Ferroptosis-Mediated Cell Death Induced by NCX4040, The Non-Steroidal Nitric Oxide Donor, in Human Colorectal Cancer Cells: Implications in Therapy.

Our recent studies show that the treatment of human ovarian tumor cells with NCX4040 results in significant depletions of cellular glutathione, the formation of reactive oxygen/nitrogen species and cell death. NCX4040 is also cytotoxic to several human colorectal cancer (CRC) cells in vitro and in vivo. Here, we examined the ferroptosis-dependent mechanism(s) of cytotoxicity of NCX4040 in HT-29 and K-RAS mutant HCT 116 colon cell lines. Ferroptosis is characterized by the accumulation of reactive oxygen species (ROS) within the cell, leading to an iron-dependent oxidative stress-mediated cell death. However, its relevance in the mechanism of NCX4040 cytotoxicity in CRCs is not known. We found that NCX4040 generates ROS in CRC cells without any depletion of cellular GSH. Combinations of NCX4040 with erastin (ER) or RSL3 (RAS-selective lethal 3), known inducers of ferroptosis, enhanced CRC death. In contrast, ferrostatin-1, an inhibitor of ferroptosis, significantly inhibited NCX4040-induced cell death. Treatment of CRC cells with NCX4040 resulted in the induction of lipid peroxidation in a dose- and time-dependent manner. NCX4040 treatment induced several genes related to ferroptosis (e.g., CHAC1, GPX4 and NOX4) in both cell lines. Metabolomic studies also indicated significant increases in both lipid and energy metabolism following the drug treatment in HT-29 and HCT 116 cells. These observations strongly suggest that NCX4040 causes the ferroptosis-mediated cell death of CRC cells. Furthermore, combinations of NCX4040 and ER or RSL3 may contribute significantly to the treatment of CRC, including those that are difficult to treat due to the presence of Ras mutations in the clinic. NCX4040-induced ferroptosis may also be a dynamic form of cell death for the treatment of other cancers.

What was old is new again: Phenotypic screening of a unique fungal library yields pyridoxatin, a promising lead against extensively resistant Acinetobacter baumannii (AB5075).

Due to the emergence of resistance, the World Health Organization considers Gram-negative pathogen Acinetobacter baumannii a top priority for therapeutic development. Using this priority pathogen and a phenotypic, agar plate-based assay, a unique library of extracts from 2,500 diverse fungi was screened for antimicrobial activity against a highly virulent, drug-resistant strain of A. baumannii (AB5075). The most potent hit from this screen was an extract from the fungus Tolypocladium sp., which was found to produce pyridoxatin. Another active extract from the fungi Trichoderma deliquescens was characterized and yielded trichokonin VII and trichokonin VIII. Evaluation of pyridoxatin against A. baumannii (AB5075) in a broth microdilution assay revealed a minimum inhibitory concentration (MIC) of 38 µM, compared to the known antibiotic levofloxacin with MIC of 28 µM. Mass spectrometry, Marfey's analysis and nuclear magnetic resonance spectroscopy analyses confirmed the structures of trichokonins VII and VIII to be consistent with previous reports. In an in vivo Galleria mellonella model, pyridoxatin tested at 150 mg/kg exhibited minimal toxicity (90% survival) and promising antimicrobial efficacy (50% survival) after 5 days. Trichokonins VII and VIII tested at 150 mg/kg were toxic to G. mellonella, with 20% survival and 40% survival after 5 days, respectively. The findings of this project suggest that pyridoxatin may serve as a lead compound for the development of antimicrobials against A. baumannii. They also demonstrate the value of the phenotypic screening approach employed herein.

Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis.

Colonization of the skin by Staphylococcus aureus is associated with exacerbation of atopic dermatitis (AD), but any direct mechanism through which dysbiosis of the skin microbiome may influence the development of AD is unknown. Here, we show that proteases and phenol-soluble modulin α (PSMα) secreted by S. aureus lead to endogenous epidermal proteolysis and skin barrier damage that promoted inflammation in mice. We further show that clinical isolates of different coagulase-negative staphylococci (CoNS) species residing on normal skin produced autoinducing peptides that inhibited the S. aureus agr system, in turn decreasing PSMα expression. These autoinducing peptides from skin microbiome CoNS species potently suppressed PSMα expression in S. aureus isolates from subjects with AD without inhibiting S. aureus growth. Metagenomic analysis of the AD skin microbiome revealed that the increase in the relative abundance of S. aureus in patients with active AD correlated with a lower CoNS autoinducing peptides to S. aureus ratio, thus overcoming the peptides' capacity to inhibit the S. aureus agr system. Characterization of a S. hominis clinical isolate identified an autoinducing peptide (SYNVCGGYF) as a highly potent inhibitor of S. aureus agr activity, capable of preventing S. aureus-mediated epithelial damage and inflammation on murine skin. Together, these findings show how members of the normal human skin microbiome can contribute to epithelial barrier homeostasis by using quorum sensing to inhibit S. aureus toxin production.

