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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.

Post-balloon expansion is considered as an appropriate procedure for adequate stent expansion for coronary bifurcation lesions. Two post-balloon expansion procedures are currently recommended: proximal optimization technique (POT)/side/POT and POT/kiss/POT. However, the effects of the two post-balloon expansion treatments are different. There is a lack of biomechanical study to quantify the difference.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 post-expansion strategies and quantified biomechanical parameters to provide a basis for clinical decisions.Based on the 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.Both post-expansion 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 mmNumerical 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 post-expansion 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 post-expansion strategies. This article is protected by copyright. All rights reserved.

Supplemantary Video 3, LLC1 cells cocultured with NG2- BMSCs

summarized excel file containing the quantitative raw data and processed values in the manuscript

Supplemantary Video 4, LLC1 cells cocultured with NG2+ Ncadherin KO BMSCs

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

Supplemantary Video 1, LLC1 cells cocultured with NG2+ BMSCs

Supplementary figures and corresponding figure legends

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

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.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).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).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.

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.

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 A 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.

Since the mapping relationship between definitized intra-interventional 2D X-ray and undefined pre-interventional 3D Computed Tomography(CT) is uncertain, auxiliary positioning devices or body markers, such as medical implants, are commonly used to determine this relationship. However, such approaches can not be widely used in clinical due to the complex realities. To determine the mapping relationship, and achieve a initializtion post estimation of human body without auxiliary equipment or markers, a cross-modal matching transformer network is proposed to matching 2D X-ray and 3D CT images directly. The proposed approach first learns skeletal features from 2D X-ray and 3D CT images. The features are then converted into 1D X-ray and CT representation vectors, which are combined using a transformer module. As a result, the well-trained network can directly predict the spatial correspondence between arbitrary 2D X-ray and 3D CT. The experimental results show that when combining our approach with the conventional approach, the achieved accuracy and speed can meet the basic clinical intervention needs, and it provides a new direction for intra-interventional registration.

In order to reduce tissue damage caused by ischemia-reperfusion injury, this study aims to investigate the protective effect and mechanism of α-lipoic acid on hepatic ischemia-reperfusion injury in rats. The bloodstream of rats was blocked in the left middle and left lateral liver lobes of the liver. Forty rats were randomly divided into two groups: treatment group and injury group. Rats were injected with either 25 mg/1 ml of α-lipoic acid (treatment group) or 1 ml of saline (injury group) into the caudal vein 15 min before hepatic ischemia-reperfusion. Rat serum alanine aminotransferase (GPT), glutathione (GSH) and superoxide dismutase (SOD) levels were examined at various time points (1, 3, 6 and 12 h) in both groups. Changes in nuclear factor kappa B P65 (NF-κB P65) expression in ischemia-reperfusion liver at various time points after reperfusion (1, 3, 6 and 12 h) were evaluated through immunohistochemistry assay. Changes in macrophage inflammatory protein-2 (MIP-2) mRNA and inducible nitric oxide synthase (iNOS) mRNA expression in ischemic reperfused rat livers were detected by RT-PCR. Serum GPT level was significantly higher in the injury group than in the treatment group (P

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of L-cysteine. Adjacent to the iron site of mammalian CDO, a post-translationally generated Cys-Tyr cofactor is present, 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 crosslinking 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 uncrosslinked human CDO due to site-specific incorporation 3,5-difluoro-L-tyrosine (F(2)-Tyr) at the crosslinking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncrosslinked F(2)-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncrosslinked F(2)-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 structural based understanding is further supported by a computational study with models built on the uncrosslinked 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 crosslink formation is proposed.

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&ndash, 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.

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.

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 crosslinked 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 (Cl(2)-Tyr) and 3,5-diflurotyrosine (F(2)-Tyr) to replace Tyr272 in the GAO(V) 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 GAO(V), Cl(2)-Tyr272, and F(2)-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 Å resolution, 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.

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. Graphical abstract

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.

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.

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.

SIGNIFICANCE Measured corneal biomechanical properties are driven by intraocular pressure, tissue thickness, and inherent material properties. We demonstrate tissue thickness as an important factor in the measurement of corneal biomechanics that can confound short-term effects due to UV riboflavin cross-linking (CXL) treatment. PURPOSE We isolate the effects of tissue thickness on the measured corneal biomechanical properties using optical coherence elastography by experimentally altering the tissue hydration state and stiffness. METHODS Dynamic optical coherence elastography was performed using phase-sensitive optical coherence tomography imaging to quantify the tissue deformation dynamics resulting from a spatially discrete, low-force air pulse (150-μm spot size; 0.8-millisecond duration

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.

