphorus concentrations since 2002 reduced productivity and algal biomass in the summer, and EpCO2 through the winter, indicating that sewage treatment improvements had improved riverine ecology. Algal blooms were limited by phosphorus, rather than silicon concentration. The value of long-term water quality data sets is discussed. The data from this monitoring programme are made freely available to the wider science community through the CEH data portal (http://gateway.ceh.ac.uk/).

Evaluation of a biological risk management tool on large western United States dairies.

Critical to changing biosecurity practices on the farm is an individual assessment of those practices contributing to disease transmission. The purpose of this project was to assess, implement, and refine a biological risk management survey for use on large western United States dairy farms. Assessment tools developed by Iowa State University Center for Food Security and Public Health (Ames, IA) were refined using a focus group process and by testing them on 40 dairy herds in California. Each question was evaluated using standard criteria and producer responses. Some survey questions required refinement for clarity and others were considered unnecessary. New questions were added based on a biosecurity literature review, resulting in a new set of questions that can be used by extension educators and food animal veterinarians to help identify disease risk areas and educate dairy producers.

Changing education to improve patient care.

Health professionals need competencies in improvement skills if they are to contribute usefully to improving patient care. Medical education programmes in the USA have not systematically taught improvement skills to residents (registrars in the UK). The Accreditation Council for Graduate Medical Education (ACGME) has recently developed and begun to deploy a competency based model for accreditation that may encourage the development of improvement skills by the 100 000 residents in accredited programmes. Six competencies have been identified for all physicians, independent of specialty, and measurement tools for these competencies have been described. This model may be applicable to other healthcare professions. This paper explores patterns that inhibit efforts to change practice and proposes an educational model to provide changes in management skills based on trainees' analysis of their own work.

Alkylamides from echinacea modulate induced immune responses in macrophages.

The ability of Echinacea and its components to alter the immune response was examined in vitro in a macrophage cell line under either basal or immunostimulated conditions. Potential immunostimulatory and inflammatory activity was determined using a nuclear transcription factor (NFkappaB) expression, tumour necrosis factor alpha (TNFalpha) and nitric oxide (NO) production as biomarkers. In the absence of alternate stimulation, the only significant effects seen were a decrease in NFkappaB expression by a 2-ene alkylamide ((2E)-N-isobutylundeca-2-ene-8,10-diynamide (1)) and a decrease in TNFalpha levels by cichoric acid and an Echinacea alkylamide fraction (EPL AA). When the cells were stimulated by lipopolysaccharide (LPS), inhibition of the increased NFkappaB expression levels was caused by cichoric acid, an Echinacea preparation (EPL), EPL AA and a 2,4-diene ((2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (2)). Increases in TNFalpha levels were inhibited by cichoric acid, EPL and EPL AA but enhanced by 1 in the presence of LPS, while only EPL AA was able to inhibit the stimulated increases in NO. When using phorbol myristate acetate to stimulate the cells, NFkappaB and NO levels were unaffected by Echinacea or its components while only cichoric acid and 2 inhibited TNFalpha levels. Although cichoric acid was found to have an effect, it is probably not an important contributor to the Echinacea modulation of the immune response in vivo, as it is not bioavailable. Echinacea appears to attenuate the response of macrophages to an immune stimulus and its combination of phytochemicals exhibits different pharmacological properties to one or more of the isolated major individual components.

Seasonal nutrient dynamics in a chalk stream: the River Frome, Dorset, UK.

Chalk streams provide unique, environmentally important habitats, but are particularly susceptible to human activities, such as water abstraction, fish farming and intensive agricultural activity on their fertile flood-meadows, resulting in increased nutrient concentrations. Weekly phosphorus, nitrate, dissolved silicon, chloride and flow measurements were made at nine sites along a 32 km stretch of the River Frome and its tributaries, over a 15 month period. The stretch was divided into two sections (termed the middle and lower reach) and mass balances were calculated for each determinand by totalling the inputs from upstream, tributaries, sewage treatment works and an estimate of groundwater input, and subtracting this from the load exported from each reach. Phosphorus and nitrate were retained within the river channel during the summer months, due to bioaccumulation into river biota and adsorption of phosphorus to bed sediments. During the autumn to spring periods, there was a net export, attributed to increased diffuse inputs from the catchment during storms, decomposition of channel biomass and remobilisation of phosphorus from the bed sediment. This seasonality of retention and remobilisation was higher in the lower reach than the middle reach, which was attributed to downstream changes in land use and fine sediment availability. Silicon showed much less seasonality, but did have periods of rapid retention in spring, due to diatom uptake within the river channel, and a subsequent release from the bed sediments during storm events. Chloride did not produce a seasonal pattern, indicating that the observed phosphorus and nitrate seasonality was a product of annual variation in diffuse inputs and internal riverine processes, rather than an artefact of sampling, flow gauging and analytical errors.