Safe delivery and newborn care practices in Sindh, Pakistan: a community-based investigation of mothers and health workers.

OBJECTIVE: to determine the prevalence of specific intrapartum practices in Sindh province, Pakistan. DESIGN: a cross-sectional, questionnaire based study. SETTING: 6 health clinics in Mirpurkhas, Sindh province, rural Pakistan. PARTICIPANTS: 225 mothers and 82 health workers. MEASUREMENTS AND FINDINGS: outcome measures were indicators of safe delivery practices and referral following an obstetric complication. Prevalence of unhygienic and unsafe practices in deliveries attended by Traditional Birth Attendants (TBAs) was common. Deliveries by skilled attendants were significantly safer but with some failures in hygienic practices. 29% of women who had experienced an obstetric complication had not received emergency obstetric care. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: safe delivery practices and newborn care needs to be improved in rural Pakistan. This may be achieved by training health workers and TBAs in safe delivery practices, using safe delivery kits and with an effective referral system.

Systematic review of effect of community-level interventions to reduce maternal mortality.

BACKGROUND: The objective was to provide a systematic review of the effectiveness of community-level interventions to reduce maternal mortality. METHODS: We searched published papers using Medline, Embase, Cochrane library, CINAHL, BNI, CAB ABSTRACTS, IBSS, Web of Science, LILACS and African Index Medicus from inception or at least 1982 to June 2006; searched unpublished works using National Research Register website, metaRegister and the WHO International Trial Registry portal. We hand searched major references.Selection criteria were maternity or childbearing age women, comparative study designs with concurrent controls, community-level interventions and maternal death as an outcome. We carried out study selection, data abstraction and quality assessment independently in duplicate. RESULTS: We found five cluster randomised controlled trials (RCT) and eight cohort studies of community-level interventions. We summarised results as odds ratios (OR) and confidence intervals (CI), combined using the Peto method for meta-analysis. Two high quality cluster RCTs, aimed at improving perinatal care practices, showed a reduction in maternal mortality reaching statistical significance (OR 0.62, 95% CI 0.39 to 0.98). Three equivalence RCTs of minimal goal-oriented versus usual antenatal care showed no difference in maternal mortality (1.09, 95% CI 0.53 to 2.25). The cohort studies were of low quality and did not contribute further evidence. CONCLUSION: Community-level interventions of improved perinatal care practices can bring about a reduction in maternal mortality. This challenges the view that investment in such interventions is not worthwhile. Programmes to improve maternal mortality should be evaluated using randomised controlled techniques to generate further evidence.

Feasibility of an exercise intervention for women with postnatal depression: a pilot randomised controlled trial.

BACKGROUND: Postnatal depression is a serious mental health problem that may be reduced by exercise. AIM: This study examined the feasibility of an exercise intervention for women with postnatal depression, and assessed which methods of recruitment are most effective. DESIGN OF STUDY: Randomised controlled trial. SETTING: General practice and the community. METHOD: Participants were recruited from various sources and randomised to an exercise intervention or usual care with follow-up at 12 weeks. As well as assessing feasibility, other trial outcomes included exercise participation and self-efficacy for exercise. Levels of depression were assessed but the study was not powered to show a difference in this. RESULTS: The recruitment rate of eligible patients was 23.1%. The highest recruitment rate was via referral from the psychiatric mother and baby unit (9/28; 32.1%), followed by invitation letters from GPs (24/93; 25.8%). Thirty-eight eligible participants were randomised. At follow-up there was no significant difference in exercise participation between groups. The intervention group reported significantly higher self-efficacy for exercise compared to usual care, but depression scores did not differ. CONCLUSION: Exercise participation over the 12-week period was not significantly increased, possibly because it is difficult to motivate women with postnatal depression to exercise, or the intervention was not sufficiently intensive. Eligible patients were recruited into this study but response rates were low. Optimum methods of recruitment in this difficult-to-reach population are required prior to a substantive trial. Further research is imperative given poorly-evidenced recommendations by the National Institute for Health and Clinical Excellence to consider this treatment.

