Trojan-horse inspired nanoblaster: X-ray triggered spot attack on radio-resistant cancer through radiodynamic therapy.

Radiotherapy as a mainstay of in-depth cervical cancer (CC) treatment suffers from its radioresistance. Radiodynamic therapy (RDT) effectively reverses radio-resistance by generating reactive oxygen species (ROS) with deep tissue penetration. However, the photosensitizers stimulated by X-ray have high toxicity and energy attenuation. Therefore, X-ray responsive diselenide-bridged mesoporous silica nanoparticles (DMSNs) are designed, loading X-ray-activated photosensitizer acridine orange (AO) for spot blasting RDT like Trojan-horse against radio-resistance cervical cancer (R-CC). DMSNs can encapsulate a large amount of AO, in the tumor microenvironment (TME), which has a high concentration of hydrogen peroxide, X-ray radiation triggers the cleavage of diselenide bonds, leading to the degradation of DMSNs and the consequent release of AO directly at the tumor site. On the one hand, it solves the problems of rapid drug clearance, adverse distribution, and side effects caused by simple AO treatment. On the other hand, it fully utilizes the advantages of highly penetrating X-ray responsive RDT to enhance radiotherapy sensitivity. This approach results in ROS-induced mitochondria damage, inhibition of DNA damage repair, cell cycle arrest and promotion of cancer cell apoptosis in R-CC. The X-ray responsive DMSNs@AO hold considerable potential in overcoming obstacles for advanced RDT in the treatment of R-CC.

Microtube self-assembly leads to conformational freezing point depression.

HYPOTHESIS: Multi-walled tubular aggregates formed by hierarchical self-assembly of beta-cyclodextrin (ß-CD) and sodium dodecylsulfate (SDS) hold a great potential as microcarriers. However, the underlying mechanism for this self-assembly is not well understood. To advance the application of these structures, it is essential to fine-tune the cavity size and comprehensively elucidate the energetic balance driving their formation: the bending modulus versus the microscopic line tension. EXPERIMENTS: We investigated temperature-induced changes in the hierarchical tubular aggregates using synchrotron small-angle X-ray scattering across a broad concentration range. Detailed analysis of the scattering patterns enabled us to determine the structural parameters of the microtubes and to construct a phase diagram of the system. FINDINGS: The microtubes grow from the outside in and melt from the inside out. We relate derived structural parameters to enthalpic changes driving the self-assembly process on the molecular level in terms of their bending modulus and microscopic line tension. We find that the conformation of the crystalline bilayer affects the saturation concentration, providing an example of a phenomenon we call conformational freezing point depression. Inspired by the colligative phenomenon of freezing point depression, well known from undergraduate physics, we model this system by including the membrane conformation, which can describe the energetics of this hierarchical system and give access to microscopic properties without free parameters.

Diagnostic Value of Chest Computed Tomography Scan for Identification of Foreign Body Aspiration in Children: A Systematic Review and Meta-analysis.

Introduction: Foreign body aspiration (FBA) is a common, life-threatening pediatric emergency and was shown to be associated with high risk of morbidity and mortality. This systematic review and meta-analysis aimed to investigate the diagnostic value of chest computed tomography (CT) scan for identification of FBA in children. Methods: From inception to May 2024, a systematic search was carried out across multiple databases including Medline, Scopus, and Web of Science, considering published papers in English language. Quality assessment of the included studies was performed using seven domains of Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). Results: The systematic literature search yielded 7203 articles. The pooled sensitivity and specificity of chest CT scan for identification of FBA were 0.99 (95% CI: 0.98-0.99) and 0.97 (95% CI: 0.96-0.98), respectively. The pooled positive likelihood ratio was 10.12 (95% CI: 4.59-22.20), and pooled negative likelihood ratio was 0.05 (95% CI: 0.02-0.1). Furthermore, the area under the summarized receiver operating characteristic (SROC) curve was 0.98. Conclusion: Our meta-analysis revealed that despite high heterogeneity, in the diagnostic characteristics of chest CT scan among studies, it has high diagnostic value in identifying FBA in suspected pediatric cases.

Clinical assessment of computed tomography for detecting ingested blister packs: A single-center retrospective study.