Screening of some Australian Flacourtiaceae species for in vitro antioxidant, cytotoxic and antimicrobial activity.

A total of 27 methanol extracts obtained from different plant parts of 10 species of rain forest trees belonging to four genera of the Flacourtiaceae and originating from Australia were investigated. In vitro cytotoxicity was measured by an ATP Lite-M assay method against the mouse P388 lymphocytic leukemia cell line. The total antioxidant activity has been assessed based on scavenging activity of stable ABTS free radicals. The minimum inhibition concentration (MIC) was determined by the dilution method performed in 96 well plates against four different microbes. The leaf extract of Casearia sp. (RB 3051), mature stem extract of Casearia grayi and stem extract of Scolopia braunii were found to have most antioxidant activity (IC50 = 2.9 microg/ml), cytotoxic activity (LC50 = 0.89 microg/ml) and antimicrobial activity against all four different microbes, respectively. The results obtained suggested that among the four genera studied Casearia is the most promising in respect of finding significant antioxidant, cytotoxic and also antimicrobial activity.

Efficacy of anti-tumor necrosis factor therapy in the treatment of spontaneous endometriosis in baboons.

OBJECTIVE: To determine the efficacy of anti-tumor necrosis factor therapy (etanercept) for treating endometriosis in baboons. DESIGN: A randomized, placebo-controlled, blinded study using the baboon endometriosis model. SETTING: Southwest National Primate Research Center. ANIMAL(S): Twelve female baboons with spontaneous peritoneal endometriosis. INTERVENTION(S): Etanercept (n = 8) or sterile water (n = 4) was administered subcutaneously three times per week. MAIN OUTCOME MEASURE(S): After 8 weeks, the number, color, and surface area of peritoneal lesions was evaluated. Revised American Society for Reproductive Medicine staging was used. RESULT(S): A statistically significant decrease in red lesion surface area in the treatment group was observed. A trend toward a decrease in the absolute number of red lesions was noted in the treatment group. White and black lesion number and total surface area slightly increased in both groups but failed to achieve statistical significance. Endometriosis was diagnosed in 60% of captive-born baboons with primary infertility. CONCLUSION(S): These results indicate that etanercept effectively reduces the amount of spontaneously occurring active endometriosis in the baboon.

Genetic control of monoterpene composition in the essential oil of Melaleuca alternifolia (Cheel).

Previous studies into the population structure of Melaleuca alternifolia by both isozyme and microsatellite analysis revealed little evidence for genetic structuring within genetic provenances. In contrast, analysis of the oil composition within these same regions showed distinct clustering of chemotypes within the provenances suggesting either that chemotype was not under genetic control, or that there is strong environmental selection for plant chemotypes. To investigate the level of genetic control of monoterpene composition in the essential oil of M. alternifolia, individuals representing the three extreme chemotypes of high terpinen-4-ol, high 1,8-cineole and high terpinolene were crossed with an individual with the commercially desirable high terpinen-4-ol oil profile. The progeny resulting from these crosses displayed oil profiles that were intermediate to that of the parent. Further analysis of the survey of oil chemotypes within the natural population also suggests that these intermediate chemotypes may arise naturally between regions containing high proportions of the extreme chemotypes. These results imply that there is a level of genetic structure for chemotype determination within the genetic provenance that is undetected by isozyme and microsatellite analysis. This information could play a vital role in the selection of appropriate genetic material to be used in future essential oil selection and breeding programs.

Changing education to improve patient care.

Health professionals need competencies in improvement skills if they are to contribute usefully to improving patient care. Medical education programmes in the USA have not systematically taught improvement skills to residents (registrars in the UK). The Accreditation Council for Graduate Medical Education (ACGME) has recently developed and begun to deploy a competency based model for accreditation that may encourage the development of improvement skills by the 100 000 residents in accredited programmes. Six competencies have been identified for all physicians, independent of specialty, and measurement tools for these competencies have been described. This model may be applicable to other healthcare professions. This paper explores patterns that inhibit efforts to change practice and proposes an educational model to provide changes in management skills based on trainees' analysis of their own work.

Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease.

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.