A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes.

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalog consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio- and stereoselective ligand for the protein transmembrane protein 97 (Tmem97/the σ2 receptor), enabling us to reconstruct the 20(S)-OHC-Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.

Light-induced generation and toxicity of docosahexaenoate-derived oxidation products in retinal pigmented epithelial cells.

Oxidative cleavage of docosahexaenoate (DHA) in retinal pigmented epithelial (RPE) cells produces 4-hydroxy-7-oxohept-5-enoic acid (HOHA) esters of 2-lysophosphatidylcholine (PC). HOHA-PC spontaneously releases a membrane-permeant HOHA lactone that modifies primary amino groups of proteins and ethanolamine phospholipids to produce 2-(ω-carboxyethyl)pyrrole (CEP) derivatives. CEPs have significant pathological relevance to age-related macular degeneration (AMD) including activation of CEP-specific T-cells leading to inflammatory M1 polarization of macrophages in the retina involved in "dry AMD" and TLR2-dependent induction of angiogenesis that characterizes "wet AMD". RPE cells accumulate DHA from shed rod photoreceptor outer segments through phagocytosis and from plasma lipoproteins secreted by the liver through active uptake from the choriocapillaris. As a cell model of light-induced oxidative damage of DHA phospholipids in RPE cells, ARPE-19 cells were supplemented with DHA, with or without the lipofuscin fluorophore A2E. In this model, light exposure, in the absence of A2E, promoted the generation HOHA lactone-glutathione (GSH) adducts, depletion of intracellular GSH and a competing generation of CEPs. While DHA-rich RPE cells exhibit an inherent proclivity toward light-induced oxidative damage, photosensitization by A2E nearly doubled the amount of lipid oxidation and expanded the spectral range of photosensitivity to longer wavelengths. Exposure of ARPE-19 cells to 1 µM HOHA lactone for 24 h induced massive (50%) loss of lysosomal membrane integrity and caused loss of mitochondrial membrane potential. Using senescence-associated ß-galactosidase (SA ß-gal) staining that detects lysosomal ß-galactosidase, we determined that exposure to HOHA lactone induces senescence in ARPE-19 cells. The present study shows that products of light-induced oxidative damage of DHA phospholipids in the absence of A2E can lead to RPE cell dysfunction. Therefore, their toxicity may be especially important in the early stages of AMD before RPE cells accumulate lipofuscin fluorophores.

4-Hydroxy-7-oxo-5-heptenoic Acid Lactone Is a Potent Inducer of the Complement Pathway in Human Retinal Pigmented Epithelial Cells.

We previously discovered that oxidative cleavage of docosahexaenoate (DHA), which is especially abundant in the retinal photoreceptor rod outer segments and retinal pigmented endothelial (RPE) cells, generates 4-hydroxy-7-oxo-5-heptenoate (HOHA) lactone, and that HOHA lactone can enter RPE cells that metabolize it through conjugation with glutathione (GSH). The consequent depletion of GSH results in oxidative stress. We now find that HOHA lactone induces upregulation of the antioxidant transcription factor Nrf2 in ARPE-19 cells. This leads to expression of GCLM, HO1, and NQO1, three known Nrf2-responsive antioxidant genes. Besides this protective response, HOHA lactone also triggers a countervailing inflammatory activation of innate immunity. Evidence for a contribution of the complement pathway to age-related macular degeneration (AMD) pathology includes the presence of complement proteins in drusen and Bruch's membrane from AMD donor eyes, and the identification of genetic susceptibility loci for AMD in the complement pathway. In eye tissues from a mouse model of AMD, accumulation of complement protein in Bruch's membrane below the RPE suggested that the complement pathway targets this interface, where lesions occur in the RPE and photoreceptor rod outer segments. In animal models of AMD, intravenous injection of NaIO3 to induce oxidative injury selectively destroys the RPE and causes secretion of factor C3 from the RPE into areas directly adjacent to sites of RPE damage. However, a molecular-level link between oxidative injury and complement activation remained elusive. We now find that sub-micromolar concentrations of HOHA lactone foster expression of C3, CFB, and C5 in ARPE-19 cells and induce a countervailing upregulation of CD55, an inhibitor of C3 convertase production and complement cascade amplification. Ultimately, HOHA lactone causes membrane attack complex formation on the plasma membrane. Thus, HOHA lactone provides a molecular-level connection between free-radical-induced oxidative cleavage of DHA and activation of the complement pathway in AMD pathology.