Activation of the human T-cell leukemia virus type 1 long terminal repeat by the ternary complex factor Elk-1.

Serum response factor (SRF) was recently shown to bind and activate the human T-cell leukemia virus type 1 (HTLV-1) promoter at bases -116 to -125 relative to the transcription start site. In addition to the SRF binding site (CArG box), serum response elements (SRE) also typically contain a binding site for a member of the ternary complex factor (TCF) family. Here we demonstrate the presence of two TCF binding sites upstream of the viral CArG box. Binding of the TCF family member Elk-1 to these sites was shown to activate transcription of the promoter. Based on these results, the position of the previously described viral SRE (vSRE) within the HTLV-1 promoter can be extended from -116 to -157 to include the two newly identified TCF sites. Purified Elk-1 bound to a probe containing the vSRE, and this complex formed a ternary complex with SRF. In addition, the complex formed by nuclear extract on this probe contained Elk-1, as shown by electrophoretic mobility shift assay supershift. Both of the predicted TCF sites independently bound Elk-1. Elk-1 activated transcription of the HTLV-1 long terminal repeat (LTR), and mutations within either of the TCF sites or the CArG box reduced responsiveness of the LTR to Elk-1. Chromatin immunoprecipitation demonstrated that Elk-1 associates with the HTLV-1 LTR in vivo. These results identify a functional SRE within the HTLV-1 LTR and suggest that both Elk-1 and SRF play important roles in regulating basal HTLV-1 gene expression.

Human T-cell leukemia virus type 1 Tax enhances serum response factor DNA binding and alters site selection.

Human T-cell leukemia virus type I (HTLV-1) is the etiological agent of adult T-cell leukemia. The viral transforming protein Tax regulates the transcription of viral and cellular genes by interacting with cellular transcription factors and coactivators. The effects of Tax on cellular gene expression have an important impact on HTLV-1-mediated cellular transformation. Expression of the c-fos cellular oncogene is regulated by serum response factor (SRF), and Tax is known to induce c-fos gene expression by activating SRF-responsive transcription. SRF activates cellular gene expression by binding to a consensus DNA sequence (CArG box) located within a serum response element (SRE). Since SRF activates transcription of many growth regulatory genes, this pathway is likely to have a significant impact on Tax-mediated transformation. Here we demonstrate that Tax interacts with SRF and enhances the binding of SRF to SREs located in the c-fos, Nur77, and viral promoters. Also, we establish that in the presence of Tax, SRF selects more divergent CArG box sequences than in the absence of Tax, revealing a novel mechanism for regulating SRF-responsive gene expression. Finally, increased association of SRF with chromatin and specific promoters was observed in Tax-expressing cells, correlating with increased c-fos and Nur77 mRNA levels in Tax-expressing cells. These results suggest that Tax activates SRF-responsive transcription by enhancing its binding affinity to multiple different SRE sequences.

The role of exercise in treating postpartum depression: a review of the literature.

There is now evidence to support the antidepressant effects of exercise in general and in clinical populations. This article reviews the evidence regarding the potential role of exercise, particularly pram walking, as an adjunctive treatment for postpartum depression. Database searches revealed two small randomised controlled trials conducted in Australia which support exercise as a useful treatment for women with postpartum depression. In addition, uncontrolled studies and observational evidence suggest that postpartum women, some of whom were depressed, report benefit from participation in exercise programmes. There are plausible mechanisms by which exercise could have such an effect. Limited evidence supports a relationship between participation in exercise and reduction in postpartum depression. Given the reluctance by some women to use antidepressant medication postpartum and the limited availability of psychological therapies, exercise as a therapeutic possibility deserves further exploration. Further research using well-designed randomised controlled trial methodologies are warranted.

An intervention involving traditional birth attendants and perinatal and maternal mortality in Pakistan.

