Fluorinated hydroxyapatite conditions a favorable osteo-immune microenvironment via triggering metabolic shift from glycolysis to oxidative phosphorylation.

BACKGROUND: Biological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects. METHODS: FPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats. RESULTS: The incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats. CONCLUSION: In summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials.

Effect of Oxyfluorination of PFA-Coated Metal Mesh with Superhydrophobic Properties on the Filtration Performance of SiO2 Microparticles.

Recently, semiconductor wastewater treatment has received much attention due to the emergence of environmental issues. Acid-resistant coatings are essential for metal prefilters used in semiconductor wastewater treatment. Perfluoroalkoxy alkane is mainly used as an acid-resistant coating agent, since PFA has inherent superhydrophobicity, water permeability is lowered. To solve this problem, the surface of the PFA-coated metal mesh was treated via an oxyfluorination method in which an injected mixed gas of fluorine and oxygen reacted with the surface functional groups. Surface analysis, water contact angle measurement, and water permeability tests were performed on the surface-treated PFA-coated mesh. Consequently, the superhydrophobic surface was effectively converted to a hydrophobic surface as the PFA coating layer was surface-modified with C-O-OH functional groups via the oxyfluorination reaction. As a result of using simulation solutions that float silica particles of various sizes, the permeability and particle removal rate of the surface-modified PFA-coated stainless-steel mesh were improved compared to those before surface modification. Therefore, the oxyfluorination treatment used in this study was suitable for improving the filtration performance of SiO2 microparticles in the PFA-coated stainless-steel mesh.

Cobalt-Catalyzed Formation of Grignard Reagents via C-O or C-S Bond Activation.

C(aryl)-OMe bond functionalization catalyzed by cobalt(II) chloride in combination with a nacnac-type ligand and magnesium as a reductant is reported. Borylation and benzoylation of aryl methoxides are demonstrated, and C(aryl)-SMe bond borylation can be achieved under similar conditions. This is the first example of achieving these transformations using cobalt catalysis. Mechanistic studies suggest that a Grignard reagent is generated as an intermediate in a rare example of a magnesiation via a C-O bond activation reaction. Indeed, an organomagnesium species could be directly observed by electrospray ionization mass spectroscopic analysis. Kinetic experiments indicate that a heterogeneous cobalt catalyst performs the C-O bond activation.

Clinical feasibility of catheter-directed selective intracoronary computed tomography angiography using an extremely low dose of iodine in patients with coronary artery disease.

OBJECTIVE: This study aimed to evaluate the clinical feasibility of catheter-directed selective computed tomography angiography (S-CTA) in patients with coronary artery disease (CAD). METHODS: We prospectively enrolled 65 patients diagnosed with CAD who underwent conventional computed tomography angiography (C-CTA). C-CTA was performed with 60-90 mL of contrast medium (370 mg iodine/mL), whereas S-CTA was performed with 15 mL of contrast medium and 17.19 mg iodine/mL. Luminal enhancement range, homogeneity of luminal enhancement, image quality, plaque volume (PV), and percent aggregate plaque volume (%APV) were measured. Paired Student's t test, Wilcoxon rank-sum test, and Pearson's correlation coefficient were used to compare two methods. RESULTS: Luminal enhancement was significantly higher on S-CTA than on C-CTA (324.4 ± 8.0 Hounsfield unit (HU) vs. 312.0 ± 8.0 HU, p < 0.0001 in the per-vessel analysis). Transluminal attenuation gradient showed a significantly slower reduction pattern on S-CTA than on C-CTA (-0.65 HU/10 mm vs. -0.89 HU/10 mm, p < 0.0001 in the per-vessel analysis). Image noise was significantly lower on S-CTA than on C-CTA (39.6 ± 10.0 HU vs. 43.9 ± 9.4 HU, p < 0.0001). There was excellent correlation between S-CTA and C-CTA with respect to PV and %APV (r = 0.99, r = 0.98, respectively). CONCLUSIONS: S-CTA might be useful in facilitating atherosclerotic plaque analysis and providing guidance for complex lesions such as chronic total occlusion, particularly in cases in which on-site procedure planning is required. KEY POINTS: • Selective computed tomography angiography (S-CTA) can serve as an intraprocedural computed tomography angiography protocol. • S-CTA was performed with low dose of iodine compared with conventional computed tomography angiography. • S-CTA enables on-site atherosclerotic plaque analysis.

