Pulmonary Arterial Hypertension Induced by Immune Checkpoint Inhibitor Combined Therapy in a Patient with Intrahepatic Cholangiocarcinoma: A Case Report.

Case: Immune Checkpoint Inhibitors (ICIs) have dramatically revolutionized the therapeutic approaches by which we treat a series of cancers accompanied by immune-related adverse events (irAEs). Herein, we reported an intrahepatic cholangiocarcinoma male patient with a history of ankylosing spondylitis developing pulmonary arterial hypertension (PAH) under ICI combined therapy with pembrolizumab and lenvatinib. The indirect measurement of cardiac ultrasound showed a pulmonary artery pressure (PAP) of 72mmHg after 21 three-week cycles of ICI combined therapy. The patient partially responded to the treatment of glucocorticoid and mycophenolate mofetil. The PAP decreased to 55mmHg 3 months after the ICI combined therapy was discontinued, but increased to 90mmHg after the ICI combined therapy was rechallenged. We treated him with adalimumab -an antitumor necrosis factor-alpha (ani-TNF-α) antibody- combined with glucocorticoid and immunosuppressants under lenvatinib monotherapy. The patient responded again with PAP decreasing to 67mmHg after 2 two-week cycles of adalimumab. Accordingly, we diagnosed him to have irAE-related PAH. Our findings supported the use of glucocorticoid disease-modifying antirheumatic drugs (DMARDs) as a treatment option in refractory PAH.

A study of balloon type on calcified coronary lesion predilation: A finite element analysis.

Calcified coronary lesions have been one of the more difficult types of lesion for interventional treatment, and angioplasty is required to break the calcification before stent implantation so that the stent can expand smoothly, however, it remains unclear which type of angioplasty is optimal for different calcified lesions. In this study, a finite element approach was used to model normal balloons, cutting balloons, and AngioSculpt balloons. In addition, calcified lesions of different degrees, thicknesses, and lengths were modeled according to Intravascular ultrasound (IVUS) calcification grade. The above three balloons were used to pretreat calcified lesions, and the brittle fracture module for calcification was used to detect fracture success, to facilitate virtual stent implantation after predilation. The simulation results showed that with a thickness of less than 0.3 mm, balloons were unable to deal with calcified plaques in lesions of less than 120°, for 180° calcified lesions the cutting balloon fractured the calcified material at 1.2 MPa, the AngioSculpt balloon produced multiple fractures at 0.8 MPa for 270° calcified plaques, but was unable to fracture calcified lesions with a thickness of 0.4 mm. Based on these results, we conclude that the length of the lesion did not affect calcification fracture, while the thickness of the lesion did. In calcified lesions of approximately 180°, the cutting balloon showed the best predilation results, while the AngioSculpt balloon was optimal for 270°. In annular calcification, all three balloons were unable to fracture the lesion.

Interferon-γ predicts the treatment efficiency of immune checkpoint inhibitors in cancer patients.

PURPOSE: Immune checkpoint inhibitors (ICIs) have improved the prognosis of cancer patients significantly with few predictive makers for treatment efficiency. Since interferon-gamma (IFN-γ) displayed its association with immunotherapy, we explored the correlation between IFN-γ and the efficacy of ICIs in tumor treatment. METHODS: We retrospectively examined cancer patients who received immune checkpoint inhibitors as first-line therapy at the Fourth Hospital of Hebei Medical University. The patients were divided into a low concentration group of IFN-γ (≤ 1.2 pg/mL) and a high concentration group (≥ 1.3 pg/mL) to evaluate the efficacy, which was indicated by the objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). RESULTS: Thirty-five patients with low IFN-γ and 56 patients with high IFN-γ were involved in the evaluation, and the DCR was significantly different between these two groups (p = 0.009) with a high group of 81.4% (95% CI 69-94%) and a low group of 51.9% (95% CI 32-72%). The subsequent Kaplan-Meier survival analysis showed that the high IFN-γ patients displayed longer median OS than that of the low IFN-γ patients (p = 0.049), while no statistical difference existed for PFS (p = 0.971). The multivariate analysis also confirmed that the high IFN-γ level was independently associated with a better prognosis (HR: 0.318 95% CI 0.113-0.894, p = 0.030). CONCLUSIONS: Basal serum IFN-γ levels were associated with the DCR and OS of cancer patients with higher IFN-γ exhibiting beneficial efficiency for ICIs treatment.