Total Synthesis Confirms the Molecular Structure Proposed for Oxidized Levuglandin D2.

Levuglandins (LG)D2 and LGE2 are γ-ketoaldehyde levulinaldehyde derivatives with prostanoid side chains produced by spontaneous rearrangement of the endoperoxide intermediate PGH2 in the biosynthesis of prostaglandins. Covalent adduction of LGs with the amyloid peptide Aß1-42 promotes formation of the type of oligomers that have been associated with neurotoxicity and are a pathologic hallmark of Alzheimer's disease. Within 1 min of their generation during the production of PGH2 by cyclooxygenation of arachidonic acid, LGs are sequestered by covalent adduction to proteins. In view of this high proclivity for covalent adduction, it is understandable that free LGs have never been detected in vivo. Recently a catabolite, believed to be an oxidized derivative of LGD2 (ox-LGD2), a levulinic acid hydroxylactone with prostanoid side chains, was isolated from the red alga Gracilaria edulis and detected in mouse tissues and in the lysate of phorbol-12-myristate-13-acetate-treated THP-1 cells incubated with arachidonic acid. Such oxidative catabolism of LGD2 is remarkable because it must be outstandingly efficient to prevail over adduction with proteins and because it requires a unique dehydrogenation. We now report a concise total synthesis that confirms the molecular structure proposed for ox-LGD2. The synthesis also produces ox-LGE2, which readily undergoes allylic rearrangement to Δ6-ox-LGE2.

Distinguish different sensorimotor performance of the hand between the individuals with diabetes mellitus and chronic kidney disease through deep learning models.

Diabetes mellitus and chronic kidney disease represent escalating global epidemics with comorbidities akin to neuropathies, resulting in various neuromuscular symptoms that impede daily performance. Interestingly, previous studies indicated differing sensorimotor functions within these conditions. If assessing sensorimotor features can effectively distinguish between diabetes mellitus and chronic kidney disease, it could serve as a valuable and non-invasive indicator for early detection, swift screening, and ongoing monitoring, aiding in the differentiation between these diseases. This study classified diverse diagnoses based on motor performance using a novel pinch-holding-up-activity test and machine learning models based on deep learning. Dataset from 271 participants, encompassing 3263 hand samples across three cohorts (healthy adults, diabetes mellitus, and chronic kidney disease), formed the basis of analysis. Leveraging convolutional neural networks, three deep learning models were employed to classify healthy adults, diabetes mellitus, and chronic kidney disease based on pinch-holding-up-activity data. Notably, the testing set displayed accuracies of 95.3% and 89.8% for the intra- and inter-participant comparisons, respectively. The weighted F1 scores for these conditions reached 0.897 and 0.953, respectively. The study findings underscore the adeptness of the dilation convolutional neural networks model in distinguishing sensorimotor performance among individuals with diabetes mellitus, chronic kidney disease, and healthy adults. These outcomes suggest discernible differences in sensorimotor performance across the diabetes mellitus, chronic kidney disease, and healthy cohorts, pointing towards the potential of rapid screening based on these parameters as an innovative clinical approach.

Cysteinyl leukotriene-like metabolites are generated in retinal pigment epithelial cells through glutathionylation/reduction of an oxidatively truncated fragment of arachidonate.

γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of arachidonyl phospholipids by radical-induced oxidative cleavage. Metabolism of HNE by Michael addition of glutathione (GSH) followed by reduction of the aldehyde carbonyl produces a GSH derivative of 1,4-dihydroxynonane (DHN)-GSH. Analogous biochemistry was anticipated to produce a GSH derivative of 5,8-dihydroxyoctanoic acid (DHOA-GSH) that has structural and functional similarity to the cysteinyl leukotriene (LT)C4. We now report that exposure of human retinal pigment epithelial cells to CoCl2, an in vitro model of hypoxia-induced oxidative stress, generates DHOA-GSH and two products of its peptidolysis, DHOA-CysGly and DHOA-Cys that resemble LTD4 and LTE4. Identification of these metabolites was confirmed by unambiguous chemical syntheses that also provided a heavy isotope labeled quantitative standard 13C2 15N-DHOA-GSH. The availability of pure samples of these arachidonate metabolites will enable assessment of their biological activities, and testing the hypothesis that øLTs promote pathological inflammation by serving as LT receptor agonists. Because LT biosynthetic enzymes, e.g., 5-lipoxygenase, are not involved in the generation of øLTs in vivo, inhibitors of LT biosynthesis, e.g., Zileuton, are not expected to prevent the generation of øLTs. On the other hand, if øLTs are leukotriene receptor agonists, then the therapeutic effects of leukotriene receptor antagonist drugs, e.g., Montelukast, may include inhibition not only of LT-induced but also øLT-induced LT receptor activation and signaling.

