Fish swim bladders as valve leaflets enhance the durability of transcatheter aortic valve replacement devices.

Transcatheter aortic valve replacement (TAVR) has emerged as an effective therapy for inoperable patients with severe aortic stenosis (AS). However, calcification-induced limited durability restricts its application. Fish swim bladders (FSB), which are resistant to calcific degeneration, offer a viable solution to this challenge. In this study, we developed a new TAVR device using FSB as the valve leaflet. Furthermore, the in vitro durability, in vivo performance, and size selection of this TAVR device were assessed by an experimental study and finite element analysis. A self-expandable TAVR device was fabricated by suturing the FSB films into a 23 mm nitinol alloy frame. Further, hemodynamic performance, such as effective orifice area, transvalvular pressure difference and regurgitant fraction, the durability was tested by the pulsatile flow test and accelerated fatigue test, according to the ISO 5840-3. The effect of release size on hydrodynamic performance was also investigated. Finally, the in vivo performance of the TAVR device were examined using a porcine implantation model. The results showed that the strength of the FSB films satisfied the requirements for valve leaflets. The hemodynamic performance of the FSB TAVR device met the requirements of ISO 5840-3 standards. After 400 million cycles, the FSB showed no fiber loss, torn, perforation, or other valve failure phenomena. In porcine models, the devices were well-positioned, functioned well with no stenosis immediately after the operation. Collectively, we successfully developed a TAVR device with FSB as valve leaflets that exhibited good fatigue resistance. STATEMENT OF SIGNIFICANCE: The source of material for the leaflets of commercialized biological heart valves (BHVs) is mainly bovine pericardium, but this material suffers the following problems: large and uneven thickness of the material, the presence of α-Gal and Neu5Gc antigens, and the susceptibility to structural valve degradation (SVD). New material for BHVs leaflets is rarely reported. In this study, we prepared a transcatheter aortic valve (TAV) and performed long-term in vitro and short-term in vivo studies using fish swim bladder (FSB) as the leaflets. The study confirmed that FSB TAV device can complete 400 million fatigue tests and maintain the good morphology of the leaflets, and that it still maintains good functionality after a certain amount of compression, indicating that FSB is a promising material for leaflets.

Highly efficient activation of periodate by a manganese-modified biochar to rapidly degrade methylene blue.

In this study, the manganese oxide/biochar composites (Mn@BC) were synthesized from Phytolacca acinosa Roxb. The Mn@BC was analyzed via techniques of Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction analysis (XRD). The results show that MnOx is successfully loaded on the surface of BC, and the load of MnOx can increase the number of surface functional groups of BC. X-ray photoelectron spectroscopy (XPS) shows that MnOx loaded on BC mainly exists in three valence forms: Mn(Ⅱ), Mn(Ⅲ), and Mn(Ⅳ). The ability of Mn@BC to activate periodate (PI) was studied by simulating the degradation of methylene blue (MB) dye. The degradation experiment results showed that the MB removal rate by the Mn@BC/PI system reached 97.4% within 30 min. The quenching experiment and electron paramagnetic resonance (EPR) analysis confirmed that Mn@BC can activate PI to produce iodate (IO3•), singlet oxygen (1O2), and hydroxyl radical (•OH), which can degrade MB during the reaction. Response surface methodology (RSM) based on Box-Behnken Design (BBD) was used to determine the interaction between pH, Mn@BC and PI concentration in the Mn@BC/PI system, and the optimum technological parameters were determined. When pH = 5.4, Mn@BC concentration 0.56 mg/L, PI concentration 1.1 mmol/L, MB removal rate can reach 98.05%. The cyclic experiments show that Mn@BC can be reused. After four consecutive runs, the removal rate of MB by the Mn@BC/PI system is still 82%, and the Mn@BC/PI system also shows high performance in treating MB in actual water bodies and degrading other pollutants. This study provides a practical method for degrading dyes in natural sewage.

Developing self-floating N-defective graphitic carbon nitride photocatalyst for efficient photodegradation of Microcystin-LR under visible light.