Bone Metastasis Initiation Is Coupled with Bone Remodeling through Osteogenic Differentiation of NG2+ Cells.

The bone microenvironment is dynamic and undergoes remodeling in normal and pathologic conditions. Whether such remodeling affects disseminated tumor cells (DTC) and bone metastasis remains poorly understood. Here, we demonstrated that pathologic fractures increase metastatic colonization around the injury. NG2+ cells are a common participant in bone metastasis initiation and bone remodeling in both homeostatic and fractured conditions. NG2+ bone mesenchymal stem/stromal cells (BMSC) often colocalize with DTCs in the perivascular niche. Both DTCs and NG2+ BMSCs are recruited to remodeling sites. Ablation of NG2+ lineage impaired bone remodeling and concurrently diminished metastatic colonization. In cocultures, NG2+ BMSCs, especially when undergoing osteodifferentiation, enhanced cancer cell proliferation and migration. Knockout of N-cadherin in NG2+ cells abolished these effects in vitro and phenocopied NG2+ lineage depletion in vivo. These findings uncover dual roles of NG2+ cells in metastasis and remodeling and indicate that osteodifferentiation of BMSCs promotes metastasis initiation via N-cadherin-mediated cell-cell interaction. SIGNIFICANCE: The bone colonization of cancer cells occurs in an environment that undergoes constant remodeling. Our study provides mechanistic insights into how bone homeostasis and pathologic repair lead to the outgrowth of disseminated cancer cells, thereby opening new directions for further etiologic and epidemiologic studies of tumor recurrences. This article is highlighted in the In This Issue feature, p. 247.

Influence of different postballoon expansion procedures: A finite element analysis.

BACKGROUND: Postballoon expansion is considered as an appropriate procedure for adequate stent expansion for coronary bifurcation lesions. Two postballoon expansion procedures are currently recommended: proximal optimization technique (POT)/side/POT and POT/kiss/POT. However, the effects of the two postballoon expansion treatments are different. There is a lack of biomechanical study to quantify the difference. PURPOSE: It is recognized that biomechanical factors influence the occurrence of Major Cardiovascular Adverse Events (MACE), which includes recurrent angina pectoris, acute myocardial infarction and coronary heart disease death. The current paper evaluated the two postexpansion strategies and quantified biomechanical parameters to provide a basis for clinical decisions. METHODS: Based on the CT angiography (CTA) data of a patient diagnosed with coronary bifurcation lesions, a personalized coronary bifurcation lesion model was constructed, and the surgical procedure after two expansions was simulated. The POT/side/POT and POT/kiss/POT expansion procedures were analyzed from the perspective of biomechanics through finite element analysis. The biomechanics factors, including the percentage of stent malapposition and stent occlusion at the side branch (SB) opening, the stent ellipse index of proximal main vessel (PMV) segment, the minimum lumen area of the stent vessel segment and the stress distribution of the vessel wall, were used to quantify clinician concerns about factors affecting patient outcomes. The factors include stent adhesion, SB open stent occlusion, poor stent deformation, patency effect of vessel stenosis, and vessel wall damage. RESULTS: Both postexpansion procedures were successfully simulated. The malapposition rate during POT/side/POT was larger (1.2% vs. 0.42%) and stent occlusion at the SB opening from the cross-section perpendicular to the SB opening after the POT/side/POT procedure was 0.20%, compared with 0.00% after POT/kiss/POT. POT/kiss/POT produced a larger PMV segment stent ellipse index. Minimum lumen area after POT/side/POT was 5.6 mm2 and after POT/kiss/POT 5.9 mm2 . POT/kiss/POT produces an effect of greater vascular stress than POT/side/POT. CONCLUSION: Numerical simulations provide a quantitative analysis to inform clinicians of the differences between preoperative planning and surgical procedures. Biomechanical analysis of the differences between the two postexpansion strategies found that the POT/kiss/POT procedure resulted in better stent fit, less occlusion of the SB open stent and better vascular patency but also resulted in poor stent deformation and caused greater vessel wall stress. The current study informs rationales for clinical understanding of postexpansion strategies.

