Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.

Ex Vivo Surface Decoration of Phenylboronic Acid onto Natural Killer Cells for Sialic Acid-Mediated Versatile Cancer Cell Targeting.

Phenylboronic acid (PBA) has been highly acknowledged as a significant cancer recognition moiety in sialic acid-overexpressing cancer cells. In this investigation, lipid-mediated biomaterial integrated PBA molecules onto the surface of natural killer (NK) cells to make a receptor-mediated immune cell therapeutic module. Therefore, a 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-conjugated di-PEG-PBA (DSPEPEG-di(PEG-PBA) biomaterial was synthesized. The DSPEPEG-di(PEG-PBA) biomaterial exhibited a high affinity for sialic acid (SA), confirmed by fluorescence spectroscopy at pH 6.5 and 7.4. DSPEPEG-di(PEG-PBA) was successfully anchored onto NK cell surfaces (PBA-NK), and this biomaterial maintains intrinsic properties such as viability, ligand availability (FasL & TRAIL), and cytokine secretion response to LPS. The anticancer efficacy of PBA-NK cells was evaluated against 2D cancer cells (MDA-MB-231, HepG2, and HCT-116) and 3D tumor spheroids of MDA-MB-231 cells. PBA-NK cells exhibited greatly enhanced anticancer effects against SA-overexpressing cancer cells. Thus, PBA-NK cells represent a new anticancer strategy for cancer immunotherapy.

Optimized Design of Hyaluronic Acid-Lipid Conjugate Biomaterial for Augmenting CD44 Recognition of Surface-Engineered NK Cells.

Triple-negative breast cancer (TNBC) presents treatment challenges due to a lack of detectable surface receptors. Natural killer (NK) cell-based adaptive immunotherapy is a promising treatment because of the characteristic anticancer effects of killing malignant cells directly by secreting cytokines and lytic granules. To maximize the cancer recognition ability of NK cells, biomaterial-mediated ex vivo cell surface engineering has been developed for sufficient cell membrane immobilization of tumor-targeting ligands via hydrophobic anchoring. In this study, we optimized amphiphilic balances of NK cell coating materials composed of CD44-targeting hyaluronic acid (HA)-poly(ethylene glycol) (PEG)-lipid to improve TNBC recognition and the anticancer effect. Changes in the modular design of our material by differentiating hydrophilic PEG length and incorporating lipid amount into HA backbones precisely regulated the amphiphilic nature of HA-PEG-lipid conjugates. The optimized biomaterial demonstrated improved anchoring into NK cell membranes and facilitating the surface presentation level of HA onto NK cell surfaces. This led to enhanced cancer targeting via increasing the formation of immune synapse, thereby augmenting the anticancer capability of NK cells specifically toward CD44-positive TNBC cells. Our approach addresses targeting ability of NK cell to solid tumors with a deficiency of surface tumor-specific antigens while offering a valuable material design strategy using amphiphilic balance in immune cell surface engineering techniques.

Crack Detection and Analysis of Concrete Structures Based on Neural Network and Clustering.

Concrete is extensively used in the construction of infrastructure such as houses and bridges. However, the appearance of cracks in concrete structures over time can diminish their sealing and load-bearing capability, potentially leading to structural failures and disasters. The timely detection of cracks allows for repairs without the need to replace the entire structure, resulting in cost savings. Currently, manual inspection remains the predominant method for identifying concrete cracks. However, in today's increasingly complex construction environments, subjective errors may arise due to human vision and perception. The purpose of this work is to investigate and design an autonomous convolutional neural network-based concrete detection system that can identify cracks automatically and use that information to calculate the crack proportion. The experiment's findings show that the trained model can classify concrete cracks with an accuracy of 99.9%. Moreover, the clustering technique applied to crack images enables the clear identification of the percentage of cracks, which facilitates the development of concrete damage level detection over time.

Networked Cluster Formation via Trigonal Lipid Modules for Augmented Ex Vivo NK Cell Priming.

