The KEAP1-NRF2 pathway regulates TFEB/TFE3-dependent lysosomal biogenesis.

The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1 (KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB (TFEB)/transcription factor binding to IGHM Enhancer 3 (TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1-NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.

AMPK CA(R)Sts a new light on amino acid sensing.

AMP-activated protein kinase (AMPK) is recognized as a critical regulator of cellular energy metabolism impacted by AMP/ATP and ADP/ATP ratios, or glucose- and fatty acid-derived metabolites. However, its ability to sense alterations in amino acid levels is poorly understood. Recent work by Yuan et al (2021) identifies a novel mechanism of AMPK regulation responsive to changes in availability of the sulfur-containing amino acid cysteine.

Risk Factors for Recent HIV Infections among Adults in 14 Countries in Africa Identified by Population-Based HIV Impact Assessment Surveys, 2015-2019.

Identifying persons who have newly acquired HIV infections is critical for characterizing the HIV epidemic direction. We analyzed pooled data from nationally representative Population-Based HIV Impact Assessment surveys conducted across 14 countries in Africa for recent infection risk factors. We included adults 15-49 years of age who had sex during the previous year and used a recent infection testing algorithm to distinguish recent from long-term infections. We collected risk factor information via participant interviews and assessed correlates of recent infection using multinomial logistic regression, incorporating each survey's complex sampling design. Compared with HIV-negative persons, persons with higher odds of recent HIV infection were women, were divorced/separated/widowed, had multiple recent sex partners, had a recent HIV-positive sex partner or one with unknown status, and lived in communities with higher HIV viremia prevalence. Prevention programs focusing on persons at higher risk for HIV and their sexual partners will contribute to reducing HIV incidence.

Awareness of and willingness to use oral pre-exposure prophylaxis (PrEP) for HIV prevention among sexually active adults in Malawi: results from the 2020 Malawi population-based HIV impact assessment.

BACKGROUND: The World Health Organization recommends Pre-Exposure Prophylaxis (PrEP) for all populations at substantial risk of HIV infection. Understanding PrEP awareness and interest is crucial for designing PrEP programs; however, data are lacking in sub-Saharan Africa. In Malawi, oral PrEP was introduced in 2018. We analyzed data from the 2020 Malawi Population-based HIV Impact Assessment (MPHIA) to assess PrEP awareness and factors associated with PrEP interest in Malawi. METHODS: MPHIA 2020 was a national cross-sectional household-based survey targeting adults aged 15 + years. Oral PrEP was first described to the survey participants as taking a daily pill to reduce the chance of getting HIV. To assess awareness, participants were asked if they had ever heard of PrEP and to assess interest, were asked if they would take PrEP to prevent HIV, regardless of previous PrEP knowledge. Only sexually active HIV-negative participants are included in this analysis. We used multivariable logistic regression to assess sociodemographic factors and behaviors associated with PrEP interest. All results were weighted. RESULTS: We included 13,995 HIV-negative sexually active participants; median age was 29 years old. Overall, 15.0%, 95% confidence interval (CI): 14.2-15.9% of participants were aware of PrEP. More males (adjusted odds ratio (aOR): 1.3, 95% CI: 1.2-1.5), those with secondary (aOR: 1.5, 95% CI: 1.2-2.0) or post-secondary (aOR: 3.4, 95% CI: 2.4-4.9) education and the wealthiest (aOR: 1.6, 95% CI: 1.2-2.0) were aware of PrEP than female, those without education and least wealthy participants, respectively. Overall, 73.0% (95% CI: 71.8-74.1%) of participants were willing to use PrEP. Being male (aOR: 1.2; 95% CI: 1.1-1.3) and having more than one sexual partner (aOR: 1.7 95% CI: 1.4-1.9), were associated higher willingness to use PrEP. CONCLUSIONS: In this survey, prior PrEP knowledge and use were low while PrEP interest was high. High risk sexual behavior was associated with willingness to use PrEP. Strategies to increase PrEP awareness and universal access, may reduce HIV transmission.

Pediatric staff and their perceptions of music therapy services.

