Hesperetin derivative 2a inhibits lipopolysaccharide-induced acute liver injury in mice via downregulation of circDcbld2.

Acute liver injury (ALI) is a complex, life-threatening inflammatory liver disease, and persistent liver damage leads to rapid decline and even failure of liver function. However, the pathogenesis of ALI is still not fully understood, and no effective treatment has been discovered. Recent evidence shows that many circular RNAs (circRNAs) are associated with the occurrence of liver diseases. In this study we investigated the mechanisms of occurrence and development of ALI in lipopolysaccharide (LPS)-induced ALI mice. We found that expression of the circular RNA circDcbld2 was significantly elevated in the liver tissues of ALI mice and LPS-treated RAW264.7 cells. Knockdown of circDcbld2 markedly alleviates LPS-induced inflammatory responses in ALI mice and RAW264.7 cells. We designed and synthesized a series of hesperidin derivatives for circDcbld2, and found that hesperetin derivative 2a (HD-2a) at the concentrations of 2, 4, 8 µM effectively inhibited circDcbld2 expression in RAW264.7 cells. Administration of HD-2a (50, 100, 200 mg/kg. i.g., once 24 h in advance) effectively relieved LPS-induced liver dysfunction and inflammatory responses. RNA sequencing analysis revealed that the anti-inflammatory and hepatoprotective effects of HD-2a were mediated through downregulating circDcbld2 and suppressing the JAK2/STAT3 pathway. We conclude that HD-2a downregulates circDcbld2 to inhibit the JAK2/STAT3 pathway, thereby inhibiting the inflammatory responses in ALI. The results suggest that circDcbld2 may be a potential target for the prevention and treatment of ALI, and HD-2a may have potential as a drug for the treatment of ALI.

Vesicoprostatic muscle reconstruction: a step further for immediate and early urinary continence.

OBJECTIVES: The vesicoprostatic muscle (VPM) is a longitudinal smooth muscle that originates from the trigone of the bladder or the opening of the ureter and is involved in urination as part of the detrusor apron. We explored the effect of VPM reconstruction on immediate and early recovery of urinary continence in patients undergoing robot-assisted laparoscopic radical prostatectomy (RALP). PATIENTS AND METHODS: A total of 523 patients with localized prostate cancer were enrolled from June 2018 to June 2020. All patients were diagnosed in our department based on magnetic resonance imaging and pathological findings on prostate biopsy. After 1:1 propensity score matching, 105 patient pairs were matched. The study was approved by our institutional review board and all surgeries were performed by three experienced high-volume surgeons. Demographic data, total operation time, pathological outcomes, the urinary continence rates of the two groups at different times after RALP, and factors influencing postoperative urinary continence after RALP were recorded. Student's t test was used to compare continuous variables and the Pearson χ2 test to compare categorical variables. Logistic regression analysis was used to identify factors affecting immediate and early postoperative urinary control. RESULTS: VPM reconstruction promoted immediate and early recovery of urinary continence (immediate continence, 66.67 vs. 40.00%, P = 0.000; 3-month continence, 80.95 vs. 64.76%, P = 0.008). CONCLUSIONS: VPM reconstruction improved immediate and early urinary continence in patients who underwent RALP.

Physiologically based pharmacokinetic modeling and simulations to inform dissolution specifications and clinical relevance of release rates on elagolix exposure.

The aim of this analysis was to use a physiologically based pharmacokinetic (PBPK) model to predict the impact of changes in dissolution rates on elagolix exposures and define clinically relevant acceptance criteria for dissolution. Varying in vitro dissolution profiles were utilized in a PBPK model to describe the absorption profiles of elagolix formulations used in Phase 3 clinical trials and for the to be marketed commercial formulations. Single dose studies of 200 mg elagolix formulations were used for model verification under fasted conditions. Additional dissolution scenarios were evaluated to assess the impact of dissolution rates on elagolix exposures. Compared to the Phase 3 clinical trial formulation, sensitivity analysis on dissolution rates suggested that a hypothetical scenario of ∼75% slower dissolution rate would result in 14% lower predicted elagolix plasma exposures, however, the predicted exposures are still within the bioequivalence boundaries of 0.8-1.25 for both Cmax and AUC. A clinically verified PBPK model of elagolix was utilized to evaluate the impact of wider dissolution specifications on elagolix plasma exposures. The simulation results indicated that a slower in vitro dissolution profile, would not have a clinically significant impact on elagolix exposures. These model results informed the setting of wider dissolution specifications without requiring in vivo studies.