The study of dormancy and reactivation of disseminated tumor cells (DTCs) in the distant organs are critical in in metastatic cancer research. During early stages of the disease, breast cancer cells have high chance to metastasize and settle in other organs such as bone. It is important to understand the spatial distribution of tumor cells under distinct microenvironments and how these niches determine the fates of tumor cells in homeostatic and pathological settings. In this work, we proposed a deep imaging systems biology (DISB) framework by integrating multiple components, including in vivo spontaneous tissue metastases models, tissue clearing, deep imaging, digital reconstruction, image quantitation, and graphic-theoretic analysis, to study the spatial distribution and pathophysiology of DTCs and related stroma cells in organs over time. Within the DISB framework, genetically engineered fluorescent reporter murine strains, which highlight specific bone marrow lineages, were used as the dimensional maps of diversified perivascular and endosteal niches. Metastases asynchronously occur in immune competent mice after resection of syngeneic primary tumors with or without adjuvant therapies, mimicking the natural course of metastatic spreading in clinics. Long bones carrying metastatic tumor cells were collected and subjected to the BABB (Benzoic Acid Benzyl Benzoate) clearing methodology. The spatial distribution of cancer and bone cells in the tumor tissue was captured by whole slide confocal imaging and digitally reconstructed. We developed deep learning image computing model to segment, quantitate, and obtain the spatial distribution and correlations among different components in the niche, such as cancer cells, vessels, and normal tissue cells over different bone specimens at various time points. We applied the DISB framework to image and trace specific bone marrow lineages (Olympus FluoView FV1000), and the results indicated that the spatial distribution of tumor cells in such spontaneous metastases models is significantly different from those in experimental metastases models. DTCs are mainly in close proximity to the endothelium, but such distribution is likely a random process. Of interest, the proliferating micrometastases were observed in close association with the endosteal vessels, suggesting the potential correlation of osteogenesis and metastatic growth in bone. In addition, adjuvant chemotherapies likely did not shift tumor cells across different bone marrow niches. Combined image-omics and graph-theoretic analysis on various cell types and vessels provide a new powerful new approach to understand the mechanism leading to diverse behaviors of tumor and various types of stroma cells in homeostatic and pathological settings in various bone marrow microenvironments under different perturbations or developmental stages. Citation Format: Tiancheng He, Weijie Zhang, Jiasong Li, Jianting Sheng, Xiang Zhang, Stephen T. Wong. Mapping spatial cellular dynamics of bone marrow niches in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 128.

The modulation of protein acetylation network is a promising strategy for life span extension and disease treatment (Sabari et al., 2016; Giblin et al., 2014) [1,2]. A variety of small molecules have been developed to target deacetylases, but extremely few of these molecules are capable of activating the mitochondrial NAD-dependent deacetylase sirtuin-3 (SIRT3) (Gertz and Steegborn, 2016; Scholz et al., 2015) [3,4]. Manganese superoxide dismutase (MnSOD) is the major superoxide scavenger in mitochondria, whose activity is regulated by SIRT3-mediated deacetylation, particularly at the Lys68 site (Chen et al., 2011) [5]. To investigate the influence of Lys68 acetylation on MnSOD activity, we produced a mutant MnSOD protein-bearing N-acetyllysine (AcK) at its Lys68 position through the genetic code expansion approach. We solved the crystal structure of this acetylated MnSOD (MnSODK68AcK), thus revealing the structural and electrostatic basis for the significant activity decrease upon Lys68 acetylation. On the basis of an assay we developed for the SIRT3-mediated deacetylation of MnSODK68AcK, we identified a novel SIRT3 activator, 7-hydroxy-3-(4'-methoxyphenyl) coumarin (C12), which binds to SIRT3 with high affinity and can promote the deacetylation and activation of MnSOD. C12 adds to the current repertoire of extremely few SIRT3 activators, which are potentially valuable for treating a wide array of diseases via modulating the cellular acetylome.

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.