Objectives: Blister pack (BP) ingestion poses serious risks, such as gastrointestinal perforation, and accurate localization by computed tomography (CT) is a common practice. However, while it has been reported in vitro that CT visibility varies with the material type of BPs, there have been no reports on this variability in clinical settings. In this study, we investigated the CT detection rates of different BPs in clinical settings. Methods: This single-center retrospective study from 2010 to 2022 included patients who underwent endoscopic foreign body removal for BP ingestion. The patients were categorized into two groups for BP components, the polypropylene (PP) and the polyvinyl chloride (PVC)/polyvinylidene chloride (PVDC) groups. The primary outcome was the comparison of CT detection rates between the groups. We also evaluated whether the BPs contained tablets and analyzed their locations. Results: This study included 61 patients (15 in the PP group and 46 in the PVC/PVDC group). Detection rates were 97.8% for the PVC/PVDC group compared to 53.3% for the PP group, a significant difference (p < 0.01). No cases of BPs composed solely of PP were detected by CT. Blister packs were most commonly found in the upper thoracic esophagus. Conclusions: Even in a clinical setting, the detection rates of PVC and PVDC were higher than that of PP alone. Identifying PP without tablets has proven challenging in clinical. Considering the risk of perforation, these findings suggest that esophagogastroduodenoscopy may be necessary, even if CT detection is negative.

Accuracy of an artificial intelligence-enabled diagnostic assistance device in recognizing normal chest radiographs: a service evaluation.

Objectives: Artificial intelligence (AI) enabled devices may be able to optimize radiologists' productivity by identifying normal and abnormal chest X-rays (CXRs) for triaging. In this service evaluation, we investigated the accuracy of one such AI device (qXR). Methods: A randomly sampled subset of general practice and outpatient-referred frontal CXRs from a National Health Service Trust was collected retrospectively from examinations conducted during November 2022 to January 2023. Ground truth was established by consensus between 2 radiologists. The main objective was to estimate negative predictive value (NPV) of AI. Results: A total of 522 CXRs (458 [87.74%] normal CXRs) from 522 patients (median age, 64 years [IQR, 49-77]; 305 [58.43%] female) were analysed. AI predicted 348 CXRs as normal, of which 346 were truly normal (NPV: 99.43% [95% CI, 97.94-99.93]). The sensitivity, specificity, positive predictive value, and area under the ROC curve of AI were found to be 96.88% (95% CI, 89.16-99.62), 75.55% (95% CI, 71.34-79.42), 35.63% (95% CI, 28.53-43.23), and 91.92% (95% CI, 89.38-94.45), respectively. A sensitivity analysis was conducted to estimate NPV by varying assumptions of the prevalence of normal CXRs. The NPV ranged from 88.96% to 99.54% as prevalence increased. Conclusions: The AI device recognized normal CXRs with high NPV and has the potential to increase radiologists' productivity. Advances in knowledge: There is a need for more evidence on the utility of AI-enabled devices in identifying normal CXRs. This work adds to such limited evidence and enables researchers to plan studies to further evaluate the impact of such devices.

Data of crystal structure of the large Stokes shift fluorescent protein tKeima.

Large Stokes shift (LSS) fluorescent proteins (FPs) are important for dual-color fluorescence cross-correlation spectroscopy and multicolor imaging. tKeima is a tetrameric LSS FP from the stony coral Montipora sp. Analyzing the tetrameric interface of tKeima is necessary to understand the oligomeric state of the Keima family and to provide insights into engineering oligomeric FPs to generate monomeric FPs, which are useful for FP-based molecular and cell biology studies. Here, detailed experimental procedures for tKeima were reported, including spontaneous crystal growth, data collection for X-ray diffraction, and structure determination. This information can be used for future experiments to obtain the high-resolution structure of tKeima, providing accurate structural information to comprehensively understand the molecular function of tKeima and the protein engineering of tetrameric FPs.

Application of improved urate analysis algorithm based on spectral parameters in Podagra: A prospective study.

OBJECTIVES: To explore whether the improved urate analysis (IUA) algorithm based on spectral parameters can reduce false positives in CT gout images compared with current urate analysis (CUA) algorithm. MATERIALS AND METHODS: This prospective study was performed from May 2022 to May 2023. Spectral feet CT images of suspected gout participants were reconstructed by IUA and CUA algorithm. Qualitative diagnosis of IUA and CUA images was recorded and compared with the reference standard (ultrasound + conventional CT). Artifacts on IUA and CUA images of non-gout participants were recorded and compared; the maximum cross-sectional area of the maximum tophi (SIT-max) on IUA and CUA images of participants with gout were measured and compared. RESULTS: There are 65 participants (mean age, 43.9 years ± 13.1 [SD]; 65 men) with 114 feet studies in the gout group, and 33 participants (mean age, 43.4 years ± 15.0 [SD]; 30 men) with 65 feet studies in the non-gout group. For all 179 feet studies, IUA images had higher specificity (19.2-86.6 % vs. 1.3-44.3 %) and accuracy (63.1-88.8 % vs. 41.3-57.0 %) than CUA images (P < 0.001). In the non-gout group, the reduction rates of artifacts from the nail bed, skin, beam hardening, vascular structures, tendons, and total artifacts on the IUA images compared to the CUA images was 40.5 %, 48.9 %, 74.3 %, 99.2 %, 99.6 %, and 80.0 %, respectively (P < 0.001). For 82 feet studies with tophi, SIT-max was higher on CUA images than IUA images (P < 0.05). CONCLUSION: The improved urate analysis algorithm based on spectral parameters can reduce image artifacts and improve diagnostic efficacy.