Applications of bio-predictive dissolution tools for the development of solid oral dosage forms: current industry experience.

Development and optimization of orally administered drug products often require bio-predictive tools to help with informing formulation and manufacturing decisions. Reliable bio-predictive dissolution toolkits not only allow rational development of target formulations without having to conduct excessive in vivo studies but also help in detecting critical material attributes (CMAs), critical formulation variables (CFVs), or critical process parameters (CPPs) that could impact a drug's in vivo performance. To provide early insights for scientists on the development of a bio-predictive method for drug product development, this review summarizes current phase-appropriate bio-predictive dissolution approaches applicable to address typical concerns on solubility-limited absorption, food effect, achlorhydria, development of extended-release formulation, clinically relevant specification, and biowaiver. The selection of an in vitro method which can capture the key rate-limiting step(s) of the in vivo dissolution and/or absorption is considered to have a better chance to produce a meaningful in vitro-in vivo correlation (IVIVC) or in vitro-in vivo relationship (IVIVR).

Amelioration of bone fragility by pulsed electromagnetic fields in type 2 diabetic KK-Ay mice involving Wnt/ß-catenin signaling.

Type 2 diabetes mellitus (T2DM) results in compromised bone microstructure and quality, and subsequently increased risks of fractures. However, it still lacks safe and effective approaches resisting T2DM bone fragility. Pulsed electromagnetic fields (PEMFs) exposure has proven to be effective in accelerating fracture healing and attenuating osteopenia/osteoporosis induced by estrogen deficiency. Nevertheless, whether and how PEMFs resist T2DM-associated bone deterioration remain not fully identified. The KK-Ay mouse was used as the T2DM model. We found that PEMF stimulation with 2 h/day for 8 wk remarkably improved trabecular bone microarchitecture, decreased cortical bone porosity, and promoted trabecular and cortical bone material properties in KK-Ay mice. PEMF stimulated bone formation in KK-Ay mice, as evidenced by increased serum levels of bone formation (osteocalcin and P1NP), enhanced bone formation rate, and increased osteoblast number. PEMF significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. PEMF exerted beneficial effects on osteoblast- and osteocyte-related gene expression in the skeleton of KK-Ay mice. Nevertheless, PEMF exerted no effect on serum biomarkers of bone resorption (TRAcP5b and CTX-1), osteoclast number, or osteoclast-specific gene expression (TRAP and cathepsin K). PEMF upregulated gene expression of canonical Wnt ligands (including Wnt1, Wnt3a, and Wnt10b), but not noncanonical Wnt5a. PEMF also upregulated skeletal protein expression of downstream p-GSK-3ß and ß-catenin in KK-Ay mice. Moreover, PEMF-induced improvement in bone microstructure, mechanical strength, and bone formation in KK-Ay mice was abolished after intragastric administration with the Wnt antagonist ETC-159. Together, our results suggest that PEMF can improve bone microarchitecture and quality by enhancing the biological activities of osteoblasts and osteocytes, which are associated with the activation of the Wnt/ß-catenin signaling pathway. PEMF might become an effective countermeasure against T2DM-induced bone deterioration.NEW & NOTEWORTHY PEMF improved trabecular bone microarchitecture and suppressed cortical bone porosity in T2DM KK-Ay mice. It attenuated T2DM-induced detrimental consequence on trabecular and cortical bone material properties. PEMF resisted bone deterioration in KK-Ay mice by enhancing osteoblast-mediated bone formation. PEMF also significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. The therapeutic potential of PEMF on T2DM-induced bone deterioration was associated with the activation of Wnt/ß-catenin signaling.

Differential skeletal response in adult and aged rats to independent and combinatorial stimulation with pulsed electromagnetic fields and mechanical vibration.