N7-methylguanosine methylation-related regulator genes as biological markers in predicting prognosis for melanoma.

The aim of this study is to find those N7-methylguanosine (m7G) methylation-related regulator genes (m7GMRRGs) which were associated with melanoma prognosis and use them to develop a prognostic prediction model. Clinical information was retrieved online from The Cancer Gene Atlas (TCGA) and the Gene Expression Omnibus (GEO). R software was used to extract m7GMRRGs by differential expression analysis. To create a prognostic risk model, univariate and multivariate Cox regression analyses were employed for the evaluation of the prognostic significance of m7G methylation modifiers. Internal validation using cohort from TCGA (training set) and external validation using cohort from GEO (validation set) of the model were carried out. The model's predictive performance was confirmed by using the Kaplan-Meier, univariate, and multivariate Cox regression, and receiver operating characteristic curve (ROC) by constructing column line plots incorporating clinical factor characteristics. Immune infiltration analyses were performed to assess the immune function of m7GMRRGs. Drug sensitivity analysis was conducted to study chemotherapeutic drug treatment cues. Prognostic models using four m7GMRRGs (EIF4E3, LARP1, NCBP3, and IFIT5) showed good prognostic power in training and validation sets. The area under the curve (AUC) at 1, 3, and 5 years for GEO-melanoma were 0.689, 0.704, and 0.726, respectively. The prediction model could distinctly classify patients with melanoma into different risk subgroups (P < 0.001 for TCGA-melanoma and P < 0.05 for GEO-melanoma). Clinical characteristics were taken into account in Cox regression and AUC analysis, which highlighted that the risk score served as an independent risk factor determining the prognosis of patients with melanoma. Immuno-infiltration analysis showed that m7GMRRGs could potentially regulate CD8+ T cells as well as regulatory T cells (Treg cells). Results of our study indicate a association between m7GMRRGs and melanoma prognosis, and the prognostic prediction model using m7GMRRGs may predict the prognosis of patients with melanoma well. Nevertheless, these results may provide a clue for potential better options of melanoma treatment but need further validation in futural studies.

Growth hormone associated with treatment efficacy of immune checkpoint inhibitors in gastric cancer patients.

Background: Immune checkpoint inhibitors (ICIs) combined with chemotherapy have been widely employed to improve the outcome of gastric cancer patients. In the present study, the impact of posttreatment growth hormone (GH) levels on the treatment efficacy of ICIs for advanced gastric cancer (AGC) patients was assessed. Methods: Seventy-five AGC patients treated with anti-PD-1 antibodies at The Fourth Hospital of Hebei Medical University were involved. We divided AGC patients into two groups as high-GH group and low-GH group based on the GH level. Immunotherapy efficacy was assessed in terms of objective response rate, disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) based on the National Comprehensive Cancer Network Guidelines. The enumeration data were compared by χ 2 test or Fisher's exact test. Survival curves were drawn by the Kaplan-Meier method, and comparisons between the curves were made using the log-rank test. Multivariate survival analysis was performed using a Cox proportional hazards model. Results: The higher GH levels were associated with a lower DCR of ICIs with a DCR of 30.0% in the high-GH group and 53.3% in the low-GH group (P = 0.046). The subsequent univariate analysis showed that a high GH level was associated with both shorter PFS (P = 0.016) and shorter OS at the borderline statistical level (P = 0.052) in AGC patients treated with ICIs. Cox model analysis also proved that the GH level was an independent risk factor for the outcome of AGC patients (PFS: P = 0.013, HR, 2.424, 95% CI, 1.202-4.890; OS: P = 0.014, HR, 3.301, 95% CI, 1.279-8.519). Conclusions: The post-treatment GH level might be a predictor for ICIs treatment in AGC patients.