Hedgehog proteins create a dynamic cholesterol interface.

During formation of the Hedgehog (Hh) signaling proteins, cooperative activities of the Hedgehog INTein (Hint) fold and Sterol Recognition Region (SRR) couple autoproteolysis to cholesterol ligation. The cholesteroylated Hh morphogens play essential roles in embryogenesis, tissue regeneration, and tumorigenesis. Despite the centrality of cholesterol in Hh function, the full structure of the Hint-SRR ("Hog") domain that attaches cholesterol to the last residue of the active Hh morphogen remains enigmatic. In this work, we combine molecular dynamics simulations, photoaffinity crosslinking, and mutagenesis assays to model cholesterolysis intermediates in the human Sonic Hedgehog (hSHH) protein. Our results provide evidence for a hydrophobic Hint-SRR interface that forms a dynamic, non-covalent cholesterol-Hog complex. Using these models, we suggest a unified mechanism by which Hh proteins can recruit, sequester, and orient cholesterol, and offer a molecular basis for the effects of disease-causing hSHH mutations.

4-Hydroxy-7-oxo-5-heptenoic acid lactone can induce mitochondrial dysfunction in retinal pigmented epithelial cells.

Oxidation of docosahexaenoate (DHA)-containing phospholipids in the cell plasma membrane leads to release of the α,ß-unsaturated aldehyde 4-hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone which is capable of inducing retinal pigmented epithelial (RPE) cell dysfunction. Previously, HOHA lactone was shown to induce apoptosis and angiogenesis, and to activate the alternative complement pathway. RPE cells metabolize HOHA lactone through enzymatic conjugation with glutathione (GSH). Competing with this process is the adduction of HOHA lactone to protein lysyl residues generating 2-(ω-carboxyethyl)pyrrole (CEP) derivatives that have pathological relevance to age-related macular degeneration (AMD). We now find that HOHA lactone induces mitochondrial dysfunction. It decreases ATP levels, mitochondrial membrane potentials, enzymatic activities of mitochondrial complexes, depletes GSH and induces oxidative stress in RPE cells. The present study confirmed that pyridoxamine and other primary amines, which have been shown to scavenge γ-ketoaldehydes formed by carbohydrate or lipid peroxidation, are ineffective for scavenging the α,ß-unsaturated aldehydes. Histidyl hydrazide (HH), that has both hydrazide and imidazole nucleophile functionalities, is an effective scavenger of HOHA lactone and it protects ARPE-19 cells against HOHA lactone-induced cytotoxicity. The HH α-amino group is not essential for this electrophile trapping activity. The Nα-acyl L-histidyl hydrazide derivatives with 2- to 7-carbon acyl groups with increasing lipophilicities are capable of maintaining the effectiveness of HH in protecting ARPE-19 cells against HOHA lactone toxicity, which potentially has therapeutic utility for treatment of age related eye diseases.

Classifying hand sensorimotor functions of the chronic kidney disease patients using novel manual tactile test and pinch-holding-up activity.