Current cytokine-based natural killer (NK) cell priming techniques have exhibited limitations such as the deactivation of biological signaling molecules and subsequent insufficient maturation of the cell population during mass cultivation processes. In this study, we developed an amphiphilic trigonal 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-polyethylene glycol (PEG) material to assemble NK cell clusters via multiple hydrophobic lipid insertions into cellular membranes. Our lipid conjugate-mediated ex vivo NK cell priming sufficiently augmented the structural modulation of clusters, facilitated diffusional signal exchanges, and finally activated NK cell population with the clusters. Without any inhibition in diffusional signal exchanges and intrinsic proliferative efficacy of NK cells, effectively prime NK cell clusters produced increased interferon-gamma, especially in the early culture periods. In conclusion, the present study demonstrates that our novel lipid conjugates could serve as a promising alternative for future NK cell mass production.

A Case of Hemophagocytic Lymphohistiocytosis following Second Dose of COVID-19 Vaccination.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, severe hyperinflammatory disease characterized by overproduction of cytokines and hemophagocytosis of hematopoietic cells, resulting in multiorgan failure. Prompt treatment initiation is essential for patient survival. The coronavirus disease 2019 (COVID-19) pandemic has led to the rapid development of several vaccines, including BNT162b2 by Pfizer-BioNTech. Few cases of immune-mediated complications of COVID-19 and its vaccines have been reported, characterized by persistent stimulation of the immune system, resembling HLH. We report the case of a 21-year-old man with secondary HLH following a second dose of the BNT162b2 vaccine. The patient did not have primary HLH or other contributors to secondary HLH and met the HLH-2004 diagnostic criteria. He was safely treated with steroid pulse therapy alone, without etoposide, cyclosporin, or immunoglobulins, which are recommended for pediatric patients. Physicians need to be aware of such severe complications following a second dose of the COVID-19 vaccine.

Deimmunizing substitutions in Pseudomonas exotoxin domain III perturb antigen processing without eliminating T-cell epitopes.

Effective adaptive immune responses depend on activation of CD4+ T cells via the presentation of antigen peptides in the context of major histocompatibility complex (MHC) class II. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity of peptides that are presented to T cells. A recombinant immunotoxin, comprising exotoxin A domain III (PE-III) from Pseudomonas aeruginosa and a cancer-specific antibody fragment, has been developed to manage cancer, but its effectiveness is limited by the induction of neutralizing antibodies. Here, we observed that this immunogenicity is substantially reduced by substituting six residues within PE-III. Although these substitutions targeted T-cell epitopes, we demonstrate that reduced conformational stability and protease resistance were responsible for the reduced antibody titer. Analysis of mouse T-cell responses coupled with biophysical studies on single-substitution versions of PE-III suggested that modest but comprehensible changes in T-cell priming can dramatically perturb antibody production. The most strongly responsive PE-III epitope was well-predicted by a structure-based algorithm. In summary, single-residue substitutions can drastically alter the processing and immunogenicity of PE-III but have only modest effects on CD4+ T-cell priming in mice. Our findings highlight the importance of structure-based processing constraints for accurate epitope prediction.

Membrane-associated androgen receptor (AR) potentiates its transcriptional activities by activating heat shock protein 27 (HSP27).

The androgen receptor (AR) is a ligand-activated nuclear receptor that plays a critical role in normal prostate physiology, as well as in the development and progression of prostate cancer. In addition to the classical paradigm in which AR exerts its biological effects in the nucleus by orchestrating the expression of the androgen-regulated transcriptome, there is considerable evidence supporting a rapid, nongenomic activity mediated by membrane-associated AR. Although the genomic action of AR has been studied in depth, the molecular events governing AR transport to the plasma membrane and the downstream AR signaling cascades remain poorly understood. In this study, we report that AR membrane transport is microtubule-dependent. Disruption of the function of kinesin 5B (KIF5B), but not of kinesin C3 (KIFC3), interfered with AR membrane association and signaling. Co-immunoprecipitation and pulldown assays revealed that AR physically interacts with KIF5B and that androgen enhances this interaction. Furthermore, we show that heat shock protein 27 (HSP27) is activated by membrane-associated AR and that HSP27 plays an important role in mediating AR-mediated membrane-to-nuclear signal transduction. Together, these results indicate that AR membrane translocation is mediated by the microtubule cytoskeleton and the motor protein KIF5B. By activating HSP27, membrane-associated AR potentiates the transcriptional activity of nuclear AR. We conclude that disruption of AR membrane translocation may represent a potential strategy for targeting AR signaling therapeutically in prostate cancer.