Knowledge-based iterative model reconstruction: comparative image quality and radiation dose with a pediatric computed tomography phantom.

BACKGROUND: CT of pediatric phantoms can provide useful guidance to the optimization of knowledge-based iterative reconstruction CT. OBJECTIVE: To compare radiation dose and image quality of CT images obtained at different radiation doses reconstructed with knowledge-based iterative reconstruction, hybrid iterative reconstruction and filtered back-projection. MATERIALS AND METHODS: We scanned a 5-year anthropomorphic phantom at seven levels of radiation. We then reconstructed CT data with knowledge-based iterative reconstruction (iterative model reconstruction [IMR] levels 1, 2 and 3; Philips Healthcare, Andover, MA), hybrid iterative reconstruction (iDose(4), levels 3 and 7; Philips Healthcare, Andover, MA) and filtered back-projection. The noise, signal-to-noise ratio and contrast-to-noise ratio were calculated. We evaluated low-contrast resolutions and detectability by low-contrast targets and subjective and objective spatial resolutions by the line pairs and wire. RESULTS: With radiation at 100 peak kVp and 100 mAs (3.64 mSv), the relative doses ranged from 5% (0.19 mSv) to 150% (5.46 mSv). Lower noise and higher signal-to-noise, contrast-to-noise and objective spatial resolution were generally achieved in ascending order of filtered back-projection, iDose(4) levels 3 and 7, and IMR levels 1, 2 and 3, at all radiation dose levels. Compared with filtered back-projection at 100% dose, similar noise levels were obtained on IMR level 2 images at 24% dose and iDose(4) level 3 images at 50% dose, respectively. Regarding low-contrast resolution, low-contrast detectability and objective spatial resolution, IMR level 2 images at 24% dose showed comparable image quality with filtered back-projection at 100% dose. Subjective spatial resolution was not greatly affected by reconstruction algorithm. CONCLUSION: Reduced-dose IMR obtained at 0.92 mSv (24%) showed similar image quality to routine-dose filtered back-projection obtained at 3.64 mSv (100%), and half-dose iDose(4) obtained at 1.81 mSv.

Sodium-metal halide and sodium-air batteries.

Impressive developments have been made in the past a few years toward the establishment of Na-ion batteries as next-generation energy-storage devices and replacements for Li-ion batteries. Na-based cells have attracted increasing attention owing to low production costs due to abundant sodium resources. However, applications of Na-ion batteries are limited to large-scale energy-storage systems because of their lower energy density compared to Li-ion batteries and their potential safety problems. Recently, Na-metal cells such as Na-metal halide and Na-air batteries have been considered to be promising for use in electric vehicles owing to good safety and high energy density, although less attention is focused on Na-metal cells than on Na-ion cells. This Minireview provides an overview of the fundamentals and recent progress in the fields of Na-metal halide and Na-air batteries, with the aim of providing a better understanding of new electrochemical systems.

Multi-scaled temporal modeling of cardiovascular disease progression: An illustration of proximal arteries in pulmonary hypertension.

The progression of cardiovascular disease is intricately influenced by a complex interplay between physiological pathways, biochemical processes, and physical mechanisms. This study aimed to develop an in-silico physics-based approach to comprehensively model the multifaceted vascular pathophysiological adaptations. This approach focused on capturing the progression of proximal pulmonary arterial hypertension, which is significantly associated with the irreversible degradation of arterial walls and compensatory stress-induced growth and remodeling. This study incorporated critical characteristics related to the distinct time scales for the deformation, thus reflecting the impact of mean pressure on artery growth and tissue damage. The in-silico simulation of the progression of pulmonary hypertension was realized based on computational code combined with the finite element method (FEM) for the simulation of disease progression. The parametric studies further explored the consequences of these irreversible processes. This computational modeling approach may advance our understanding of pulmonary hypertension and its progression.

Identifying Coronary Artery Calcification Using Chest X-ray Radiographs and Machine Learning: The Role of the Radiomics Score.