PURPOSE: To reveal the benefits, needs, and limitations of music therapy observed by clinical pediatric staff at a children's hospital in the United States of America. DESIGN AND METHODS: Researchers developed an electronic 13-question survey and distributed the survey in the fall of 2022. Questions included demographics, Likert-type scale, and open-ended queries. Data was collected via Qualtrics and analyzed using descriptive statistics and content analysis. RESULTS: A total of 83 pediatric staff completed the survey. Staff observed positive benefits, where the highest reported areas were opportunities for dealing with anxiety/stress (94.7%), opportunities for social interaction (93.3%), and quality of life (89.3%). Analysis of free-response questions suggest that staff expect expertise and a nuanced understanding of the needs of each of their clinical units. CONCLUSION: Results suggest that staff possess an overall positive attitude toward music therapy in all settings served. Music therapists may be valuable for psychosocial and rehabilitative support to hospitalized children and their families. PRACTICE IMPLICATIONS: Nursing staff may utilize music therapists to improve patient outcomes and reduce the negative effects of hospitalization.

Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth.

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap-/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap-/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.

Active Hexose-Correlated Compound Restores Gene Expression and Protein Secretion of Protective Cytokines of Immune Cells in a Murine Stress Model during Chlamydia muridarum Genital Infection.

Chlamydia trachomatis genital infection is the most common bacterial sexually transmitted disease worldwide. Previously, we reported that cold-induced stress results in immune suppression of mice that subsequently leads to increased intensity of Chlamydia muridarum genital infection. Furthermore, we demonstrated that stressed mice orally fed with active hexose-correlated compound (AHCC) have reduced shedding of C. muridarum from the genital tract. However, the mechanism of AHCC in reducing the organ load and changing the immune response in the stress model is not well known. This study evaluated infection and changes in immunological parameters of stressed AHCC-fed mice with or without C. muridarum genital infection. We hypothesized that AHCC feeding to stressed mice restores protective immune function and reduces susceptibility to C. muridarum genital infection. The results show that oral feeding of stressed mice with AHCC resulted in decreased shedding of C. muridarum from the genital tract, reduced production of plasma catecholamines, increased expression of T-bet and reduced GATA-3 in CD4+ T cells, increased production of interleukin-12 (IL-12) and interferon gamma (IFN-γ) and reduced production of IL-4 in CD4+ T cells, and enhanced expression of surface markers and costimulatory molecules of CD4+ T cells, bone marrow-derived dendritic cells (BMDCs), and natural killer cells. Coculturing of mature BMDCs with splenic CD4+ T cells led to the increased and decreased production of T helper 1 and T helper 2 cytokines, respectively. Overall, our results show that AHCC fosters the restoration of Th1 cytokine production while reducing Th2 cytokine production, which would promote C. muridarum clearance in the murine stress model.

Human Immunodeficiency Virus Infection in Adolescents and Mode of Transmission in Southern Africa: A Multinational Analysis of Population-Based Survey Data.

BACKGROUND: Adolescents aged 10-19 years living with human immunodeficiency virus (HIV) (ALHIV), both perinatally infected adolescents (APHIV) and behaviorally infected adolescents (ABHIV), are a growing population with distinct care needs. We characterized the epidemiology of HIV in adolescents included in Population-based HIV Impact Assessments (2015-2017) in Zimbabwe, Malawi, Zambia, Eswatini, and Lesotho. METHODS: Adolescents were tested for HIV using national rapid testing algorithms. Viral load (VL) suppression (VLS) was defined as VL <1000 copies/mL, and undetectable VL (UVL) as VL <50 copies/mL. Recent infection (within 6 months) was measured using a limiting antigen avidity assay, excluding adolescents with VLS or with detectable antiretrovirals (ARVs) in blood. To determine the most likely mode of infection, we used a risk algorithm incorporating recency, maternal HIV and vital status, history of sexual activity, and age at diagnosis. RESULTS: HIV prevalence ranged from 1.6% in Zambia to 4.8% in Eswatini. Of 707 ALHIV, 60.9% (95% confidence interval, 55.3%-66.6%) had HIV previously diagnosed, and 47.1% (41.9%-52.3%) had VLS. Our algorithm estimated that 72.6% of ALHIV (485 of 707) were APHIV, with HIV diagnosed previously in 69.5% of APHIV and 39.4% of ABHIV, and with 65.3% of APHIV and 33.5% of ABHIV receiving ARV treatment. Only 67.2% of APHIV and 60.5% of ABHIV receiving ARVs had UVL. CONCLUSIONS: These findings suggest that two-thirds of ALHIV were perinatally infected, with many unaware of their status. The low prevalence of VLS and UVL in those receiving treatment raises concerns around treatment effectiveness. Expansion of opportunities for HIV diagnoses and the optimization of treatment are imperative.