The frequent occurrence of algal blooms in water bodies leads to a significant accumulation of microcystin-LR (MC-LR). In this study, we developed a porous foam-like self-floating N-deficient g-C3N4 (SFGN) photocatalyst for efficient photocatalytic degradation of MC-LR. Both the characterization results and DFT calculations indicate that the surface defects and floating state of SFGN synergistically enhance light harvesting and photogenerated carrier migration rate. The photocatalytic process achieved a nearly 100 % removal rate of MC-LR within 90 min, while the self-floating state of SFGN maintained good mechanical strength. ESR and radical capture experiments revealed that the primary active species responsible for the photocatalytic process was OH. This finding confirmed that the fragmentation of MC-LR occurs as a result of OH attacking the MC-LR ring. LC-MS analysis indicated that majority of the MC-LR molecules were mineralized into small molecules, allowing us to infer possible degradation pathways. Furthermore, after four consecutive cycles, SFGN exhibited remarkable reusability and stability, highlighting the potential of floating photocatalysis as a promising technique for MC-LR degradation.

Biomimetic polymeric transcatheter heart valve leaflets with low calcification and good regenerative ability.

Transcatheter aortic valve replacement (TAVR) technology is quickly advancing in clinic, however, as it expands to low-risk populations and younger patients (age <65 years), device durability is becoming a major challenge. Tissue-engineered heart valves (TEHVs) are a potential alternative. In this study, a bionic tri-layer tissue-engineered heart valve was constructed using poly (L-lactate-co-ε-caprolactone) (PLCL), gelatin (GEL), hyaluronic acid (HA) and silk fibroin (SF), to simulate the fibrosa, spongiosa and ventricular layer of natural heart valves. To obtain a scaffold with sufficient strength and regenerative capacity, we optimized the ratio of components of each layer. The physical and mechanical properties were tested, and the cytocompatibility, calcification deposition and regeneration potential were tested in a rat model of subcutaneous implantation. Finally, the hydrodynamic function of the new TAVR device was verified. The results demonstrated that the strength of the tri-layer valve could reach up to 10 MPa, significantly higher than that of the PLCL and mono-layer groups. Most importantly, calcification related gene expression was down-regulated in the TEHV groups compared to valvular interstitial cells (VICs) treated with calcification induced medium, and calcification levels of TEHVs in in vivo assay were below 0.5 µg mg-1. Besides, we found HA in the middle layer was very conducive to rapid cell infiltration and good angiogenesis, which ultimately promoted host tissue regeneration at 8 weeks after implantation. Collectively, we provide a bionic tri-layer electrospun leaflet with appropriate mechanical strength, low calcification and good regenerative capacity, which has great potential as a TEHV leaflet.

A Thermostable Type I Collagen from Swim Bladder of Silver Carp (Hypophthalmichthys molitrix).

As a major component of the extracellular matrix, collagen has been used as a biomaterial for many purposes including tissue engineering. Commercial collagen derived from mammals is associated with a risk of prion diseases and religious restrictions, while fish-derived collagen can avoid such issues. In addition, fish-derived collagen is widely available and low-cost; however, it often suffers from poor thermal stability, which limits its biomedical application. In this study, collagen with a high thermal stability was successfully extracted from the swim bladder of silver carp (Hypophthalmichthys molitrix) (SCC). The results demonstrated that it was a type I collagen with high purity and well-preserved triple-helix structure. Amino acid composition assay showed that the amounts of threonine, methionine, isoleucine and phenylalanine in the collagen of swim bladder of silver carp were higher than those of bovine pericardium. After adding salt solution, swim-bladder-derived collagen could form fine and dense collagen fibers. In particular, SCC exhibited a higher thermal denaturation temperature (40.08 °C) compared with collagens from the swim bladder of grass carp (Ctenopharyngodon idellus) (GCC, 34.40 °C), bovine pericardium (BPC, 34.47 °C) and mouse tail (MTC, 37.11 °C). Furthermore, SCC also showed DPPH radical scavenging ability and reducing power. These results indicate that SCC presents a promising alternative source of mammalian collagen for pharmaceutical and biomedical applications.

The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2.

Centromere protein A (CENP-A) nucleosomes containing the centromere-specific histone H3 variant CENP-A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing the CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and the RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression.

Immunogenicity assessment of swim bladder-derived biomaterials.

