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Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules in E. coli persister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field. IMPORTANCE The recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies identified multiple genetic pathways involved in persister formation but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study, we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely differing results based on poorly controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis for future studies on the formation and eradication of antibiotic-tolerant persisters. The recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies identified multiple genetic pathways involved in persister formation but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study, we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely differing results based on poorly controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis for future studies on the formation and eradication of antibiotic-tolerant persisters.

PurposeTo compare the diagnostic performance of contrast-enhanced computed tomography (CE-CT), magnetic resonance imaging (MRI) and combined fluorodeoxyglucose/positron emission tomography/computed tomography (FDG-PET/CT) for detection of colorectal liver metastases (CRLM) in patients eligible for local treatment.Materials and methodsThis health-research ethics-committee-approved prospective consecutive diagnostic accuracy study, with written informed consent, included 80 cases (76 patients, four participating twice) between 29 June 2015 and 7 February 2017. Prior chemotherapy or local treatment did not exclude participation. Combined FDG-PET/CT including CE-CT and MRI was performed within 0–3 days shortly before local treatment. CE-CT and MRI images were read independently by two readers for each modality. The combined FDG-PET/CT images were read independently by two pairs of readers. A composite reference standard was used. Sensitivities, specificities and area under the receiver operating characteristic curves (AUCROC) were calculated and compared.ResultsIn total, 260 CRLMs were confirmed. The MRI readers had significantly higher per-lesion sensitivity (85.9% and 83.8%) than both CE-CT readers (69.1% and 62.3%) and both PET/CT reader pairs (72.0% and 72.1%) (p<0.001). There were no significant differences in per-lesion specificity. MRI readers had significantly higher AUCROC (0.92 and 0.88) than both CE-CT readers (0.80 and 0.82) (p≤0.001). AUCROC for MR reader 1 was higher than that of both PET/CT reader pairs (0.83 and 0.84) (p≤0.0001).ConclusionMRI performed significantly better than both CE-CT and combined FDG-PET/CT for detection of CRLM in consecutive patients eligible for local treatment irrespective of prior chemotherapy or local treatment.Key Points• Patients eligible for local treatment of colorectal liver-metastases require optimal imaging.• In 80 consecutive patients, MRI had superior per lesion diagnostic performance.• Findings were independent of prior treatment and type of planned local treatment.• Equally, MRI had superior diagnostic performance on per segment basis.

Control of magnetization and electric polarization is attractive in relation to tailoring materials for data storage and devices such as sensors or antennae. In magnetoelectric materials, these degrees of freedom are closely coupled, allowing polarization to be controlled by a magnetic field, and magnetization by an electric field, but the magnitude of the effect remains a challenge in the case of single-phase magnetoelectrics for applications. We demonstrate that the magnetoelectric properties of the mixed-anisotropy antiferromagnet LiNi 1− x Fe x PO 4 are profoundly affected by partial substitution of Ni 2+ ions with Fe 2+ on the transition metal site. This introduces random site-dependent single-ion anisotropy energies and causes a lowering of the magnetic symmetry of the system. In turn, magnetoelectric couplings that are symmetry-forbidden in the parent compounds, LiNiPO 4 and LiFePO 4 , are unlocked and the dominant coupling is enhanced by almost two orders of magnitude. Our results demonstrate the potential of mixed-anisotropy magnets for tuning magnetoelectric properties. In magnetoelectric materials, the magnetization can be controlled by the application of an electric field, making it comparatively easy to switch magnetization, which is attractive for data storage and other proposed devices. Unfortunately, the effect in single-phase materials is typically fairly weak. Here Fogh et al. demonstrate a two orders of magnitude enhancement of the magnetoelectric coupling in LiNi 0.8 Fe 0.2 PO 4 compared to the parent compounds.

Many bacteria encode multiple toxin–antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the Escherichia coli hipBA system, HipA, is a kinase that inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli O127:H6 encodes the hipBA -like, tripartite TA system; hipBST , in which the HipT toxin specifically targets the tryptophanyl-tRNA synthetase, TrpS. Notably, in the tripartite system, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is inhibited remain poorly understood. Here, we show that HipBST is structurally different from E. coli HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, inhibits the kinase through insertion of a conserved Trp residue into the active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve different roles and affect the ability of HipS to neutralize HipT. Finally, solution structural studies show how phosphorylation affects overall TA complex flexibility.