Image-to-image translation of label-free molecular vibrational images for a histopathological review using the UNet+/seg-cGAN model.

Translating images generated by label-free microscopy imaging, such as Coherent Anti-Stokes Raman Scattering (CARS), into more familiar clinical presentations of histopathological images will help the adoption of real-time, spectrally resolved label-free imaging in clinical diagnosis. Generative adversarial networks (GAN) have made great progress in image generation and translation, but have been criticized for lacking precision. In particular, GAN has often misinterpreted image information and identified incorrect content categories during image translation of microscopy scans. To alleviate this problem, we developed a new Pix2pix GAN model that simultaneously learns classifying contents in the images from a segmentation dataset during the image translation training. Our model integrates UNet+ with seg-cGAN, conditional generative adversarial networks with partial regularization of segmentation. Technical innovations of the UNet+/seg-cGAN model include: (1) replacing UNet with UNet+ as the Pix2pix cGAN's generator to enhance pattern extraction and richness of the gradient, and (2) applying the partial regularization strategy to train a part of the generator network as the segmentation sub-model on a separate segmentation dataset, thus enabling the model to identify correct content categories during image translation. The quality of histopathological-like images generated based on label-free CARS images has been improved significantly.

Artificial intelligence-augmented, label-free molecular imaging method for tissue identification, cancer diagnosis, and cancer margin detection.

Label-free high-resolution molecular and cellular imaging strategies for intraoperative use are much needed, but not yet available. To fill this void, we developed an artificial intelligence-augmented molecular vibrational imaging method that integrates label-free and subcellular-resolution coherent anti-stokes Raman scattering (CARS) imaging with real-time quantitative image analysis via deep learning (artificial intelligence-augmented CARS or iCARS). The aim of this study was to evaluate the capability of the iCARS system to identify and differentiate the parathyroid gland and recurrent laryngeal nerve (RLN) from surrounding tissues and detect cancer margins. This goal was successfully met.

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor.

Cysteamine dioxygenase (ADO) plays a vital role in regulating thiol metabolism and preserving oxygen homeostasis in humans by oxidizing the sulfur of cysteamine and N-terminal cysteine-containing proteins to their corresponding sulfinic acids using O2 as a cosubstrate. However, as the only thiol dioxygenase that processes both small-molecule and protein substrates, how ADO handles diverse substrates of disparate sizes to achieve various reactions is not understood. The knowledge gap is mainly due to the three-dimensional structure not being solved, as ADO cannot be directly compared with other known thiol dioxygenases. Herein, we report the first crystal structure of human ADO at a resolution of 1.78 Å with a nickel-bound metal center. Crystallization was achieved through both metal substitution and C18S/C239S double mutations. The metal center resides in a tunnel close to an entry site flanked by loops. While ADO appears to use extensive flexibility to handle substrates of different sizes, it also employs proline and proline pairs to maintain the core protein structure and to retain the residues critical for catalysis in place. This feature distinguishes ADO from thiol dioxygenases that only oxidize small-molecule substrates, possibly explaining its divergent substrate specificity. Our findings also elucidate the structural basis for ADO functioning as an oxygen sensor by modifying N-degron substrates to transduce responses to hypoxia. Thus, this work fills a gap in structure-function relationships of the thiol dioxygenase family and provides a platform for further mechanistic investigation and therapeutic intervention targeting impaired oxygen sensing.

The Progress of Label-Free Optical Imaging in Alzheimer's Disease Screening and Diagnosis.