Fish swim bladder-derived biomaterials are prospective cardiovascular materials due to anti-calcification, adequate mechanical properties, and good biocompatibility. However, their immunogenic safety profile, which primarily determines their feasibility as medical devices in clinical practice, remains unknown. Herein, the immunogenicity of glutaraldehyde crosslinked fish swim bladder (Bladder-GA) and un-crosslinked swim bladder (Bladder-UN) samples was examined using in vitro and in vivo assays according to ISO 10993-20. The in vitro splenocyte proliferation assay showed that cell growth was lower in the extract medium of Bladder-UN and Bladder-GA, compared to the LPS-or Con A-treated group. Similar results were obtained in in vivo assays. In the subcutaneous implantation model, the thymus coefficient, spleen coefficient and ratio of immune cell subtypes showed no significant difference between the bladder groups and the sham group. In terms of the humoral immune response, the total IgM concentration was lower in the Bladder-GA and Bladder-UN groups (988 ± 238 µg ml-1 and 1095 ± 296 µg ml-1, respectively) than that in the sham group (1329 ± 132 µg ml-1) at 7 days. The total IgG concentrations were 422 ± 78 µg ml-1 in Bladder-GA and 469 ± 172 µg ml-1 in Bladder-UN at 30 days, which were slightly higher than that in the sham group (276 ± 95 µg ml-1) but there was no significant difference compared with Bovine-GA (468 ± 172 µg ml-1), indicating that these materials did not elicit a strong humoral immune response. Systemic immune response-related cytokines and C-reactive protein were stable during implantation, while IL-4 levels increased with time. The classical foreign body response was not observed around all the implants, and the ratio of CD163+/iNOS macrophages in Bladder-GA and Bladder-UN was higher than that in the Bovine-GA group at the implanted site at 7 and 30 days. Finally, no organ toxicity was observed in any of the groups. Collectively, the swim bladder-derived material did not elicit significant aberrant immune responses in vivo, giving strong confidence for its application in tissue engineering or medical devices. Furthermore, more dedicated research on immunogenic safety assessment in large animal models is encouraged to facilitate the clinical practice of swim bladder-derived materials.

Nitrogen and Phosphorus Co-doped Porous Carbon for High-Performance Supercapacitors.

As one of the most promising fast energy storage devices, supercapacitor has been attracting intense attention for many emerging applications. However, how to enhance the electrochemical performance of electrode materials is still the main issue among various researches. In this paper, hierarchical porous carbons derived from Eleocharis dulcis has been prepared by chemical activation process with the aid of KOH at elevated temperature. Results show that the N, P co-doped porous carbon exhibits excellent electrochemical performance, it owns a specific capacitance of 340.2 F/g at 1 A/g, and obtains outstanding cycling stability of 96.9% of capacitance retention at 10 A/g after 5,000 cycles in a three-electrode system. Moreover, in the two-electrode system, the product still maintains a high specific capacitance of 227.2 F/g at 1 A/g, and achieves good electrochemical cycle stability (94.2% of capacitance retention at 10 A/g after 10,000 cycles); besides, its power/energy density are 3694.084 and 26.289 Wh/kg, respectively. Therefore, the combination of facile synthesis strategy and excellent electrochemical performance makes Eleocharis dulcis-based porous carbon as a promising electrode material for supercapacitor.

Effects of Enhanced Recovery After Surgery in Total Knee Arthroplasty for Patients Older Than 65 Years.