BACKGROUND: There are approximately 4 million neonatal deaths and half a million maternal deaths worldwide each year. There is limited evidence from clinical trials to guide the development of effective maternity services in developing countries. METHODS: We performed a cluster-randomized, controlled trial involving seven subdistricts (talukas) of a rural district in Pakistan. In three talukas randomly assigned to the intervention group, traditional birth attendants were trained and issued disposable delivery kits; Lady Health Workers linked traditional birth attendants with established services and documented processes and outcomes; and obstetrical teams provided outreach clinics for antenatal care. Women in the four control talukas received usual care. The primary outcome measures were perinatal and maternal mortality. RESULTS: Of the estimated number of eligible women in the seven talukas, 10,114 (84.3 percent) were recruited in the three intervention talukas, and 9443 (78.7 percent) in the four control talukas. In the intervention group, 9184 women (90.8 percent) received antenatal care by trained traditional birth attendants, 1634 women (16.2 percent) were seen antenatally at least once by the obstetrical teams, and 8172 safe-delivery kits were used. As compared with the control talukas, the intervention talukas had a cluster-adjusted odds ratio for perinatal death of 0.70 (95 percent confidence interval, 0.59 to 0.82) and for maternal mortality of 0.74 (95 percent confidence interval, 0.45 to 1.23). CONCLUSIONS: Training traditional birth attendants and integrating them into an improved health care system were achievable and effective in reducing perinatal mortality. This model could result in large improvements in perinatal and maternal health in developing countries.

Proximity of first intercourse to menarche and the risk of human papillomavirus infection: a longitudinal study.

Cross-sectional studies have suggested that compared with women who delay the start of their sexual career, those who first have intercourse soon after menarche are more susceptible to cervical human papillomavirus (HPV) infection and thus have a greater risk of cervical neoplasia. We describe, using longitudinal observations, how the risk of infection with HPV varies with the interval between menarche and first intercourse in 474 women aged 15-19 recruited within 12 months of first intercourse and before the acquisition of a second sexual partner. One hundred forty-five women became HPV-positive; the cumulative risk of HPV infection 3 years after first intercourse was 45.0% (95% CI = 37.9-51.2). In univariate analyses, the hazards ratio (HR) of HPV infection increased significantly with age at first intercourse (HR = 1.212 per year; 95% CI = 1.050-1.398), partner age (HR = 1.084 per year; 95% CI = 1.045-1.125) and when women reported a sexually experienced partner (HR = 2.794; 95% CI = 1.804-4.326); the interval between menarche and first intercourse was a significant predictor of infection, with an increase in the HR of 12.9% for every year of increase in this interval (95% CI = 2.1%-24.9%). In a multivariate analysis, compared with women who first had intercourse within 3 years of menarche, those who postponed first intercourse beyond this time had a greater risk of infection (HR = 1.581; 95% CI = 1.113-2.245) after controlling for age and sexual experience of partner.

Human papillomavirus type 18 and rapidly progressing cervical intraepithelial neoplasia.

BACKGROUND: Human papillomavirus type 18 (HPV-18) is the second most frequent of the HPV types detected when squamous-cell cancer is diagnosed and the type most strongly associated with adenocarcinoma of the cervix. However, in cross-sectional studies, HPV-18 is rarely detected at the time of diagnosis of high-grade cervical intraepithelial neoplasia (CIN). We used a longitudinal study design to describe the occurrence of cytological abnormality after incident HPV-18 and HPV-16 infections. METHODS: The analysis was based on 1075 women aged 15-19 years, who had normal cytology and were negative for HPV at recruitment from a single family-planning clinic, and who had further follow-up. The women reattended every 6 months, and samples were taken for cytological and virological examination. FINDINGS: The relative risk of a cytological diagnosis of borderline nuclear abnormality after exposure to HPV-18 was 2.06 (95% CI 1.24-3.43) and that after exposure to HPV-16 was 1.99 (1.32-3.01). The relative risks of mild dyskaryosis were 3.11 (1.86-5.18) and 4.76 (3.15-7.18), and the relative risks of moderate or severe dyskaryosis were 0.80 (0.24-2.65) and 2.85 (1.36-5.97). Time to acquisition of cytological abnormality was unrelated to the infecting type (p=0.88). INTERPRETATION: Our findings do not support the long-held view that the reason why HPV-18 infection is under-represented at the time of diagnosis of high-grade CIN is because HPV-18-associated disease rapidly progresses through the preinvasive stages of neoplasia. We suggest that the cytological changes detected after HPV-18 infection might understate the severity of underlying disease. This feature could compromise the effectiveness of screening programmes in reducing the frequency of HPV-18-associated cancers.

High incidence of cervical human papillomavirus infection in women during their first sexual relationship.

The prevalence of cervical human papillomavirus increases with increasing numbers of sexual partners, leaving the impression that this infection is acquired only as a result of high risk sexual behaviour. Using longitudinal data from 242 women who had only had one sexual partner, we found that the risk of acquiring cervical human papillomavirus infection was 46% (95% CI 28-64) at three years after first intercourse and that the median time from first intercourse to first detection of human papillomavirus was only three months.