PURPOSE: To evaluate the elastic anisotropy of porcine corneas at different intraocular pressures (IOPs) using a noncontact optical coherence elastography (OCE) technique. METHODS: A focused air-pulse induced low amplitude (≤ 10 µ m) elastic waves in fresh porcine corneas (n = 7) in situ in the whole eye globe configuration. A home-built phase-stabilized swept source optical coherence elastography (PhS-SSOCE) system imaged the elastic wave propagation at different stepped radial directions. A closed-loop feedback system was used to artificially control the IOP and the OCE measurements were repeated as the IOP was incrementally increased from 15 to 30 mm Hg in 5-mm Hg increments. RESULTS: The OCE measurements demonstrated that the stiffness of the cornea increased as a function of IOP and elastic anisotropy of the cornea became more pronounced at higher IOPs. The standard deviation of the modified planar anisotropy coefficient increased from 0.72 ± 0.42 at an IOP of 15 mm Hg to 1.58 ± 0.40 at 30 mm Hg. CONCLUSIONS: The presented noncontact OCE method was capable of detecting and assessing the corneal elastic anisotropy as a function of IOP. Due to the noninvasive nature and small amplitude of the elastic wave, this method may be able to provide further information about corneal health and integrity in vivo. [ J Refract Surg. 2016;32(8):562–567.]

Acute glomerulonephritis caused by antiglomerular basement membrane marked by high mortality. The primary reason for this is delayed diagnosis via blood examination, urine analysis, tissue biopsy, or ultrasound and X-ray computed tomography imaging. Blood, urine, and tissue-based diagnoses can be time consuming, while ultrasound and CT imaging have relatively low spatial resolution, with reduced sensitivity. Optical coherence tomography is a noninvasive and high-resolution imaging technique that provides superior spatial resolution (micrometer scale) as compared to ultrasound and CT. Changes in tissue properties can be detected based on the optical metrics analyzed from the OCT signals, such as optical attenuation and speckle variance. Furthermore, OCT does not rely on ionizing radiation as with CT imaging. In addition to structural changes, the elasticity of the kidney can significantly change due to nephritis. In this work, OCT has been utilized to quantify the difference in tissue properties between healthy and nephritic murine kidneys. Although OCT imaging could identify the diseased tissue, its classification accuracy is clinically inadequate. By combining optical metrics with elasticity, the classification accuracy improves from 76% to 95%. These results show that OCT combined with OCE can be a powerful tool for identifying and classifying nephritis. Therefore, the OCT/OCE method could potentially be used as a minimally invasive tool for longitudinal studies during the progression and therapy of glomerulonephritis as well as complement and, perhaps, substitute highly invasive tissue biopsies. Elastic-wave propagation in mouse healthy and nephritic kidneys.

Accurate non-invasive assessment of tissue elasticity in vivo is required for early diagnostics of many tissue abnormalities. We have developed a focused air-pulse system that produces a low-pressure and short-duration air stream, which can be used to excite transient surface waves (SWs) in soft tissues. System characteristics were studied using a high-resolution analog pressure transducer to describe the excitation pressure. Results indicate that the excitation pressure provided by the air-pulse system can be easily controlled by the air source pressure, the angle of delivery, and the distance between the tissue surface and the port of the air-pulse system. Furthermore, we integrated this focused air-pulse system with phase-sensitive optical coherence tomography (PhS-OCT) to make non-contact measurements of tissue elasticity. The PhS-OCT system is used to assess the group velocity of SW propagation, which can be used to determine Young's modulus. Pilot experiments were performed on gelatin phantoms with different concentrations (10%, 12% and 14% w/w). The results demonstrate the feasibility of using this focused air-pulse system combined with PhS-OCT to estimate tissue elasticity. This easily controlled non-contact technique is potentially useful to study the biomechanical properties of ocular and other tissues in vivo.

Several methods have been proposed to assess changes in corneal biomechanical properties due to various factors, such as degenerative diseases, intraocular pressure, and therapeutic interventions (e.g. corneal collagen crosslinking). However, the effect of the corneal tissue hydration state on corneal stiffness is not well understood. In this work, we induce low amplitude (< 10 μm) elastic waves with a focused micro air-pulse in fresh in situ rabbit corneas (n = 10) in the whole eye-globe configuration at an artificially controlled intraocular pressure. The waves were then detected with a phase-stabilized swept source optical coherence elastography system. Baseline measurements were taken every 20 minutes for an hour while the corneas were hydrated with 1X PBS. After the measurement at 60 minutes, a 20% dextran solution was topically instilled to dehydrate the corneas. The measurements were repeated every 20 minutes again for an hour. The results showed that the elastic wave velocity decreased as the corneal thickness decreased. Finite element modeling (FEM) was performed using the corneal geometry and elastic wave propagation speed to assess the stiffness of the samples. The results show that the stiffness increased from ~430 kPa during hydration with PBS to ~500 kPa after dehydration with dextran, demonstrating that corneal hydration state, apart from geometry and intraocular pressure, can change the stiffness of the cornea.