OBJECTIVES: To explore the safety and efficacy of the enhanced recovery after surgery (ERAS) program for elderly total knee arthroplasty (TKA) patients. METHODS: A prospective controlled study was conducted for patients older than 65 years, who would undergo unilateral TKA with a minimum follow-up of 2 years. Patients were divided into an ERAS group (n = 106) and a traditional group (n = 141) based on the patients' willingness to participate in the ERAS program. Baseline parameters of American Society of Anesthesiologists classification and comorbidity were recorded. Complication, mortality, knee function assessment using knee society score and knee range of motion, and perioperative clinical outcomes were compared between the two groups. RESULTS: There were no significant differences between the two groups in terms of baseline parameters. Although no significant differences were found in postoperative nausea and vomiting, urinary tract infection, deep venous thrombosis, pulmonary embolism, wound delayed healing, superficial infection, and deep infection, there were significantly fewer total complications in the ERAS group (26/106 vs 52/141; P = 0.039). No significant difference was found in short-term mortality (1/106 vs 3/141; P = 0.836) between the two groups. There were no significant differences in preoperative visual analogue scale (VAS), knee society score (KSS), and range of motion (ROM) between the two groups. Lower VAS scores were found in the ERAS group at time of postoperative day (POD) 1 (P = 0.012) and POD 5 (P = 0.020); no significant differences were observed at time of postoperative month (POM) 1 and final follow-up. Higher KSS scores were found in the ERAS group at time of POD 1 (P = 0.013), and POD 5 (P = 0.011), no significant differences were observed at time of POM 1 and final follow-up. Increased ROM degree was found in the ERAS group at time of POD 1 (P = 0.021); no significant differences were observed at time of POD 5, POM 1 and final follow-up. Decreased intraoperative blood loss (P < 0.001), total blood loss (P < 0.001), transfusion rate (P = 0.004), and length of stay (P < 0.001) were found in the ERAS group; no significant differences were found in operative time and hospitalization costs between the two groups. CONCLUSION: The ERAS program is safer and more efficacious in elderly TKA patients compared to the traditional pathway. It could effectively relieve perioperative pain and improve joint function, and reduce blood transfusion, length of stay, and total complications without increasing short-term mortality.

In vitro study of anti-ER positive breast cancer effect and mechanism of 1,2,3,4-6-pentyl-O-galloyl-beta-d-glucose (PGG).

Breast cancer is one of the most common malignancies and the leading cause of women's death, most of breast cancers are estrogen receptor-positive (ER+) breast cancer which can develop into advanced stage from early stage with treatment resistance. The purpose of this study was to investigate anti-ER+ breast cancer effects of 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) and its possible mechanisms. Cell viability was analyzed by MTT assay. The cell cycle distribution and apoptosis were detected by Flow cytometry. The expressions of cell proliferation- and apoptosis-related proteins were determined by western blot and immunofluorescence staining. The results showed PGG induced cytotoxicity and decreased viability of ER+ breast cancer T-47D and BT-474 cells. Flow cytometry analysis revealed that cell cycle was blocked in S phase at lower dose (25 µM PGG), and G1 phase at higher dose (50 or 75 µM PGG). One of the underlying mechanisms of the anti-cancer effect exerted by PGG was owed to inhibition of the expression of HURP, an up-regulated protein in human hepatocellular carcinoma which is closely related to tumor proliferation, invasion and metastasis. PGG affected cell cycle- or apoptosis-related proteins such as cyclin D1, Bcl-2 and Bax. These data suggest that PGG exerts anti-ER+ breast cancer effects. In this sense, our study provides new alternative therapies to treat breast cancer.

Change of miRNA expression profiles in patients with knee osteoarthritis before and after celecoxib treatment.

BACKGROUND: This study aimed to investigate the change of circulating miRNA expression profiles in knee osteoarthritis (OA) patients before and after celecoxib treatment. METHODS: Two hundred and eighteen knee OA patients underwent celecoxib treatment for 6 weeks were enrolled. Plasma samples were obtained at baseline (W0) and at W6, and treatment efficacy were assessed by WOMAC index. In the exploration stage, miRNA expression profiles in plasma before and after treatment from 6 patients were detected by microarray. Subsequently, in the validation stage, 10 top differentially expressed miRNAs (DEMs) after and before treatment in microarray were further validated in all 218 patients by qPCR. RESULTS: In the exploration stage, patients after treatment could be distinguished from them before treatment by miRNAs expression profiles by PCA plot and heatmap analysis, and 45 up-regulated and 48 down-regulated miRNAs were identified by volcano plot. In the validation stage, miR-126-5p and miR-320a levels increased at W6 compared to W0, while miR-155-5p and miR-146a-5p levels decreased. WOMAC pain/stiffness/physical function scores were all decreased at W6 compared to W0, and 71% of patients achieved clinical response. The increase of miR-126-5p expression (W6-W0) in clinical responders was much larger compared to nonclinical responders. And miRNA-320a level declined in nonclinical responders while increased in clinical responders. Conversely, miRNA-146a-5p level increased in nonclinical responders while decreased in clinical responders. CONCLUSION: Circulating miRNA expression profiles act as important roles in knee OA patients underwent celecoxib treatment, and miR-126-5p, miR-320a as well as miR-146a-5p might correlate with treatment response to celecoxib.