As the major neurodegenerative disease of dementia, Alzheimer's disease (AD) has caused an enormous social and economic burden on society. Currently, AD has neither clear pathogenesis nor effective treatments. Positron emission tomography (PET) and magnetic resonance imaging (MRI) have been verified as potential tools for diagnosing and monitoring Alzheimer's disease. However, the high costs, low spatial resolution, and long acquisition time limit their broad clinical utilization. The gold standard of AD diagnosis routinely used in research is imaging AD biomarkers with dyes or other reagents, which are unsuitable for in vivo studies owing to their potential toxicity and prolonged and costly process of the U.S. Food and Drug Administration (FDA) approval for human use. Furthermore, these exogenous reagents might bring unwarranted interference to mechanistic studies, causing unreliable results. Several label-free optical imaging techniques, such as infrared spectroscopic imaging (IRSI), Raman spectroscopic imaging (RSI), optical coherence tomography (OCT), autofluorescence imaging (AFI), optical harmonic generation imaging (OHGI), etc., have been developed to circumvent this issue and made it possible to offer an accurate and detailed analysis of AD biomarkers. In this review, we present the emerging label-free optical imaging techniques and their applications in AD, along with their potential and challenges in AD diagnosis.

The effect of platelet G proteins on platelet extravasation and tumor growth in the murine model of ovarian cancer.

We and other investigators have shown that platelets promote metastasis and the growth of tumors. Our rationale for conducting this study is that platelets' prometastatic and progrowth effects depend on a close encounter between platelets and cancer cells. This interaction occurs inside blood vessels with circulating tumor cells and outside blood vessels with cancer cells residing in the tumor parenchyma. Our hypothesis was that platelet extravasation is required for the effect of platelets on tumor growth. Platelets respond to environmental stimuli by activation of G protein-coupled receptors on their surface. We investigated the impact of various platelet G proteins on the growth of ovarian cancer tumors and platelet extravasation. We used mice with platelet-specific deficiency of Gαi2 (Gi), Gα13 (G13), or Gαq (Gq) in a syngeneic ovarian cancer model. We measured the total weight of tumor nodules resected from tumor-bearing mice. We developed methods for automated whole-slide image acquisition and unbiased computerized image analysis to quantify extravasated platelets. We compared the number of platelets inside tumor nodules of platelet G protein-deficient tumor-bearing mice. We found that deficiency of Gi and G13, but not Gq, in platelets resulted in smaller tumors compared with those in corresponding littermates. Deficiency of Gi and G13 in platelets reduced the number of extravasated platelets by >90%, but deficiency of Gq did not reduce the number of extravasated platelets significantly. The lack of Gi or G13 in platelets reduced platelet extravasation into the tumor and tumor growth.

The bone microenvironment invigorates metastatic seeds for further dissemination.

Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.

Heme Binding to HupZ with a C-Terminal Tag from Group A Streptococcus.

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV-vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

Formation of Monofluorinated Radical Cofactor in Galactose Oxidase through Copper-Mediated C-F Bond Scission.

Galactose oxidase (GAO) contains a Cu(II)-ligand radical cofactor. The cofactor, which is autocatalytically generated through the oxidation of the copper, consists of a cysteine-tyrosine radical (Cys-Tyr•) as a copper ligand. The formation of the cross-linked thioether bond is accompanied by a C-H bond scission on Tyr272 with few details known thus far. Here, we report the genetic incorporation of 3,5-dichlorotyrosine (Cl2-Tyr) and 3,5-difluorotyrosine (F2-Tyr) to replace Tyr272 in the GAOV previously optimized for expression through directed evolution. The proteins with an unnatural tyrosine residue are catalytically competent. We determined the high-resolution crystal structures of the GAOV, Cl2-Tyr272, and F2-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 Šresolution, respectively. The structural data showed only one halogen remained in the cofactor, indicating that an oxidative carbon-chlorine/fluorine bond scission has occurred during the autocatalytic process of cofactor biogenesis. Using hydroxyurea as a radical scavenger, the spin-coupled hidden Cu(II) was observed by EPR spectroscopy. Thus, the structurally defined catalytic center with genetic unnatural tyrosine substitution is in the radical containing form as in the wild-type, i.e., Cu(II)-(Cl-Tyr•-Cys) or Cu(II)-(F-Tyr•-Cys). These findings illustrate a previously unobserved C-F/C-Cl bond cleavage in biology mediated by a mononuclear copper center.

Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine.

Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of l-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of l-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-l-tyrosine (F2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both l-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidation reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the cross-link formation is proposed.

Significant expansion and red-shifting of fluorescent protein chromophore determined through computational design and genetic code expansion.

ABSTRACT: Fluorescent proteins (FPs) with emission wavelengths in the far-red and infrared regions of the spectrum provide powerful tools for deep-tissue and super-resolution imaging. The development of red-shifted FPs has evoked widespread interest and continuous engineering efforts. In this article, based on a computational design and genetic code expansion, we report a rational approach to significantly expand and red-shift the chromophore of green fluorescent protein (GFP). We applied computational calculations to predict the excitation and emission wavelengths of a FP chromophore harboring unnatural amino acids (UAA) and identify in silico an appropriate UAA, 2-amino-3-(6-hydroxynaphthalen-2-yl)propanoic acid (naphthol-Ala). Our methodology allowed us to formulate a GFP variant (cpsfGFP-66-Naphthol-Ala) with red-shifted absorbance and emission spectral maxima exceeding 60 and 130 nm, respectively, compared to those of GFP. The GFP chromophore is formed through autocatalytic post-translational modification to generate a planar 4-(p-hydroxybenzylidene)-5-imidazolinone chromophore. We solved the crystal structure of cpsfGFP-66-naphthol-Ala at 1.3 Å resolution and demonstrated the formation of a much larger conjugated π-system when the phenol group is replaced by naphthol. These results explain the significant red-shifting of the excitation and emission spectra of cpsfGFP-66-naphthol-Ala.

Quantifying the effects of hydration on corneal stiffness with noncontact optical coherence elastography.

PURPOSE: To quantify the effects of the hydration state on the Young's modulus of the cornea. SETTING: Biomedical Optics Laboratory, University of Houston, Houston, Texas, USA. DESIGN: Experimental study. METHODS: Noncontact, dynamic optical coherence elastography (OCE) measurements were taken of in situ rabbit corneas in the whole eye-globe configuration (n = 10) and at an artificially controlled intraocular pressure of 15 mm Hg. Baseline OCE measurements were taken by topically hydrating the corneas with saline for 1 hour. The corneas were then dehydrated topically with a 20% dextran solution for another hour, and the OCE measurements were repeated. A finite element method was used to quantify the Young's modulus of the corneas based on the OCE measurements. RESULTS: The thickness of the corneas shrank considerably after topical addition of the 20% dextran solution (∼680 µm to ∼370 µm), and the OCE-measured elastic-wave speed correspondingly decreased (∼3.2 m/s to ∼2.6 m/s). The finite element method results showed an increase in Young's modulus (500 kPa to 800 kPa) resulting from dehydration and subsequent thinning. CONCLUSION: Young's modulus increased significantly as the corneas dehydrated and thinned, showing that corneal geometry and hydration state are critical factors for accurately quantifying corneal biomechanical properties.

Cleavage of a carbon-fluorine bond by an engineered cysteine dioxygenase.

Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93-Tyr157 cross-linked cofactor. Here, we investigated this Cys-Tyr cross-linking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and 19F NMR, these data indicated that the enzyme could catalyze oxidative C-F or C-Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys-Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon-halogen bond activation.

Cofactor Biogenesis in Cysteamine Dioxygenase: C-F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine.

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein-derived cofactor. Reported herein is the discovery and elucidation of a Cys-Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C-S) bond. By genetically incorporating an unnatural amino acid, 3,5-difluoro-tyrosine (F2 -Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon-fluorine bond activation and fluoride release were identified by mass spectrometry and 19 F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low-resolution techniques. Finally, a new sequence motif, C-X-Y-Y(F), is proposed for identifying the Cys-Tyr crosslink.