PURPOSE: To evaluate the ability of radiomics score (RS)-based machine learning to identify moderate to severe coronary artery calcium (CAC) on chest x-ray radiographs (CXR). MATERIALS AND METHODS: We included 559 patients who underwent a CAC scan with CXR obtained within 6 months and divided them into training (n = 391) and validation (n = 168) cohorts. We extracted radiomic features from annotated cardiac contours in the CXR images and developed an RS through feature selection with the least absolute shrinkage and selection operator regression in the training cohort. We evaluated the incremental value of the RS in predicting CAC scores when combined with basic clinical factor in the validation cohort. To predict a CAC score ≥100, we built an RS-based machine learning model using random forest; the input variables were age, sex, body mass index, and RS. RESULTS: The RS was the most prominent factor for the CAC score ≥100 predictions (odds ratio = 2.33; 95% confidence interval: 1.62-3.44; P < 0.001) compared with basic clinical factor. The machine learning model was tested in the validation cohort and showed an area under the receiver operating characteristic curve of 0.808 (95% confidence interval: 0.75-0.87) for a CAC score ≥100 predictions. CONCLUSIONS: The use of an RS-based machine learning model may have the potential as an imaging marker to screen patients with moderate to severe CAC scores before diagnostic imaging tests, and it may improve the pretest probability of detecting coronary artery disease in clinical practice.

Simultaneous Viability Assessment and Invasive Coronary Angiography Using a Therapeutic CT System in Chronic Myocardial Infarction Patients.

PURPOSE: In a preclinical study using a swine myocardial infarction (MI) model, a delayed enhancement (DE)-multi-detector computed tomography (MDCT) scan was performed using a hybrid system alongside diagnostic invasive coronary angiography (ICA) without the additional use of a contrast agent, and demonstrated an excellent correlation in the infarct area compared with histopathologic specimens. In the present investigation, we evaluated the feasibility and diagnostic accuracy of a myocardial viability assessment by DE-MDCT using a hybrid system comprising ICA and MDCT alongside diagnostic ICA without the additional use of a contrast agent. MATERIALS AND METHODS: We prospectively enrolled 13 patients (median age: 67 years) with a previous MI (>6 months) scheduled to undergo ICA. All patients underwent cardiac magnetic resonance (CMR) imaging before diagnostic ICA. MDCT viability scans were performed concurrently with diagnostic ICA without the use of additional contrast. The total myocardial scar volume per patient and average transmurality per myocardial segment measured by DE-MDCT were compared with those from DE-CMR. RESULTS: The DE volume measured by MDCT showed an excellent correlation with the volume measured by CMR (r=0.986, p<0.0001). The transmurality per segment by MDCT was well-correlated with CMR (r=0.900, p<0.0001); the diagnostic performance of MDCT in differentiating non-viable from viable myocardium using a 50% transmurality criterion was good with a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 87.5%, 99.5%, 87.5%, 99.5%, and 99.1%, respectively. CONCLUSION: The feasibility of the DE-MDCT viability assessment acquired simultaneously with conventional ICA was proven in patients with chronic MI using DE-CMR as the reference standard.

Artificial intelligence-enhanced automation for M-mode echocardiographic analysis: ensuring fully automated, reliable, and reproducible measurements.

To enhance M-mode echocardiography's utility for measuring cardiac structures, we developed and evaluated an artificial intelligence (AI)-based automated analysis system for M-mode images through the aorta and left atrium [M-mode (Ao-LA)], and through the left ventricle [M-mode (LV)]. Our system, integrating two deep neural networks (DNN) for view classification and image segmentation, alongside an auto-measurement algorithm, was developed using 5,958 M-mode images [3,258 M-mode (LA-Ao), and 2,700 M-mode (LV)] drawn from a nationwide echocardiographic dataset collated from five tertiary hospitals. The performance of view classification and segmentation DNNs were evaluated on 594 M-mode images, while automatic measurement accuracy was tested on separate internal test set with 100 M-mode images as well as external test set with 280 images (140 sinus rhythm and 140 atrial fibrillation). Performance evaluation showed the view classification DNN's overall accuracy of 99.8% and segmentation DNN's Dice similarity coefficient of 94.3%. Within the internal test set, all automated measurements, including LA, Ao, and LV wall and cavity, resonated strongly with expert evaluations, exhibiting Pearson's correlation coefficients (PCCs) of 0.81-0.99. This performance persisted in the external test set for both sinus rhythm (PCC, 0.84-0.98) and atrial fibrillation (PCC, 0.70-0.97). Notably, automatic measurements, consistently offering multi-cardiac cycle readings, showcased a stronger correlation with the averaged multi-cycle manual measurements than with those of a single representative cycle. Our AI-based system for automatic M-mode echocardiographic analysis demonstrated excellent accuracy, reproducibility, and speed. This automated approach has the potential to improve efficiency and reduce variability in clinical practice.