Pulsed electromagnetic fields (PEMFs) and whole-body vibration (WBV) are proved to partially preserve bone mass/strength in hindlimb-unloaded and ovariectomized animals. However, the potential age-dependent skeletal response to either PEMF or WBV has not been fully investigated. Moreover, whether the coupled "mechano-electro-magnetic" signals can induce greater osteogenic potential than single stimulation remains unknown. Herein, 5-month-old or 20-month-old rats were assigned to the Control, PEMF, WBV, and PEMF + WBV groups. After 8-week treatment, single PEMF/WBV enhanced bone mass, strength, and anabolism in 5-month-old rats, but not in 20-month-old rats. PEMF + WBV induced greater increase of bone quantity, quality, and anabolism than single PEMF/WBV in young adult rats. PEMF + WBV also inhibited bone loss in elderly rats by primarily improving osteoblast and osteocyte activity, but had no effects on bone resorption. PEMF + WBV upregulated the expression of various canonical Wnt ligands and downstream molecules (p-GSK-3ß and ß-catenin), but had no impacts on noncanonical Wnt5a expression in aged skeleton, revealing the potential involvement of canonical Wnt signaling in bone anabolism of PEMF + WBV. This study not only reveals much weaker responsiveness of aged skeleton to single PEMF/WBV relative to young adult skeleton, but also presents a novel noninvasive approach based on combinatorial treatment with PEMF + WBV for improving bone health and preserving bone quantity/quality (especially for age-related osteoporosis) with stronger anabolic effects.

Spatiotemporal characterization of microdamage accumulation and its targeted remodeling mechanisms in diabetic fatigued bone.

The skeleton of type 1 diabetes mellitus (T1DM) has deteriorated mechanical integrity and increased fragility, whereas the mechanisms are not fully understood. Load-induced microdamage naturally occurs in bone matrix and can be removed by initiating endogenous targeted bone remodeling. However, the microdamage accumulation in diabetic skeleton and the corresponding bone remodeling mechanisms remain poorly understood. Herein, streptozotocin-induced T1DM rats and age-matched non-diabetic rats were subjected to daily uniaxial ulnar loading for 1, 4, 7, and 10 days, respectively. The SPECT/CT and basic fuchsin staining revealed significant higher-density spatial accumulation of linear and diffuse microdamage in diabetic ulnae than non-diabetic ulnae. Linear microcracks increased within 10-day loading in diabetic bone, whereas peaked at Day 7 in non-diabetic bone. Moreover, diabetic fatigued ulnae had more severe disruptions of osteocyte canaliculi around linear microcracks. Immunostaining results revealed that diabetes impaired targeted remodeling in fatigued bone at every key stage, including increased apoptosis of bystander osteocytes, decreased RANKL secretion, reduced osteoclast recruitment and bone resorption, and impaired osteoblast-mediated bone formation. This study characterizes microdamage accumulation and abnormal remodeling mechanisms in the diabetic skeleton, which advances our etiologic understanding of diabetic bone deterioration and increased fragility from the aspect of microdamage accumulation and bone remodeling.

The interactions between mTOR and NF-κB: A novel mechanism mediating mechanical stretch-stimulated osteoblast differentiation.

Mechanical stretch is known to promote osteoblast differentiation in vitro and accelerate bone regeneration in vivo, whereas the relevant mechanism remains unclear. Recent studies have shown the importance of reciprocal interactions between mammalian target of rapamycin (mTOR) and nuclear factor kappa B (NF-κB; two downstream molecules of Akt) in the regulation of tumor cells. Thus, we hypothesize that mTOR and NF-κB as well as their interconnection play a critical role in mediating stretch-induced osteogenic differentiation in osteoblasts. We herein found that mechanical stretch (10% elongation at six cycles/min) significantly promoted the expression of osteoblast differentiation-related markers (including ALP, BMP2, Col1α, OCN, and Runx2) in osteoblast-like MG-63 cells, accompanied by increased mTOR phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. Blockade of mTOR by antagonist or small interfering RNA suppressed osteogenesis-related gene expression in response to mechanical stretch, whereas inhibition of NF-κB further increased stretch-induced osteoblast differentiation. Moreover, inhibition of mTOR decreased the phosphorylation of NF-κB, and blockade of NF-κB reduced the mTOR activation in MG63 cells under mechanical stretch. Coinhibition of mTOR and NF-κB abolishes the alteration of osteogenic differentiation induced by single mTOR or NF-κB inhibition under mechanical stretch, which is equivalent to the noninhibition level for osteoblasts under mechanical stretch. The expression levels of osteogenic differentiation in osteoblasts after inhibition of Akt were similar to those after co-inhibition of mTOR and NF-κB under mechanical stretch. This study for the first time reveals the reciprocal interconnection between mTOR and NF-κB in osteoblasts under mechanical stretch and indicates that mTOR and NF-κB as well as their interactions play a key role in the regulation of cellular homeostasis of osteoblasts in response to mechanical stretch. These findings are helpful for enriching our basic knowledge of the molecular mechanisms of osteoblast mechanotransduction, and also providing insight into the clinical therapeutic modality associated with mechanical stretch (e.g., distraction osteogenesis).