Spatial-temporal dynamics of carbon emissions and carbon sinks in economically developed areas of China: a case study of Guangdong Province.

This study analysed spatial-temporal dynamics of carbon emissions and carbon sinks in Guangdong Province, South China. The methodology was based on land use/land cover data interpreted from continuous high-resolution satellite images and energy consumption statistics, using carbon emission/sink factor method. The results indicated that: (1) From 2005 to 2013, different land use/land cover types in Guangdong experienced varying degrees of change in area, primarily the expansion of built-up land and shrinkage of forest land and grassland; (2) Total carbon emissions increased sharply, from 76.11 to 140.19 TgC yr-1 at the provincial level, with an average annual growth rate of 10.52%, while vegetation carbon sinks declined slightly, from 54.52 to 53.20 TgC yr-1. Both factors showed significant regional differences, with Pearl River Delta and North Guangdong contributing over 50% to provincial carbon emissions and carbon sinks, respectively; (3) Correlation analysis showed social-economic factors (GDP per capita and permanent resident population) have significant positive impacts on carbon emissions at the provincial and city levels; (4) The relationship between economic growth and carbon emission intensity suggests that carbon emission efficiency in Guangdong improves with economic growth. This study provides new insight for Guangdong to achieve carbon reduction goals and realize low-carbon development.

The Prevalence and Distribution of Vertebral Pedicles in Adolescent Idiopathic Scoliosis in Chinese people: A Computed Tomography-Based Study of 2958 Vertebral Pedicles.

OBJECTIVE: To explore the prevalence and distribution of abnormal vertebral pedicles in adolescent idiopathic scoliosis (AIS) in Chinese people. METHODS: We retrospectively reviewed AIS patients at a single institution between 2011 and 2017. Transverse pedicle widths from T1 to L5 were measured carefully using computed tomography, including cancellous and cortical channels. Pedicle morphology was classified as: type A, a cancellous channel larger than 4 mm; type B, a cancellous channel measuring 2-4 mm; type C, a cancellous channel smaller than 2 mm with an entirely cortical channel of 2 mm or greater; or type D, a cortical channel smaller than 2 mm. Types B, C, and D were defined as abnormal. Prevalence and distribution of abnormal pedicles were assessed. RESULTS: Eighty-seven patients with AIS, with a total of 2958 vertebral pedicles, were carefully measured and classified. The total prevalence of abnormal vertebral pedicles was as high as 65%, with type B comprising 40%, type C comprising 23%, and type D comprising 2%. Pedicles were located between T2 and T10 in 84% of type C and 91% of type D cases. Female sex, proximal thoracic location, major curve greater than 70 degrees, and concave pedicle may be risk factors for type C and D pedicles. CONCLUSIONS: There is a significantly high prevalence of abnormal pedicles in AIS in Chinese people, with a total prevalence of 65%. Female sex, proximal thoracic location, major curve greater than 70 degrees, and concave pedicle may be risk factors for type C and D pedicles.

Influence of screw density on thoracic kyphosis restoration in hypokyphotic adolescent idiopathic scoliosis.

BACKGROUND: Previous studies have reported that rod composition and diameter, as well as the correction technique are key factors associated with thoracic kyphosis (TK) restoration. However, few study has analyzed the correlation between screw density and TK restoration in hypokyphotic adolescent idiopathic scoliosis (AIS). METHODS: Fifty-seven thoracic AIS patients with preoperative TK < 10° treated with all pedicle screw fixation with a minimum 2-year follow-up were recruited. Preoperative and postoperative radiographic measurements, and information of posterior instrumentation were reviewed. Pearson and Spearman correlation coefficient analysis were used to assess relationships between change in TK and number of variables. Then, the included patients were classified into two groups (Group 1: postoperative TK ≥ 20°; Group 2: postoperative TK < 20°) to evaluate the influence factors of TK restoration. RESULTS: The average preoperative TK was 4.75°, which was significantly restored to 17.30° (P < 0.001). Significant correlations were found between change in TK and flexibility of major thoracic curve (r = 0.357, P = 0.006), preoperative TK (r = -0.408, P = 0.002), and screw density of concave side (r = 0.306, P = 0.021), respectively. In the subgroup comparison, 17 patients (29.8%) maintain the postoperative TK ≥ 20°, increased flexibility of major thoracic curve (P < 0.001), screw number of concave side (P = 0. 029), and cobalt chromium rods (P = 0.041) were found in the group of postoperative TK ≥ 20°. CONCLUSIONS: TK restoration remains a challenge for AIS patients with hypokyphosis, especially for the poor flexibility ones. Except for thicker and cobalt chromium rods, screw density of concave side might be another positive predictor of restoring normal kyphosis, which provides a stronger corrective force on the sagittal plane with more pedicle screws.