The exponential growth of bacterial populations is punctuated by long or short periods of starvation lasting from the point of nutrient exhaustion until nutrients are replenished. To understand the consequences of long-term starvation for Escherichia coli cells, we performed month-long carbon and phosphorus starvation experiments and measured three key phenotypes of the cultures, namely, the survival of the cells, the time needed for them to resume growth after nutrient replenishment, and the levels of intact rRNA preserved in the cultures. The stationary phase is the general term for the state a bacterial culture reaches when no further increase in cell mass occurs due to exhaustion of nutrients in the growth medium. Depending on the type of nutrient that is first depleted, the metabolic state of the stationary phase cells may vary greatly, and the subsistence strategies that best support cell survival may differ. As ribosomes play a central role in bacterial growth and energy expenditure, ribosome preservation is a key element of such strategies. To investigate the degree of ribosome preservation during long-term starvation, we compared the dynamics of rRNA levels of carbon-starved and phosphorus-starved Escherichia coli cultures for up to 28 days. The starved cultures’ contents of full-length 16S and 23S rRNA decreased as the starvation proceeded in both cases, and phosphorus starvation resulted in much more rapid rRNA degradation than carbon starvation. Bacterial survival and regrowth kinetics were also quantified. Upon replenishment of the nutrient in question, carbon-starved cells resumed growth faster than cells starved for phosphate for the equivalent amount of time, and for both conditions, the lag time increased with the starvation time. While these results are in accordance with the hypothesis that cells with a larger ribosome pool recover more readily upon replenishment of nutrients, we also observed that the lag time kept increasing with increasing starvation time, also when the amount of rRNA per viable cell remained constant, highlighting that lag time is not a simple function of ribosome content under long-term starvation conditions. IMPORTANCE The exponential growth of bacterial populations is punctuated by long or short periods of starvation lasting from the point of nutrient exhaustion until nutrients are replenished. To understand the consequences of long-term starvation for Escherichia coli cells, we performed month-long carbon and phosphorus starvation experiments and measured three key phenotypes of the cultures, namely, the survival of the cells, the time needed for them to resume growth after nutrient replenishment, and the levels of intact rRNA preserved in the cultures. The starved cultures’ concentration of rRNA dropped with starvation time, as did cell survival, while the lag time needed for regrowth increased. While all three phenotypes were more severely affected during starvation for phosphorus than for carbon, our results demonstrate that neither survival nor lag time is correlated with ribosome content in a straightforward manner.

Introduction: Spontaneous cervical artery dissection (CeAD) is an important cause of ischemic strokes in young adults. The pathophysiology as well as risk factors are largely unknown. Recurrences are considered rare and primarily to occur within the first 3 months. The frequency of asymptomatic recurrences varies in recent studies. This study seeks to determine the risk of all recurrent dissections in an unselected consecutive patient population after initial diagnosis. Methods: Of 218 patients referred between October 2014 and December 2024, 102 were included in the final analysis. Patients’ medical records were reviewed to determine demographics, including associated risk factors and antithrombotic treatments used. Reports from relevant imaging at routine follow-up and at repeat admissions were acquired to determine recurrence rates of CeAD and new cases of stroke. Results: Seven (6.9%) patients had CeAD recurrences after the index event, of which 6 occurred after 6 months and 2 (28.6% of recurrences) were asymptomatic. A family history of dissections (RR: 6.9, CI: 1.7; 27.3, p = 0.006) or radiologically verified tortuous cervical arteries (RR: 9.8, CI: 2.8; 34.3, p = 0.0003) were significantly associated with recurrence. By 1-year follow-up, 56 patients had persisting vessel sequelae from the index CeAD. Stroke occurrences after the index CeAD was 2.9% (n = 3) and did not occur in patients with CeAD recurrence. All patients received antithrombotic treatment for at least a year. Conclusion: Recurrence of CeAD and stroke occurrence were low after the index event. CeAD were often asymptomatic and occurred later than previously reported. Long-term, regular follow-up and stroke-preventive treatments are essential to reduce morbidity from repeat CeAD and strokes, especially in patients with relevant family history or known artery tortuosity.