Structural insights into molecular-targeting helix-loop-helix peptide against vascular endothelial growth factor-A.

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

X-ray coronary angiography background subtraction by adaptive weighted total variation regularized online RPCA.

Coronary X-ray angiograms (XCA) are widely used in diagnosing and treating cardiovascular diseases. Various structures with independent motion patterns in the background of XCA images and limitations in the dose of injected contrast agent have resulted in low-contrast XCA images. Background subtraction methods have been developed to enhance the visibility and contrast of coronary vessels in XCA sequences, consequently reducing the requirement for excessive contrast agent injections. The current study proposes an adaptive weighted total variation regularized online RPCA (WTV-ORPCA) method, which is a low-rank and sparse subspaces decomposition approach to subtract the background of XCA sequences. In the proposed method, the images undergo initial preprocessing using morphological operators to eliminate large-scale background structures and achieve image homogenization. Subsequently, the decomposition algorithm decomposes the preprocessed images into background and foreground subspaces. This step applies an adaptive weighted total variation (TV) constraint to the foreground subspace to ensure the spatial coherency of the finally extracted coronary vessel images. To evaluate the effectiveness of the proposed background subtraction method, some qualitative and quantitative experiments are conducted on two clinical and synthetic low-contrast XCA datasets containing videos from 21 patients. The obtained results are compared with six state-of-the-art methods employing three different assessment criteria. By applying the proposed method to the clinical dataset, the mean values of the global contrast-to-noise ratio (GCNR), local contrast-to-noise ratio (LCNR), structural similarity index (SSIM), and reconstruction error (RE) are obtained as 5.976, 3.173, 0.987, and 0.026, respectively. These criteria over the low-contrast synthetic dataset were 4.851, 2.942, 0.958, and 0.034, respectively. The findings demonstrate the superiority of the proposed method in improving the contrast and visibility of coronary vessels, preserving the integrity of the vessel structure, and minimizing reconstruction errors without imposing excessive computational complexity. .

Colloidal platinum nanoparticles enhance resin-dentin bonding durability.

OBJECTIVES: This study aims to investigate the effect of colloidal platinum nanoparticles (CPN) on the durability of resin-dentin bonding performance with contemporary adhesives. METHODS: Sixty non-carious human maxillary premolars were subjected to microtensile bond strength (µTBS) testing and divided into two main groups: CPN-treated and untreated. Within each group, specimens were randomly allocated to Clearfil Megabond 2 (MB2), Scotchbond Universal Plus Adhesive with self-etch mode (SE-SUP), and etch-and-rinse mode (ER-SUP) subgroups (n = 10/group). CPN was applied to dentin in the MB2 and SE-SUP groups for 20 s, followed by rinsing before adhesive application. In the ER-SUP group, CPN was applied after etch-and-rinse. The µTBS was tested after 24 h, 6 months, and 1 year, and the fracture modes were observed using SEM. The µTBS data were analyzed using a two-way ANOVA and post-hoc Tukey HSD test (α = 0.05). An additional twelve premolars underwent TEM/STEM/EDX for ultra-morphological observations. RESULTS: The application of CPN significantly prevented a decline in the µTBS of both the MB2 and SE-SUP groups. No significant decrease was observed in the ER-SUP group, either with aging or CPN application. Ultra-morphological images revealed platinum nanoparticles attaching to the collagen fibrils of the hybrid layer regardless of aging. It was highlighted that the nanoparticles attached to the banded collagen in the aging groups were observed. SIGNIFICANCE: CPN exhibits the potential in enhancing the longevity of resin-dentin bonding in SE mode.

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than ß-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to ß-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

An Integrated Approach using YOLOv8 and ResNet, SeResNet & Vision Transformer (ViT) Algorithms based on ROI Fracture Prediction in X-ray Images of the Elbow.