Coronavirus Disease 2019 (COVID-19) Mitigation Efforts and Testing During an In-Person Training Event-Uganda, 12-29 October 2020.

Large public-health training events may result in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Universal SARS-CoV-2 testing during trainings for the Uganda Population-based HIV Impact Assessment identified 28 of 475 (5.9%) individuals with coronavirus disease 2019 (COVID-19) among attendees; most (89.3%) were asymptomatic. Until COVID-19 vaccine is readily available for staff and participants, effective COVID-19 mitigation measures, along with SARS-CoV-2 testing, are recommended for in-person trainings, particularly when trainees will have subsequent contact with survey participants.

Screening for HIV Among Patients at Tuberculosis Clinics - Results from Population-Based HIV Impact Assessment Surveys, Malawi, Zambia, and Zimbabwe, 2015-2016.

The World Health Organization and national guidelines recommend HIV testing and counseling at tuberculosis (TB) clinics for all patients, regardless of TB diagnosis (1). Population-based HIV Impact Assessment (PHIA) survey data for 2015-2016 in Malawi, Zambia, and Zimbabwe were analyzed to assess HIV screening at TB clinics among persons who had positive HIV test results in the survey. The analysis was stratified by history of TB diagnosis* (presumptive versus confirmed†), awareness§ of HIV-positive status, antiretroviral therapy (ART)¶ status, and viral load suppression among HIV-positive adults, by history of TB clinic visit. The percentage of adults who reported having ever visited a TB clinic ranged from 4.7% to 9.7%. Among all TB clinic attendees, the percentage who reported that they had received HIV testing during a TB clinic visit ranged from 48.0% to 62.1% across the three countries. Among adults who received a positive HIV test result during PHIA and who did not receive a test for HIV at a previous TB clinic visit, 29.4% (Malawi), 21.9% (Zambia), and 16.2% (Zimbabwe) reported that they did not know their HIV status at the time of the TB clinic visit. These findings represent missed opportunities for HIV screening and linkage to HIV care. In all three countries, viral load suppression rates were significantly higher among those who reported ever visiting a TB clinic than among those who had not (p<0.001). National programs could strengthen HIV screening at TB clinics and leverage them as entry points into the HIV diagnosis and treatment cascade (i.e., testing, initiation of treatment, and viral load suppression).

Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis.

Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Patient experiences in behavioral health integrated primary care settings: the role of stigma in shaping patient outcomes over time.

Behavioral health integration (BHI) models seek to improve patient experience and outcomes by bridging physical and behavioral health services. Past BHI research has not focused on stigma in these settings, which has been previously found to impact patient engagement and outcomes. We surveyed patients over a two year period at 12 integrated clinics in Oregon using measures developed by a Patient Advisory Team. Over a quarter of respondents reported stigmatization (26.81%). Compared to non-stigmatized patients, those who reported stigma had five times the odds of reporting unmet health needs (OR=5.14, p<0.0001), three times the odds reporting issues accessing care (OR=2.93, p<0.0001), six times the odds reporting hassle to get care (OR=6.49, p<0.0001), and three times the odds of reporting poor communication between providers (OR=3.45, p<0.0001). After examining the interaction between stigmatization and time, we found that stigmatized patients had lower odds at year two of reporting unmet health needs (OR=0.68, p=0.0034), issues accessing care (OR=0.77, p=0.0400), hassle getting care (OR=0.57, p=0.0001), and poor provider communication (OR=0.77, p=0.0544). We found that stigma remained prevalent for patients seeking care in the integrated clinics studied despite integration. Systems should consider integration efforts and reducing stigmatizing experiences in tandem to truly improve patient outcomes.