Aminoacylated (charged) transfer RNA isoacceptors read different messenger RNA codons for the same amino acid. The concentration of an isoacceptor and its charged fraction are principal determinants of the translation rate of its codons. A recent theoretical model predicts that amino‐acid starvation results in ‘selective charging’ where the charging levels of some tRNA isoacceptors will be low and those of others will remain high. Here, we developed a microarray for the analysis of charged fractions of tRNAs and measured charging for all Escherichia coli tRNAs before and during leucine, threonine or arginine starvation. Before starvation, most tRNAs were fully charged. During starvation, the isoacceptors in the leucine, threonine or arginine families showed selective charging when cells were starved for their cognate amino acid, directly confirming the theoretical prediction. Codons read by isoacceptors that retain high charging can be used for efficient translation of genes that are essential during amino‐acid starvation. Selective charging can explain anomalous patterns of codon usage in the genes for different families of proteins.

ObjectiveProgrammed death ligand 1 (PD-L1) expression affects tumor evasion of immune surveillance. The prognostic value and relationship of PD-L1 expression to T-cell–inflamed immune signatures in ovarian cancer are unclear. The purpose of this study is to evaluate the impact of PD-L1 on overall survival and its correlation with an immune-mediated gene expression profile in patients with advanced ovarian cancer.MethodsPD-L1 expression in tumor and immune cells was assessed by immunohistochemistry, and PD-L1–positive expression was defined as a combined positive score ≥1; a T-cell–inflamed gene expression profile containing interferon γ response genes was evaluated using extracted RNA from surgical samples. Associations between PD-L1 expression, gene expression profile status, and overall survival were analyzed using the Kaplan-Meier method, log-rank test, and multivariate Cox proportional hazards regression models.ResultsA total of 376 patients with advanced epithelial ovarian, primary peritoneal, or fallopian tube cancer treated by cytoreductive surgery and platinum-based therapy were included. PD-L1–positive expression was observed in 50.5% of patients and associated with more advanced stage (p=0.047), more aggressive histologic subtype (p=0.001), and platinum sensitivity defined by increasing treatment-free interval from first platinum-based chemotherapy to next systemic treatment (p=0.027). PD-L1–positive expression was associated with longer overall survival in multivariate analyses (adjusted HR 0.72, 95% CI 0.56 to 0.93). In subgroup analyses, this association was most pronounced in patients with partially platinum-sensitive disease (treatment-free interval ≥6 to <12 months). T-cell–inflamed gene expression profile status correlated with PD-L1 expression (Spearman, ρ=0.712) but was not an independent predictor of overall survival.ConclusionPD-L1 expression is associated with longer overall survival among advanced ovarian cancer patients. PD-L1 expression may be an independent prognostic biomarker.

The Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) clinical trial, including 1,873 patients found an increased risk for cancer with lipid-lowering therapy with ezetimibe/simvastatin 10/40 mg/day, relative to placebo. In a registry-based follow-up study over 21 months from the conclusion of the SEAS trial, new incident cancer and total mortality were investigated in the SEAS study cohort from Denmark, Finland, Norway, Sweden, and the United Kingdom. Among 1,359 subjects eligible for follow-up (73% of the original total cohort), 1,194 had no history of cancer (primary follow-up cohort). New cancers and deaths were identified in the national cancer and mortality registries and classified by an Expert Review Committee. Data were analyzed using Cox proportional-hazards models of new cancers and mortality during follow-up according to treatment group assigned in the SEAS base study and with age, gender, smoking history, and previous cancers as covariates. The primary follow-up cohort had 12 patients with new cancers in the ezetimibe/simvastatin group and 22 in the placebo group (hazard ratio 0.55, 95% confidence interval 0.27 to 1.11), indicating no significant difference between the treatment groups. During follow-up, 43 patients assigned to ezetimibe/simvastatin and 33 assigned to placebo died (hazard ratio 1.29, 95% confidence interval 0.82 to 2.03). In conclusion, in this registry-based observational follow-up study of the original SEAS study patient population, treatment with ezetimibe/simvastatin was not associated with an increased risk for cancer or mortality in the 21-month period after the completion of the original SEAS study.