Application of Thermally Fluorinated Multi-Wall Carbon Nanotubes as an Additive to an Li4Ti5O12 Lithium Ion Battery.

In this study, multi-walled carbon nanotubes (MWCNTs) were modified by thermal fluorination to improve dispersibility between MWCNTs and Li4Ti5O12 (LTO) and were used as additives to compensate for the disadvantages of LTO anode materials with low electronic conductivity. The degree of fluorination of the MWCNTs was controlled by modifying the reaction time at constant fluorination temperature; the clear structure and surface functional group changes in the MWCNTs due to the degree of fluorination were determined. In addition, the homogeneous dispersion in the LTO was improved due to the strong electronegativity of fluorine. The F-MWCNT conductive additive was shown to exhibit an excellent electrochemical performance as an anode for lithium ion batteries (LIBs). In particular, the optimized LTO with added fluorinated MWCNTs not only exhibited a high specific capacity of 104.8 mAh g-1 at 15.0 C but also maintained a capacity of ~116.8 mAh g-1 at a high rate of 10.0 C, showing a capacity almost 1.4 times higher than that of LTO with the addition of pristine MWCNTs and an improvement in the electrical conductivity. These results can be ascribed to the fact that the semi-ionic C-F bond of the fluorinated MWCNTs reacts with the Li metal during the charge/discharge process to form LiF, and the fluorinated MWCNTs are converted into MWCNTs to increase the conductivity due to the bridge effect of the conductive additive, carbon black, with LTO.

Trimethylsilyl Compounds for the Interfacial Stabilization of Thiophosphate-Based Solid Electrolytes in All-Solid-State Batteries.

Argyrodite-type Li6 PS5 Cl (LPSCl) has attracted much attention as a solid electrolyte for all-solid-state batteries (ASSBs) because of its high ionic conductivity and good mechanical flexibility. LPSCl, however, has challenges of translating research into practical applications, such as irreversible electrochemical degradation at the interface between LPSCl and cathode materials. Even for Li-ion batteries (LIBs), liquid electrolytes have the same issue as electrolyte decomposition due to interfacial instability. Nonetheless, current LIBs are successfully commercialized because functional electrolyte additives give rise to the formation of stable cathode-electrolyte interphase (CEI) and solid-electrolyte interphase (SEI) layers, leading to supplementing the interfacial stability between electrolyte and electrode. Herein, inspired by the role of electrolyte additives for LIBs, trimethylsilyl compounds are introduced as solid electrolyte additives for improving the interfacial stability between sulfide-based solid electrolytes and cathode materials. 2-(Trimethylsilyl)ethanethiol (TMS-SH), a solid electrolyte additive, is oxidatively decomposed during charge, forming a stable CEI layer. As a result, the CEI layer derived from TMS-SH suppresses the interfacial degradation between LPSCl and LiCoO2 , thereby leading to the excellent electrochemical performance of Li | LPSCl | LiCoO2 , such as superior cycle life over 2000 cycles (85.0% of capacity retention after 2000 cycles).

Operando Spatial Pressure Mapping Analysis for Prototype Lithium Metal Pouch Cells Under Practical Conditions.

Monitoring and diagnosing the battery status in real-time are of utmost importance for clarifying failure mechanism, improving battery performance, and ensuring safety, particularly under fast charging conditions. Recently, advanced operando techniques have been developed to observe changes in the microstructures of lithium deposits using laboratory-scale cell designs, focusing on understanding the nature of Li metal electrodes. However, the macroscopic spatial inhomogeneity of lithium electroplating/stripping in the prototype pressurized pouch cells has not been measured in real-time under practical conditions. Herein, a new noninvasive operando technique, spatial pressure mapping analysis, is introduced to macroscopically and quantitatively measure spatial pressure changes in a pressurized pouch cell during cycling. Moreover, dynamic spatial changes in the macroscopic morphology of the lithium metal electrode are theoretically visualized by combining operando pressure mapping data with mechanical analyses of cell components. Additionally, under fast charging conditions, the direct correlation between abrupt capacity fading and sudden increases in spatial pressure distribution inhomogeneity is demonstrated through comparative analysis of pouch cells under various external pressures, electrolyte species, and electrolyte weight to cell capacity (e/c) ratios. This operando technique provides insights for assessing the current battery status and understanding the complex origin of cell degradation behavior in pressurized pouch cells.