Effects of mechanical vibration on cell morphology, proliferation, apoptosis, and cytokine expression/secretion in osteocyte-like MLO-Y4 cells exposed to high glucose.

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well-recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO-Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO-Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG-induced apoptosis in MLO-Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG-incubated MLO-Y4 cells were observed after mechanical vibration via the F-actin fluorescence staining. The real-time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, ß-catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor-κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme-linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E2 and OPG, and inhibited the secretion of tumor necrosis factor-α and RANKL in the supernatant of HG-treated MLO-Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.

Pulsed electromagnetic fields regulate osteocyte apoptosis, RANKL/OPG expression, and its control of osteoclastogenesis depending on the presence of primary cilia.

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).

Development and evaluation of a direct TaqMan qPCR assay for the rapid detection of diverse carnivore amdoparvoviruses.

Amdoparvoviruses infect carnivore species, including mink, raccoon dog, fox, skunk, and red panda. Amdoparvovirus infection is a major cause of morbidity and mortality in farmed minks. Here, we developed a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using three primers and one probe based on the conserved VP2 gene. The detection limit for Aleutian mink disease virus (AMDV) and Raccoon dog and arctic fox amdoparvovirus (RFAV) were 4.06 × 101 copies/µl and 2.93 × 101 copies/µl, respectively. Both intra- and inter-assay variability were less than 2%. Among 74 carnivore samples, the positive rates for amdoparvoviruses were 62.2% (46/74) by direct TaqMan qPCR, while only 40.5% (30/74) by SYBR Green I qPCR. This result suggests that the direct TaqMan qPCR was more sensitive than the SYBR Green I qPCR. Additionally, the direct TaqMan qPCR is a rapid and sensitive method for liquid samples at microliter level as the assay employed the direct alkaline lysis method to obtain viral DNA and, therefore, eliminated the cumbersome steps in extracting DNA. Overall, the direct TaqMan qPCR assay possessed high specificity, sensitivity, and reproducibility, indicating that it can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses in epidemiological and pathogenesis studies.

Investigating the proteomic expression profile of tobacco (Nicotiana tabacum) leaves during four growth stages using the iTRAQ method.

Despite the importance of tobacco (Nicotiana tabacum) in agriculture and model organism investigations, the proteomic changes that occur in the tobacco leaf as it matures remain to be explored. In this study, an isobaric tags for relative and absolute quantification (iTRAQ) strategy was applied to investigate the proteomic profiles of K326 and Honghua Dajinyuan (HD) tobacco leaves at four growth stages. The proteomic profile varied with growth stage in both K326 and HD. Gene ontology (GO) classification was used to identify the biological processes that showed the greatest changes in protein expression between growth stages of HD and K326. Moreover, the number of differentially expressed proteins was greater in HD than in K326, especially during the rosette growth stage and the fast-growing stage. The galactose metabolism and glycosphingolipid biosynthesis-globo series pathways appeared only during the rosette growth stage of HD. It therefore appears that these pathways may be correlated with tobacco mosaic disease. The identification of these pathways should prove useful in investigations of the pathogenesis of tobacco mosaic virus. Graphical abstract ᅟ.

rs401681 and rs402710 confer lung cancer susceptibility by regulating TERT expression instead of CLPTM1L in East Asian populations.