Status of HIV Epidemic Control Among Adolescent Girls and Young Women Aged 15-24 Years - Seven African Countries, 2015-2017.

In 2016, an estimated 1.5 million females aged 15-24 years were living with human immunodeficiency virus (HIV) infection in Eastern and Southern Africa, where the prevalence of HIV infection among adolescent girls and young women (3.4%) is more than double that for males in the same age range (1.6%) (1). Progress was assessed toward the Joint United Nations Programme on HIV/AIDS (UNAIDS) 2020 targets for adolescent girls and young women in sub-Saharan Africa (90% of those with HIV infection aware of their status, 90% of HIV-infected persons aware of their status on antiretroviral treatment [ART], and 90% of those on treatment virally suppressed [HIV viral load <1,000 HIV RNA copies/mL]) (2) using data from recent Population-based HIV Impact Assessment (PHIA) surveys in seven countries. The national prevalence of HIV infection in adolescent girls and young women aged 15-24 years, the percentage who were aware of their status, and among those persons who were aware, the percentage who had achieved viral suppression were calculated. The target for viral suppression among all persons with HIV infection is 73% (the product of 90% x 90% x 90%). Among all seven countries, the prevalence of HIV infection among adolescent girls and young women was 3.6%; among those in this group, 46.3% reported being aware of their HIV-positive status, and 45.0% were virally suppressed. Sustained efforts by national HIV and public health programs to diagnose HIV infection in adolescent girls and young women as early as possible to ensure rapid initiation of ART should help achieve epidemic control among adolescent girls and young women.

Sequence analysis of mutations and translocations across breast cancer subtypes.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Can knowledge exchange support the implementation of a health-promoting schools approach? Perceived outcomes of knowledge exchange in the COMPASS study.

BACKGROUND: Despite the potential population-level impact of a health-promoting schools approach, schools face challenges in implementation, indicating a gap between school health research and practice. Knowledge exchange provides an opportunity to reduce this gap; however, there has been limited evaluation of these initiatives. This research explored researchers' and knowledge users' perceptions of outcomes associated with a knowledge exchange initiative within COMPASS, a longitudinal study of Canadian secondary students and schools. Schools received annual tailored summaries of their students' health behaviours and suggestions for action and were linked with knowledge brokers to support them in taking action to improve student health. METHODS: Qualitative semi-structured interviews were conducted with COMPASS researchers (n = 13), school staff (n = 13), and public health stakeholders (n = 4) to explore their experiences with COMPASS knowledge exchange. Key issues included how knowledge users used school-specific findings, perceived outcomes of knowledge exchange, and suggestions for change. RESULTS: Outcomes for both knowledge users and researchers were identified; interestingly, knowledge users attributed more outcomes to using school-specific findings than knowledge brokering. School and public health participants indicated school-specific findings informed their programming and planning. Importantly, knowledge exchange provided a platform for partnerships between researchers, schools, and public health units. Knowledge brokering allowed researchers to gain feedback from knowledge users to enhance the study and a better understanding of the school environment. Interestingly, COMPASS knowledge exchange outcomes aligned with Samdal and Rowling's eight theory-driven implementation components for health-promoting schools. Hence, knowledge exchange may provide a mechanism to help schools implement a health-promoting schools approach. CONCLUSIONS: This research contributes to the limited literature regarding outcomes of knowledge brokering in public health and knowledge exchange in school health research. However, since not all schools engaged in knowledge brokering, and not all schools that engaged discussed these outcomes, further research is needed to determine the amount of engagement required for change and examine the process of COMPASS knowledge brokering to consider how to increase school engagement.