MBP-11901 Inhibits Tumor Growth of Hepatocellular Carcinoma through Multitargeted Inhibition of Receptor Tyrosine Kinases.

Hepatocellular carcinomas (HCCs) are aggressive tumors with a poor prognosis. Approved first-line treatments include sorafenib, lenvatinib, and a combination of atezolizumab and bevacizumab; however, they do not cure HCC. We investigated MBP-11901 as a drug candidate for HCC. Cell proliferation and cytotoxicity were evaluated using normal and cancer human liver cell lines, while Western blotting and flow cytometry evaluated apoptosis. The anticancer effect of MBP-11901 was verified in vitro through migration, invasion, colony formation, and JC-1 MMP assays. In mouse models, the tumor volume, tumor weight, and bodyweight were measured, and cancer cell proliferation and apoptosis were analyzed. The toxicity of MBP-11901 was investigated through GOT/GPT and histological analyses in the liver and kidney. The signaling mechanism of MBP-11901 was investigated through kinase assays, phosphorylation analysis, and in silico docking simulations. Results. MBP-11901 was effective against various human HCC cell lines, leading to the disappearance of most tumors when administered orally in animal models. This effect was dose-dependent, with no differences in efficacy according to administration intervals. MBP-11901 induced anticancer effects by targeting the signaling mechanisms of FLT3, VEGFR2, c-KIT, and PDGFRß. MBP-11901 is suggested as a novel therapeutic agent for the treatment of advanced or unresectable liver cancer.

The Synthesis, Characterization, Molecular Docking and In Vitro Antitumor Activity of Benzothiazole Aniline (BTA) Conjugated Metal-Salen Complexes as Non-Platinum Chemotherapeutic Agents.

Here, we describe the synthesis, characterization, and in vitro biological evaluation of a series of transition metal complexes containing benzothiazole aniline (BTA). We employed BTA, which is known for its selective anticancer activity, and a salen-type Schiff-based ligand to coordinate several transition metals to achieve selective and synergistic cytotoxicity. The compounds obtained were characterized by NMR spectroscopy, mass spectrometry, Fourier transform infrared spectroscopy, and elemental analysis. The compounds L, MnL, FeL, CoL, and ZnL showed promising in vitro cytotoxicity against cancer cells, and they had a lower IC50 than that of the clinically used cisplatin. In particular, MnL had synergistic cytotoxicity against liver, breast, and colon cancer cells. Moreover, MnL, CoL, and CuL promoted the production of reactive oxygen species in HepG2 tumor cell lines. The lead compound of this series, MnL, remained stable in physiological settings, and docking results showed that it interacted rationally with the minor groove of DNA. Therefore, MnL may serve as a viable alternative to platinum-based chemotherapy.

Synthesis, Characterization, and Anticancer Activity of Benzothiazole Aniline Derivatives and Their Platinum (II) Complexes as New Chemotherapy Agents.

We describe the synthesis, characterization, molecular modeling, and in vitro anticancer activity of three benzothiazole aniline (BTA) ligands and their corresponding platinum (II) complexes. We designed the compounds based on the selective antitumor properties of BTA, along with three types of metallic centers, aiming to take advantage of the distinctive and synergistic activity of the complexes to develop anticancer agents. The compounds were characterized using nuclear magnetic resonance spectrometry, Fourier transform infrared spectroscopy, mass spectrometry, elemental analysis, and tested for antiproliferative activity against multiple normal and cancerous cell lines. L1, L2, and L1Pt had better cytotoxicity in the liver, breast, lung, prostate, kidney, and brain cells than clinically used cisplatin. Especially, L1 and L1Pt demonstrated selective inhibitory activities against liver cancer cells. Therefore, these compounds can be a promising alternative to the present chemotherapy drugs.

Assessment of Image Quality for Selective Intracoronary Contrast-Injected CT Angiography in a Hybrid Angio-CT System: A Feasibility Study in Swine.