Hand function deterioration brings about inconvenience to the daily lives of the chronic kidney disease patients. However, a full spectrum of hand function examination is absent. Therefore, this study aimed to classify the hand sensorimotor functions of the chronic kidney disease patients using the novel sensorimotor assessment tools, manual tactile test (MTT) and pinch-holding-up activity (PHUA) test, and explore the feasibility in comparison with traditional evaluations in the clinical practice. 68 stage-5 chronic kidney disease patients and 50 healthy subjects were recruited in this study. A series of conventional evaluations and two novel hand function tools, manual tactile test and pinch-holding-up activity test were conducted from the perspective of hand dexterity, sensory input threshold, force generation and sensorimotor control. Independent t-test was used to find out group differences and the receiver operating characteristic curve was used to determine accuracy of the tests. In our results, significant reduction of hand dexterity, sensory input, force generation and sensorimotor control was found in patients from an overall perspective. This trend was discovered to be the same when dividing the subjects into the old and young age group. From the receiver operator characteristic curves, nearly all the areas under the curve of all tests were over 0.8. The novel evaluation tools, the manual tactile test and pinch-holding-up activity, were found to have comparable or even better accuracy than the traditional ones. The shape and weight subtests of the manual tactile test displayed the highest accuracy. To sum up, by incorporating the novel and conventional assessment tests, this study built up the fundamental understanding of the hand functions in multiple dimensions and consolidate the clinical merits of applying the two novel tools, manual tactile test and pinch-holding-up activity, on chronic kidney disease patients.

Kinetic analysis of canine gait on the effect of failure tendon repair and tendon graft.

Kinetic analysis of canine gait has been extensively studied, including normal and abnormal gait. However, no research has looked into how flexor tendon injury and further treatment would affect the walking pattern comparing to the uninjured state. Therefore, this study was aimed to utilize a portable pressure walkway system, which has been commonly used for pedobarographic and kinetic analysis in the veterinary field, to examine the effect of a failed tendon repair and tendon graft reconstruction on canine digit kinetics during gait. 12 mixed breed (mongrel) hound-type female dogs were included in this study and 2nd and 5th digits were chosen to undergo flexor tendon repair and graft surgeries. Kinetic parameters from the surgery leg in stance phase were calculated. From the results, after tendon failure repair, decrease of weight bearing was seen in the affected digits and weight bearing was shifted to the metacarpal pad. After tendon graft reconstruction, weight bearing returned to the affected digits and metacarpal pads. Slight alteration in peak pressure and instant of peak force were identified, but it was estimated to have little influence on post-reconstruction gait. This study could serve as a reference in evaluating canine digit function in flexor tendon injury for future studies.

Strengthening injectable thermo-sensitive NIPAAm-g-chitosan hydrogels using chemical cross-linking of disulfide bonds as scaffolds for tissue engineering.

In the present study, we fabricated non-toxic, injectable, and thermo-sensitive NIPAAm-g-chitosan (NC) hydrogels with thiol modification for introduction of disulfide cross-linking strategy. Previously, NIPAAm and chitosan copolymer has been proven to have excellent biocompatibility, biodegradability and rapid phase transition after injection, suitable to serve as cell carriers or implanted scaffolds. However, weak mechanical properties significantly limit their potential for biomedical fields. In order to overcome this issue, we incorporated thiol side chains into chitosan by covalently conjugating N-acetyl-cysteine (NAC) with carbodiimide chemistry to strengthen mechanical properties. After oxidation of thiols into disulfide bonds, modified NC hydrogels did improve the compressive modulus over 9 folds (11.4 kPa). Oscillatory frequency sweep showed a positive correlation between storage modulus and cross-liking density as well. Additionally, there was no cytotoxicity observed to mesenchymal stem cells, fibroblasts and osteoblasts. We suggested that the thiol-modified thermo-sensitive polysaccharide hydrogels are promising to be a cell-laden biomaterial for tissue regeneration.

Comprehensive simulation on morphological and mechanical properties of trigger finger - A cadaveric model.

Trigger finger has long been a common disorder in hand orthopedics. To clarify the unknown causative factors regarding the disease, numerous experiments were done on human cadavers, including tendon forces, tendon moment arm, mechanical properties of the pulley, gliding resistance, etc. However, most of these studies were conducted on normal fingers. As the etiology of trigger finger is still controversial on whether it is an outcome of tendon nodule or pulley scarring, in this study, a trigger finger model was built combining both the nodule created by silicone gel injection and pulley constriction by external compression. Indentation and gliding resistance tests were performed on cadaveric specimens to verify the model. Results showed that after silicone gel injection into the tendon, a significant increase in thickness was found. In addition, no significant difference was found in the toe region compressive modulus of the tendon after injection. Moreover, maximum, drop of gliding resistance and work of extension were all found to be significantly larger as the severity of triggering increased. Our results indicated we have developed a feasible cadaver model simulating trigger finger nodule which could be utilized for further experiments to elucidate other causative factors and biomechanical features of trigger finger in the future.