In this study the dynamic behavior of a layered viscoelastic medium in response to the harmonic and impulsive acoustic radiation force applied to its surface was investigated both theoretically and experimentally. An analytical solution for a layered viscoelastic compressible medium in frequency and time domains was obtained using the Hankel transform. A special incompressible case was considered to model soft biological tissues. To verify our theoretical model, experiments were performed using tissue-like gel-based phantoms with varying mechanical properties. A 3.5 MHz single-element focused ultrasound transducer was used to apply the radiation force at the surface of the phantoms. A phase-sensitive optical coherence tomography (OCT) system was used to track the displacements of the phantom surface. Theoretically predicted displacements were compared with experimental measurements. The role of the depth dependence of the elastic properties of a medium in its response to an acoustic pulse at the surface was studied. It was shown that the low-frequency vibrations at the surface are more sensitive to the deep layers than high-frequency ones. Therefore, the proposed model in combination with spectral analysis can be used to evaluate depth-dependent distribution of the mechanical properties based on the measurements of the surface deformation.

Tissue optical clearing is an emerging technique for dynamically modifying tissue optical properties to increase imaging depth, which is useful in applications such as imaging and functional diagnostics of many diseases. For example, optical clearing of cartilage allowed imaging of subchondral bone that is used to assess orthopedic diseases. However, the effect of the clearing processes on tissue elastic properties has not been investigated yet. In this study we report the first use of phase-stabilized swept source optical coherence elastography (PhS-SSOCE) to quantitatively monitor the change in elasticity of hyaline cartilage during the optical clearing process noninvasively. The results showed that PhS-SSOCE was able to assess the increase in cartilage stiffness during the clearing process over time and with different concentrations of glucose. In addition, the results demonstrated that the elasticity of the cartilage was reversed once the clearing agent was replaced with saline. To verify the results obtained from the PhS-SSOCE measurements, benchmark mechanical testing was performed using a uniaxial mechanical compression frame. Both methods demonstrated the same trend of the elasticity change of the cartilage immersed in glucose solution. The data show that during the transition from phosphate buffered saline to the clearing agent, the cartilage stiffness decreases significantly, which indicates that the clearing agent diffused into the cartilage extracellular matrix and decreased the tissue elasticity due to dehydration. Therefore, the proposed optical coherence elastography can dynamically assess the effects of optical clearing and associated changes in tissue biomechanical properties noninvasively and nondestructively. This technique may be potentially useful in orthopedic studies such as early detection and monitoring of osteoarthritic diseases.

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.

PURPOSE To evaluate the capability of a novel, coaligned focused ultrasound and phase-sensitive optical coherence elastography (US-OCE) system to assess age-related changes in biomechanical properties of the crystalline lens in situ. METHODS Low-amplitude elastic deformations in young and mature rabbit lenses were measured by an US-OCE system consisting of a spectral-domain optical coherence tomography (OCT) system coaligned with a focused ultrasound system used to produce a transient force on the lens surface. Uniaxial compressional tests were used to validate the OCE data. RESULTS The OCE measurements showed that the maximum displacements of the young rabbit lenses were significantly larger than those of the mature lenses, indicating a gradual increase of the lens stiffness with age. Temporal analyses of the displacements also demonstrate a similar trend of elastic properties in these lenses. The stress-strain measurements using uniaxial mechanical tests confirmed the results obtained by the US-OCE system. CONCLUSIONS The results demonstrate that the US-OCE system can be used for noninvasive analysis and quantification of lens biomechanical properties in situ and possibly in vivo.

While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y) redox potential and pKa to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Through the genetic incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we recapitulated important features of cytochrome c oxidase (CcO) into this small soluble protein, which exhibits selective O2 reduction activity while generating small amount of reactive oxygen species (ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the active site is important for CcO function. Moreover, we elucidated the structural basis for the genetic incorporation of OMeY into proteins, by solving the X-ray structure of OMeY specific aminoacyl-tRNA synthetase in complex with OMeY.