Exposure to volatile organic compounds and airway inflammation.

BACKGROUND: Exposure to low levels of volatile organic compounds (VOCs) in ordinary life is suspected to be related to oxidative stress and decreased lung function. This study evaluated whether exposure to ambient VOCs in indoor air affects airway inflammation. METHODS: Thirty-four subjects from the hospital that had moved to a new building were enrolled. Symptoms of sick building syndrome, pulmonary function tests, and fractional exhaled nitric oxide (FeNO) were evaluated, and random urine samples were collected 1 week before and after the move. Urine samples were analyzed for VOC metabolites, oxidative stress biomarkers, and urinary leukotriene E4 (uLTE4) levels. RESULTS: The level of indoor VOCs in the new building was higher than that in the old building. Symptoms of eye dryness and eye irritation, as well as the level of a xylene metabolite (o-methylhippuric acid) increased after moving into the new building (p = 0.012, p = 0.008, and p < 0.0001, respectively). For the inflammatory markers, FeNO decreased (p = 0.012 and p = 0.04, respectively) and the uLTE4 level increased (p = 0.005) after the move. CONCLUSION: Exposure to a higher level of VOCs in everyday life could affect airway inflammation.

Effect of Nasogastric Tube on Aspiration Risk: Results from 147 Patients with Dysphagia and Literature Review.

Nasogastric tube (NGT) is a common feeding strategy for patients at risk of endotracheal aspiration with an oral diet. With NGT feeding, however, swallowing of small amounts saliva cannot be avoided. We investigated whether the aspiration rate when swallowing 1 mL of fluid increased in patients using an NGT in different dysphagia severities. One hundred forty-seven patients who had been receiving NGT feeding underwent a videofluoroscopic swallowing study (VFSS). During VFSS, subjects were offered 1 mL of fluid twice: initially, with the tube inserted (NGT-in) and, subsequently, with the tube removed (NGT-out). Aspiration depth was determined using the 8-point Penetration-Aspiration Scale (PAS) (0 points, no aspiration/penetration; 8 points, aspiration passing the vocal cords with no ejection efforts). PAS-diff was computed (PASNGT-in - PASNGT-out), and a positive PAS-diff (PAS-diff > 0) meant increased aspiration depth in the presence of NGT. After VFSS, diet recommendations were made according to dysphagia severity assessment: non-oral feeding (n = 59), diet modification (n = 74), and diet as tolerated (n = 13). Cognitive level (mini-mental state examination, MMSE) and general functional level (Modified Barthel Index, MBI) were compared between the PAS-diff > 0 and PAS-diff ≤ 0 groups. Aspiration severity did not significantly change after NGT removal (PASNGT-in, 2.45 ± 2.40; PASNGT-out, 2.57 ± 2.58; P = .50). Regardless of recommended diet, PAS-diff values were not significantly different (P = .49). MMSE and MBI were not significantly different (P = .23 and .94) between subjects with PAS-diff > 0 (n = 25) and PAS-diff ≤ 0 (n = 121). In conclusion, the risk of aspirating a small amount of fluid was not significantly different before and after NGT removal, regardless of swallowing function, cognitive level, or general functional level.

A Metabolomic Approach for the Discrimination of Red Ginseng Root Parts and Targeted Validation.