PURPOSE: To compare image quality in selective intracoronary contrast-injected computed tomography angiography (Selective-CTA) with that in conventional intravenous contrast-injected CTA (IV-CTA). MATERIALS AND METHODS: Six pigs (35 to 40 kg) underwent both IV-CTA using an intravenous injection (60 mL) and Selective-CTA using an intracoronary injection (20 mL) through a guide-wire during/after percutaneous coronary intervention. Images of the common coronary artery were acquired. Scans were performed using a combined machine comprising an invasive coronary angiography suite and a 320-channel multi-slice CT scanner. Quantitative image quality parameters of CT attenuation, image noise, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), mean lumen diameter (MLD), and mean lumen area (MLA) were measured and compared. Qualitative analysis was performed using intraclass correlation coefficient (ICC), which was calculated for analysis of interobserver agreement. RESULTS: Quantitative image quality, determined by assessing the uniformity of CT attenuation (399.06 vs. 330.21, p<0.001), image noise (24.93 vs. 18.43, p<0.001), SNR (16.43 vs. 18.52, p=0.005), and CNR (11.56 vs. 13.46, p=0.002), differed significantly between IV-CTA and Selective-CTA. MLD and MLA showed no significant difference overall (2.38 vs. 2.44, p=0.068, 4.72 vs. 4.95, p=0.078). The density of contrast agent was significantly lower for selective-CTA (13.13 mg/mL) than for IV-CTA (400 mg/mL). Agreement between observers was acceptable (ICC=0.79±0.08). CONCLUSION: Our feasibility study in swine showed that compared to IV-CTA, Selective-CTA provides better image quality and requires less iodine contrast medium.

Age-related isomerization of Asp in human immunoglobulin G kappa chain.

Isomerization of aspartate (Asp) is a common non-enzymatic posttranslational modification. Isomerized residues accumulate in proteins associated with age-related human disorders such as cataract and are well known to affect protein structure and function. We previously detected d-Asp-containing peptides in human serum. In this study, we investigated whether isomerized Asp residues are present in human immunoglobulin G (IgG) kappa chain by a qualitative d-amino acid analysis based on diastereomer formation and liquid chromatography tandem mass spectrometry (LC-MS/MS). We also investigated the d/l ratio of Asp residues in the IgG kappa chain in serum from donors aged 25, 37, 41, 54 and 67 years. As a result, two isomerized Asp residues, Asp151 and Asp170, were detected in the IgG kappa chain, and the d/l ratio of these residues was found to increase with aging. To assess the effects of this isomerization, we synthesized four isomeric peptides of IgG kappa chain containing lα-, lß-, dα-, or dß-Asp at position 170, and compared their secondary structures by CD spectroscopy. Peptide containing normal lα-Asp170 showed type II ß-turn structure, while the other isomeric peptides showed random structure, clearly indicating that substitution of a single Asp isomer alters the secondary structure of the peptide. Because IgG is a main component of humoral immunity, Asp isomerization in IgG may reflect changes of structure and decrease in immune function. Proteome research on serum from the standpoint of racemization might enable us to develop new kinds of biomarker and new directions to study the aging process.

Site-specific rapid deamidation and isomerization in human lens αA-crystallin in vitro.

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens-specific αA-crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA-crystallin with Asn by using site-directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA-crystallin was subjected to enzymatic digestion followed by liquid chromatography-MS/MS to evaluate the ratio of modifications in Asn151-containing peptides. The Asp151Asn αA-crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit-subunit interactions between αA-crystallin and αB-crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA-crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L-Asp to D-Asp residues in vivo.

Iodine staining as a useful probe for distinguishing insulin amyloid polymorphs.

It is recently suggested that amyloid polymorphism, i.e., structural diversity of amyloid fibrils, has a deep relationship with pathology. However, its prompt recognition is almost halted due to insufficiency of analytical methods for detecting polymorphism of amyloid fibrils sensitively and quickly. Here, we propose that iodine staining, a historically known reaction that was firstly found by Virchow, can be used as a method for distinguishing amyloid polymorphs. When insulin fibrils were prepared and iodine-stained, they exhibited different colors depending on polymorphs. Each of the colors was inherited to daughter fibrils by seeding reactions. The colors were fundamentally represented as a sum of three absorption bands in visible region between 400 and 750 nm, and the bands showed different titration curves against iodine, suggesting that there are three specific iodine binding sites. The analysis of resonance Raman spectra and polarization microscope suggested that several polyiodide ions composed of I3- and/or I5- were formed on the grooves or the edges of ß-sheets. It was concluded that the polyiodide species and conformations formed are sensitive to surface structure of amyloid fibrils, and the resultant differences in color will be useful for detecting polymorphism in a wide range of diagnostic samples.