Predictors of Percutaneous Endoscopic Gastrostomy Placement in Acute Ischemic Stroke.

BACKGROUND: Dysphagia is a common complication of stroke and can have a lasting impact on morbidity and mortality; yet there are no standards to guide dysphagia management in stroke patients. We assessed predictors of percutaneous endoscopic gastrostomy (PEG) placement in an ischemic stroke cohort and sought to determine the utility of an objective scale in predicting PEG placement in a high-risk sub-set. METHODS: Consecutive cases of ischemic stroke were retrospectively, identified and demographic and clinical variables were collected. Penetration-Aspiration (PAS) scores (1-2 normal; 3-5 penetration; 6-8 aspiration) were calculated for patients undergoing Fiberoptic Endoscopic Evaluation of Swallowing (FEES) or Modified Barium Swallowing Studies (MBSS). Multiple logistic regression analysis was used to assess predictors of PEG placement. RESULTS: Among 724 patients, 131 underwent PEG placement. In univariate analysis of the overall cohort, sex, age, insured payer status, arrival National Institute of Health Stroke Scale (NIHSS), NIHSS level of consciousness severity, NIHSS dysarthria severity, diabetes mellitus, and prior International Conference for Harmonization (ICH) were all significantly associated with PEG placement. Among 197 high-risk patients undergoing FEES or MBSS, the multivariate logistic regression analysis showed that PAS scores 6-8 versus 1-2 (odds ratio [OR] 13.2; 95% confidence interval [CI] 4.58, 38.2), PAS score 3-5 versus 1.2 (OR 33.8; 95% CI 11.6, 98.3), Hispanic race (OR, 5.73; 95% CI 1.82, 18.0), male sex (OR, 2.59; 95% CI 1.05, 6.34), and arrival NIHSS (OR, 1.11; 95% CI 1.05, 1.18) were associated with PEG placement. CONCLUSIONS: Use of an objective dysphagia scale simplified the prediction model among acute ischemic stroke patients undergoing instrumental assessments of dysphagia with FEES or MBSS. Male sex and Hispanic race were also significantly associated with PEG placement in this analysis. These findings support the need for rigorously designed prospective studies to assess biological and social factors that influence PEG placement and to determine, how to best evaluate and manage patients with dysphagia.

"Now What?" Perceived Factors Influencing Knowledge Exchange in School Health Research.

Increasing the uptake of school health research into practice is pivotal for improving adolescent health. COMPASS, a longitudinal study of Ontario and Alberta secondary students and schools (2012-2021), used a knowledge exchange process to enhance schools' use of research findings. Schools received annual summaries of their students' health behaviors and suggestions for action and were linked with a knowledge broker to support them in making changes to improve student health. The current research explored factors that influenced COMPASS knowledge exchange activities. Semistructured interviews were conducted with researchers (n = 13), school staff (n = 13), and public health stakeholders (n = 4). Interestingly, knowledge users focused more on factors that influenced their use of COMPASS findings than factors that influenced knowledge brokering. The factors identified by participants are similar to those that influence implementation of school health interventions (e.g., importance of school champions, competing priorities, inadequate resources). While knowledge exchange offers a way to reduce the gap between research and practice, schools that need the most support may not engage in knowledge exchange; hence, we must consider how to increase engagement of these schools to ultimately improve student health.

Biocatalytic Synthesis of Chiral N-Functionalized Amino Acids.

N-Functionalized amino acids are important building blocks for the preparation of diverse bioactive molecules, including peptides. The development of sustainable manufacturing routes to chiral N-alkylated amino acids remains a significant challenge in the pharmaceutical and fine-chemical industries. Herein we report the discovery of a structurally diverse panel of biocatalysts which catalyze the asymmetric synthesis of N-alkyl amino acids through the reductive coupling of ketones and amines. Reactions have been performed on a gram scale to yield optically pure N-alkyl-functionalized products in high yields.

A human fatty acid synthase inhibitor binds ß-ketoacyl reductase in the keto-substrate site.

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the ß-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

Type IIA topoisomerase inhibition by a new class of antibacterial agents.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.