INTRODUCTION: In this study, we harnessed three cutting-edge algorithms' capabilities to refine the elbow fracture prediction process through X-ray image analysis. Employing the YOLOv8 (You only look once) algorithm, we first identified Regions of Interest (ROI) within the X-ray images, significantly augmenting fracture prediction accuracy. METHODS: Subsequently, we integrated and compared the ResNet, the SeResNet (Squeeze-and-Excitation Residual Network) ViT (Vision Transformer) algorithms to refine our predictive capabilities. Furthermore, to ensure optimal precision, we implemented a series of meticulous refinements. This included recalibrating ROI regions to enable finer-grained identification of diagnostically significant areas within the X-ray images. Additionally, advanced image enhancement techniques were applied to optimize the X-ray images' visual quality and structural clarity. RESULTS: These methodological enhancements synergistically contributed to a substantial improvement in the overall accuracy of our fracture predictions. The dataset utilized for training, testing & validation, and comprehensive evaluation exclusively comprised elbow X-ray images, where predicting the fracture with three algorithms: Resnet50; accuracy 0.97, precision 1, recall 0.95, SeResnet50; accuracy 0.97, precision 1, recall 0.95 & ViTB- 16 with high accuracy of 0.99, precision same as the other two algorithms, with a recall of 0.95. CONCLUSION: This approach has the potential to increase the precision of diagnoses, lessen the burden of radiologists, easily integrate into current medical imaging systems, and assist clinical decision-making, all of which could lead to better patient care and health outcomes overall.

Uranium Repartitioning during Microbial Driven Reductive Transformation of U(VI)-Sorbed Schwertmannite and Jarosite.

This study exposes U(VI)-sorbed schwertmannite and jarosite to biotic reductive incubations under field-relevant conditions and examines the changes in aqueous and solid-phase speciation of U, Fe, and S as well as associated microbial communities over 180 days. The chemical, X-ray absorption spectroscopy, X-ray diffraction, and microscopic data demonstrated that the U(VI)-sorbed schwertmannite underwent a rapid reductive dissolution and solid-phase transformation to goethite, during which the surface-sorbed U(VI) was partly reduced and mostly repartitioned to monomeric U(VI)/U(IV) complexes by carboxyl and phosphoryl ligands on biomass or organic substances. Furthermore, the microbial data suggest that these processes were likely driven by the consecutive developments of fermentative and sulfate- and iron- reducing microbial communities. In contrast, the U(VI)-sorbed jarosite only stimulated the growth of some fermentative communities and underwent very limited reductive dissolution and thus, remaining in its initial state with no detectable mineralogical transformation and solid-phase U reduction/repartitioning. Accordingly, these two biotic incubations did not induce increased risk of U reliberation to the aqueous phase. These findings have important implications for understanding the interactions of schwertmannite/jarosite with microbial communities and colinked behavior and fate of U following the establishment of reducing conditions in various acidic and U-rich settings.

Structural characterization of green fluorescent protein in the I-state.

Green fluorescent protein (GFP) is widely utilized as a fluorescent tag in biochemical fields. Whereas the intermediate (I) state has been proposed in the photoreaction cycle in addition to the A and B states, until now the structure of I has only been estimated by computational studies. In this paper, we report the crystal structures of the I stabilizing variants of GFP at high resolutions where respective atoms can be observed separately. Comparison with the structures in the other states highlights the structural feature of the I state. The side chain of one of the substituted residues, Val203, adopts the gauche- conformation observed for Thr203 in the A state, which is different from the B state. On the other hand, His148 interacts with the chromophore by ordinary hydrogen bonding with a distance of 2.85 Å, while the weaker interaction by longer distances is observed in the A state. Therefore, it was indicated that it is possible to distinguish three states A, B and I by the two hydrogen bond distances Oγ-Thr203···Oη-chromophore and Nδ1-His148···Oη-chromophore. We discuss the characteristics of the I intermediate of wild-type GFP on the bases of the structure estimated from the variant structures by quantum chemical calculations.

Uncertainty quantification in multi-class image classification using chest X-ray images of COVID-19 and pneumonia.

This paper investigates uncertainty quantification (UQ) techniques in multi-class classification of chest X-ray images (COVID-19, Pneumonia, and Normal). We evaluate Bayesian Neural Networks (BNN) and the Deep Neural Network with UQ (DNN with UQ) techniques, including Monte Carlo dropout, Ensemble Bayesian Neural Network (EBNN), Ensemble Monte Carlo (EMC) dropout, across different evaluation metrics. Our analysis reveals that DNN with UQ, especially EBNN and EMC dropout, consistently outperform BNNs. For example, in Class 0 vs. All, EBNN achieved a UAcc of 92.6%, UAUC-ROC of 95.0%, and a Brier Score of 0.157, significantly surpassing BNN's performance. Similarly, EMC Dropout excelled in Class 1 vs. All with a UAcc of 83.5%, UAUC-ROC of 95.8%, and a Brier Score of 0.165. These advanced models demonstrated higher accuracy, better discriaminative capability, and more accurate probabilistic predictions. Our findings highlight the efficacy of DNN with UQ in enhancing model reliability and interpretability, making them highly suitable for critical healthcare applications like chest X-ray imageQ6 classification.