Ginsenosides are used as existing markers of red ginseng (RG) quality, and ginsenoside ratios are also indicative of the different components of red ginseng. For the analysis and classification of ginsenoside content, red ginseng was separated into three parts, namely, main roots, lateral roots, and fine roots, and each extract was subjected to ultra-performance liquid chromatography quadruple time-of-flight mass spectrometry (UPLC-QToF-MS) with multivariate statistical analysis. Principal component analysis (PCA) showed a clear discrimination between the extracts of main roots and fine roots and suggested discrimination markers (four for the main roots and five for the fine roots). The fine root markers were identified as ginsenoside. We identified two markers for the main roots of red ginseng in this study. Moreover, the contents of 22 ginsenosides were analyzed in all three components of red ginseng. Fine roots have the highest protopanaxadiol (PPD)/protopanaxatriol (PPT) ratio. The PPD group of ginsenosides, which is quantitatively dominant in fine roots, clearly distinguishes the main roots from the other parts.

Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain.

Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic nonbiodegradable systems. Using a state-of-the-art biodegradable polymer, poly(ß-amino ester), colloidally stable sub-100 nm DNA nanoparticles are engineered with a nonadhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles are able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases.

Farmers' Cohort for Agricultural Work-Related Musculoskeletal Disorders (FARM) Study: Study Design, Methods, and Baseline Characteristics of Enrolled Subjects.

BACKGROUND: The ongoing Farmers' Cohort for Agricultural Work-related Musculoskeletal Disorders (FARM) study was developed to evaluate health status and related factors in farmers. METHODS: Farmers in Kangwon Province, South Korea, were recruited. Baseline characteristics were determined using questionnaires about sociodemographic and health characteristics and agricultural work-related factors. In addition, laboratory examinations (lumbar spinal radiography and serologic testing) were conducted. RESULTS: The FARM study covers eight rural areas and recruited 1013 subjects (534 women; mean [standard deviation {SD}] age, 57.2 [7.5] years). Musculoskeletal pain in multiple areas was reported by 925 subjects (91.3%), and low back pain (63.8%) was the most frequent site of pain. Farmer's Stress Inventory (mean [SD], 77.7 [10.2]; range, 28-112] and subjective stress index (mean [SD], 5.3 [2.4]; range, 0-10) were above median scale values, reflecting a stressful condition, while the EuroQol-5D-3L index and the EuroQol-Visual Analog Scale scores were high (mean [SD], 0.9 [0.1]; range -0.171-1 and mean [SD], 67.7 [18.7]; range 0-100, respectively), reflecting good life quality. In total, 53% of participants had worked in farming for more than 30 years, and workers involved in dry-field farming comprised the largest subgroup (41.5%). Most participants (94.3%) had no more than a high school education, and families with annual income below 20 million won constituted the largest subgroup (36.3%). CONCLUSIONS: The FARM study may provide data on the current health status and related sociodemographic and agricultural work-related risk factors in Korean farmers, with the goal of providing a scientific basis for developing coping interventions and preventive strategies.

The Association between Trunk Body Composition and Spinal Bone Mineral Density in Korean Males versus Females: a Farmers' Cohort for Agricultural Work-Related Musculoskeletal Disorders (FARM) Study.

The purpose of this study was proposed to identify the association of trunk body composition with spinal bone mineral density (BMD) in Korean male and female farmers. A total of 523 Korean farmers (259 males, 44 premenopausal females, and 220 postmenopausal females) were recruited. Computed tomography scans were acquired at the mid-L4 vertebral level, and total trunk muscle mass (TMM, cm³), back muscle mass (BMM), and abdominal wall muscle mass (AMM), total trunk fat mass (TFM), visceral fat mass (VFM), and subcutaneous fat mass (SFM) were assessed. Spinal BMD (g/cm²) was estimated from dual-energy X-ray absorptiometry at the L4 level. In terms of muscle mass, spinal BMD was significantly correlated with all the components of the trunk muscle mass (r = 0.171-0.360; P < 0.05, P < 0.001) in female farmers, while only with AMM (r = 0.181; P < 0.01) in male farmers. In terms of fat mass, spinal BMD was significantly correlated with all components of the trunk fat mass (r = 0.142-0.424; P < 0.05, P < 0.001) in male and premenopausal female farmers, while only with VFM (r = 0.132; P < 0.05) in postmenopausal females. Adjusted multivariate regression analysis showed that AMM in male and post-menopausal female farmers was closely associated with spinal BMD. There may be positive associations between trunk muscle and fat mass and spinal BMD with sexual dimorphism, and abdominal wall muscle mass was an explanatory variable closely related to spinal BMD in Korean farmers. Registered at the Clinical Research Information Service (CRIS, http://cris.nih.go.kr), number KCT0000829.