Comparison of low density and high density pedicle screw instrumentation in Lenke 1 adolescent idiopathic scoliosis.

BACKGROUND: The correlation between implant density and deformity correction has not yet led to a precise conclusion in adolescent idiopathic scoliosis (AIS). The aim of this study was to evaluate the effects of low density (LD) and high density (HD) pedicle screw instrumentation in terms of the clinical, radiological and Scoliosis Research Society (SRS)-22 outcomes in Lenke 1 AIS. METHODS: We retrospectively reviewed 62 consecutive Lenke 1 AIS patients who underwent posterior spinal arthrodesis using all-pedicle screw instrumentation with a minimum follow-up of 24 months. The implant density was defined as the number of screws per spinal level fused. Patients were then divided into two groups according to the average implant density for the entire study. The LD group (n = 28) had fewer than 1.61 screws per level, while the HD group (n = 34) had more than 1.61 screws per level. The radiographs were analysed preoperatively, postoperatively and at final follow-up. The perioperative and SRS-22 outcomes were also assessed. Independent sample t tests were used between the two groups. RESULTS: Comparisons between the two groups showed no significant differences in the correction of the main thoracic curve and thoracic kyphosis, blood transfusion, hospital stay, and SRS-22 scores. Compared with the HD group, there was a decreased operating time (278.4 vs. 331.0 min, p = 0.004) and decreased blood loss (823.6 vs. 1010.9 ml, p = 0.048), pedicle screws needed (15.1 vs. 19.6, p < 0.001), and implant costs ($10,191.0 vs. $13,577.3, p = 0.003) in the LD group. CONCLUSIONS: Both low density and high density pedicle screw instrumentation achieved satisfactory deformity correction in Lenke 1 AIS patients. However, the operating time and blood loss were reduced, and the implant costs were decreased with the use of low screw density constructs.

Solvothermal synthesis of hydroxyapatite nanorods with assistance of green polymer.

Development of low cost, environmental friendly crystal growth modifying reagents that are effective to HAp's microstructure regulation is highly crucial. Herein, sodium lignin sulfonate (SS), a byproduct of the pulp process, was used for preparing HAp nanorods. Results showed that both stirring time of reaction solution and the concentration of SS affected crystals' structure and aspect ratio. The growth mechanism was discussed on the basis of the results. And we hope this new synthetic strategy will offer an attractive route for the design of functional HAp nano-/micro- materials with fine structure.

Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin ß2 on Monocytes via NADPH Oxidase.

Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin ß2. Flow cytometry validated the activation of LFA-1 (αLß2) and Mac-1 (αMß2), two subfamilies of integrin ß2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

Active noise cancellation using MEMS accelerometers for motion-tolerant wearable bio-sensors.

An active noise cancellation method using a MEMS accelerometer is developed for recovering corrupted wearable sensor signals due to body motion. The method is developed for a finger ring PPG sensor, the signal of which is susceptive to the hand motion of the wearer. A MEMS accelerometer (ACC) imbedded in the PPG sensor detects the hand acceleration, and is used for recovering the corrupted PPG signal. The correlation between the acceleration and the distorted PPG signal is analyzed, and a low-order FIR model relating the signal distortion to the hand acceleration is obtained. The model parameters are identified in real time with a recursive least square method. Experiments show that the active noise cancellation method can recover ring PPG sensor signals corrupted with 2G of acceleration in the longitudinal direction of the digital artery.