Antoine Béclère (1856-1939): His Great Contribution to Radiotherapy.

After Wilhelm Conrad Röntgen discovered X-rays in 1895, French physician Antoine Louis Gustave Béclère pioneered the development of radiology in the late 1800s. Béclère recognized the enormous potential of radiation both diagnostically and therapeutically. His radiotherapy techniques quickly gained international renown. In 1897, he founded the world's first radiology teaching lab, the Hospital Radiology Laboratory at Tenon Hospital in Paris. As a hospital physician and researcher, Béclère also had endocrinology, immunology, and virology expertise and published several important papers on various diseases, including many articles on cancer treatment.

Wafer-Sized CsPbBr3/CsPbCl3 Heterojunction: Breaking the Trade-Off between Sensitivity and Dark Current for Efficient X-ray Detector.

Polycrystalline lead halide perovskite finds promising use in fabricating X-ray detectors with a large lateral size, adjustable thickness, and diverse synthesis processes. However, a large dark current hinders its development for weak signal detection. Herein, we propose a multistep pressing strategy for manufacturing a CsPbBr3/CsPbCl3 heterojunction wafer for a reduced dark current X-ray detector, and the device keeps a high sensitivity value after the insertion of a barrier by heterojunction; thus, the trade-off between sensitivity and dark current can be broken. The X-ray detector with a metal-semiconductor-metal structure yields a sensitivity of 6.32 × 104 µC Gyair-1 cm-2 at a bias of 12 V, a 1/f noise of 1.02 × 10-13 A/Hz-1/2, and a detection limit of 66.58 nGy s-1. These performance parameters are considerably better than those of a similar X-ray detector based on the single-structure wafer. The improved device performance of the heterostructure X-ray detector is ascribed to the suppressed carrier recombination, enhanced carrier transportation of the heterojunction, and strong X-ray attenuation of the CsPbCl3 layer. The pixel array device is further used in imaging applications. Hence, this study provides an efficient strategy for fabricating heterostructure polycrystalline lead halide perovskite wafers for use in high-performance wafer-based X-ray detectors.

Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Distinguishing Protein Corona from Nanoparticle Aggregate Formation in Complex Biological Media Using X-ray Photon Correlation Spectroscopy.

In biological systems, nanoparticles interact with biomolecules, which may undergo protein corona formation that can result in noncontrolled aggregation. Therefore, comprehending the behavior and evolution of nanoparticles in the presence of biological fluids is paramount in nanomedicine. However, traditional lab-based colloid methods characterize diluted suspensions in low-complexity media, which hinders in-depth studies in complex biological environments. Here, we apply X-ray photon correlation spectroscopy (XPCS) to investigate silica nanoparticles (SiO2) in various environments, ranging from low to high complex biological media. Interestingly, SiO2 revealed Brownian motion behavior, irrespective of the complexity of the chosen media. Moreover, the SiO2 surface and media composition were tailored to underline the differences between a corona-free system from protein corona and aggregates formation. Our results highlighted XPCS potential for real-time nanoparticle analysis in biological media, surpassing the limitations of conventional techniques and offering deeper insights into colloidal behavior in complex environments.

Experimental localization of metal-binding sites reveals the role of metal ions in type II DNA topoisomerases.

Metal ions have important roles in supporting the catalytic activity of DNA-regulating enzymes such as topoisomerases (topos). Bacterial type II topos, gyrases and topo IV, are primary drug targets for fluoroquinolones, a class of clinically relevant antibacterials requiring metal ions for efficient drug binding. While the presence of metal ions in topos has been elucidated in biochemical studies, accurate location and assignment of metal ions in structural studies have historically posed significant challenges. Recent advances in X-ray crystallography address these limitations by extending the experimental capabilities into the long-wavelength range, exploiting the anomalous contrast from light elements of biological relevance. This breakthrough enables us to confirm experimentally the locations of Mg2+ in the fluoroquinolone-stabilized Streptococcus pneumoniae topo IV complex. Moreover, we can unambiguously identify the presence of K+ and Cl- ions in the complex with one pair of K+ ions functioning as an additional intersubunit bridge. Overall, our data extend current knowledge on the functional and structural roles of metal ions in type II topos.