Incidence of Pneumonia After Videofluoroscopic Swallowing Study and Associated Factors.

Pneumonia after videofluoroscopic swallow study (VFSS) is sometimes considered to be caused by aspiration during VFSS; however, to our knowledge, a relationship between these events has not been clearly investigated. The aim of this study was to assess the incidence of VFSS-related pneumonia and related factors. Overall, 696 VFSS cases were retrospectively reviewed. Cases in which blood culture was performed within 3 days after VFSS due to newly developed infectious signs were considered as post-VFSS infection cases. Pneumonia was suspected when there was some evidence of respiratory infectious signs in clinical, radiological, and laboratory findings. The underlying disease, clinical signs, and VFSS findings of the pneumonia group were assessed. Among 696 cases, pneumonia was diagnosed in 15 patients. The patients in the pneumonia group tended to be older and had higher aspiration rate on VFSS than those in the non-pneumonia group. In the pneumonia group, 2 patients showed no aspiration during VFSS. In 6 patients, pneumonia developed after massive aspiration of gastric content in 5 patients and inappropriate oral feeding with risk of aspiration before VFSS in 1 patient. Only 7 patients (1.0 %) were finally determined as having VFSS-related pneumonia. In conclusion, the 72-h incidence of VFSS-related pneumonia was 1.0 %. Old age and severity of swallowing difficulty are associated with occurrence of pneumonia.

Metabolomic Approach for Discrimination of Four- and Six-Year-Old Red Ginseng (Panax ginseng) Using UPLC-QToF-MS.

Panax ginseng C.A. MEYER is one of the most popular medicinal herbs in Asia and the chemical constituents are changed by processing methods such as steaming or sun drying. Metabolomic analysis was performed to distinguish age discrimination of four- and six-year-old red ginseng using ultra-performance liquid chromatography quadruple time of flight mass spectrometry (UPLC-QToF-MS) with multivariate statistical analysis. Principal component analysis (PCA) showed clear discrimination between extracts of red ginseng of different ages and suggest totally six discrimination markers (two for four-year-old and four for six-year-old red ginseng). Among these, one marker was isolated and the structure determined by NMR spectroscopic analysis was 13-cis-docosenamide (marker 6-1) from six-year-old red ginseng. This is the first report of a metabolomic study regarding the age differentiation of red ginseng using UPLC-QToF-MS and determination of the structure of the marker. These results will contribute to the quality control and standardization as well as provide a scientific basis for pharmacological research on red ginseng.

Structural characterization of human cholesterol 7α-hydroxylase.

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.

Structural characterization of a new N-substituted pantothenamide bound to pantothenate kinases from Klebsiella pneumoniae and Staphylococcus aureus.

Pantothenate kinase (PanK) is the rate-limiting enzyme in Coenzyme A biosynthesis, catalyzing the ATP-dependent phosphorylation of pantothenate. We solved the co-crystal structures of PanKs from Staphylococcus aureus (SaPanK) and Klebsiella pneumonia (KpPanK) with N-[2-(1,3-benzodioxol-5-yl)ethyl] pantothenamide (N354-Pan). Two different N354-Pan conformers interact with polar/nonpolar mixed residues in SaPanK and aromatic residues in KpPanK. Additionally, phosphorylated N354-Pan is found at the closed active site of SaPanK but not at the open active site of KpPanK, suggesting an exchange of the phosphorylated product with a new N354-Pan only in KpPanK. Together, pantothenamides conformational flexibility and binding pocket are two key considerations for selective compound design.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system.

In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1--the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.