Ultrasound elastography for carpal tunnel pressure measurement: A cadaveric validation study.

Carpal tunnel pressure is a key factor in the etiology of carpal tunnel syndrome. Numerous approaches have been conducted to measure carpal tunnel pressure. However, most techniques are invasive and take time and effort. We have developed an innovative approach to noninvasively assess the tunnel pressure by using the ultrasound surface wave elastography (USWE) technique. In a previous study it was shown that the shear wave speed in a tendon increased linearly with increasing tunnel pressure enclosed the tendon in a simple tendon model. This study aimed to examine the relationship between the carpal tunnel pressure and the shear wave speeds inside and outside the carpal tunnel in a human cadaveric model. The result showed that the shear wave speed inside the carpal tunnel increased linearly with created carpal tunnel pressure, while the shear wave speed outside the carpal tunnel remained constant. These findings suggest that noninvasive measurement of carpal tunnel pressure is possible by measuring the shear wave speed in the tendon. After fully establishing this technology and being applicable in clinic, it would be useful in the diagnosis of carpal tunnel syndrome. For that reason, further validation with this technique in both healthy controls and patients with carpal tunnel syndrome is required. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:477-483, 2018.

The quantitative evaluation of the relationship between the forces applied to the palm and carpal tunnel pressure.

Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy occurring in upper limbs. The etiology, however, has not been fully understood yet. Median nerve could be compressed by either increase of carpal tunnel pressure (CTP) or direct impingement when it is forced toward to carpal ligament especially in wrist flexion leading to CTS development. Thus, the increase of carpal tunnel pressure is considered an important role in CTS development. It has been identified that forces applied to the palm would affect the CTP. However, the quantitative relationship between palmar contact force and CTP is not known. The purpose of this study was to quantitatively evaluate the relationship between palmar contact force and CTP. Eight human cadaveric hands were used. The CTP was measured with a diagnostic catheter-based pressure transducer inserted into the carpal tunnel. A custom made device was used to apply forces to the palm for the desired CTP. Palmar contact forces corresponding to the determined CTP level were recorded respectively. The testing was repeated with different ranges of tension applied to the flexor digitorum superficialis tendon of the third finger. The tensions were constant at 50 g for the other flexor tendons and median nerve. The results showed that CTP increased linearly with the force applied to the palm. When CTP was 30 mmHg, mean values of the contact force to the palm was 293 g (SD: 15.2) including all tensions. These results would help to understand the effect of daily activities with hands on CTP.

Comparison of two ways of altering carpal tunnel pressure with ultrasound surface wave elastography.

Higher carpal tunnel pressure is related to the development of carpal tunnel syndrome. Currently, the measurement of carpal tunnel pressure is invasive and therefore, a noninvasive technique is needed. We previously demonstrated that speed of wave propagation through a tendon in the carpal tunnel measured by ultrasound elastography could be used as an indicator of carpal tunnel pressure in a cadaveric model, in which a balloon had to be inserted into the carpal tunnel to adjust the carpal tunnel pressure. However, the method for adjusting the carpal tunnel pressure in the cadaveric model is not applicable for the in vivo model. The objective of this study was to utilize a different technique to adjust carpal tunnel pressure via pressing the palm and to validate it with ultrasound surface wave elastography in a human cadaveric model. The outcome was also compared with a previous balloon insertion technique. Results showed that wave speed of intra-carpal tunnel tendon and the ratio of wave speed of intra-and outer-carpal tunnel tendons increased linearly with carpal tunnel pressure. Moreover, wave speed of intra carpal tunnel tendon via both ways of altering carpal tunnel pressure showed similar results with high correlation. Therefore, it was concluded that the technique of pressing the palm can be used to adjust carpal tunnel pressure, and pressure changes can be detected via ultrasound surface wave elastography in an ex vivo model. Future studies will utilize this technique in vivo to validate the usefulness of ultrasound surface wave elastography for measuring carpal tunnel pressure.