Lung cancer is a common cancer in human and has presented significant genetic predisposition. Previous genome-wide association study observed that rs401681 within CLPTM1L (CLPTM1 like) was significantly associated with lung cancer. By analyzing 1000 genomes data for East Asian, we identified only one SNP in nearby region, rs402710, in high linkage disequilibrium with rs401681, which was also associated with lung cancer. However, the real causal SNP and mechanism for the association were still not clear. The following plasmid construction, mutagenesis, transient transfection and luciferase reading indicated that both SNPs could regulate gene expression in lung/bronchial epithelium Beas-2B cell line. By chromosome conformation capture, it was identified that the segment containing these two SNPs could interact with TERT (telomerase reverse transcriptase) promoter, thus indicating that these SNPs confer lung cancer risk by regulating TERT expression instead of CLPTM1L. Through chromatin immunoprecipitation, the transcript factors HNF4A (hepatocyte nuclear factor 4 alpha) and MAF1 (MAF1 homolog, negative regulator of RNA polymerase III) were recognized for the regions spanning rs401681 and rs402710, respectively. Our results uncovered a complete link between these two SNPs and lung cancer.

Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling.

In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. (1) In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 µm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 µm i.d. nano-LC packing column with 3 µm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. (1) Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.

On-orbit radiometric calibration of Suomi NPP VIIRS reflective solar bands using the Moon and solar diffuser.

Radiometric calibration of the Suomi National Polar-orbiting Partnership Visible Infrared Imaging Radiometer Suite (VIIRS) reflective solar bands relies mainly on the onboard solar diffuser (SD) observations. The SD reflectance degrades over time due to the exposure to solar ultraviolet radiation. The uncertainties embedded in characterizing the SD bidirectional reflectance distribution function (BRDF) directly affect the accuracy of sensor radiometric calibration coefficients, such as F-factors, which are proxies of detector gain. The Moon-based radiometric calibration provides an independent way of validating and correcting the SD-based calibration. This study focuses on the comparison of the long-term SD F-factors with lunar F-factors by using two independent lunar irradiance models, i.e., Miller and Turner (MT) model and the Global Space-based Inter-Calibration System Implementation of ROLO (GIRO) model. To monitor the long-term detector response changes, the lunar F-factor differences are matched to the SD F-factors by applying the best fit scaling factors. Overall, the two lunar F-factors agree well, within 2% of one sigma standard deviation in the reflective solar bands compared to the SD F-factors. The lifetime standard deviations of difference between the GIRO-based lunar and SD F-factors show better long-term match than that of MT-based lunar F-factors. The GIRO-based lunar F-factors show increasing differences over time in comparison with the SD F-factors especially for bands M1 to M4, which indicates the underestimation of the VIIRS detector degradation by SD F-factors for these bands. Using standard SD calibration method and the GIRO-based lunar model, long-term difference between the lunar and SD F-factors shows there are 1.6%, 1.3%, 1.0%, and 0.9% increases in lunar F-factor trend for bands M1 to M4 at the end of year 2015. To mitigate these time-dependent biases, NOAA Ocean Color (OC) group and NASA VIIRS characterization support team (VCST) developed lunar correction methods and applied them to their specific products. However, the amounts of band-dependent lunar corrections are not consistent between these two teams, especially in the short-wavelength bands from M1 to M4, depending on the versions of lunar models and SD F-factor calculation algorithms. Using the standard SD F-factor algorithm and the multi-agency endorsed GIRO model, we derived lunar correction factors based on the quadratic fits between the SD and lunar F-factors. The differences with the NOAA OC group and NASA VCST team are compared and described in this study.

A capillary column packed with a zirconium(IV)-based organic framework for enrichment of endogenous phosphopeptides.

A zirconium(IV)-based metal organic framework (Zr-MOF) was deposited on polydopamine-coated silica microspheres to form microspheres of type SiO2@PDA@Zr-MOF. These were packed into capillary columns for enrichment of phosphopeptides. The column was off-line coupled to both matrix-assisted laser desorption/ionization time of flight mass spectrometry and LC-ESI-MS/MS. The method has a detection limit as low as 4 fmol of ß-casein digest and a selectivity as high as 1:1000 (molar ratio of ß-casein and BSA digest). It was applied to the analysis of human saliva. In total, 240 endogenous phosphopeptides were identified in only 25 µL human saliva. Graphical abstract A zirconium-based metal organic framework (Zr-MOF) was modified outside of polydopamine-coated silica microspheres to form microspheres named SiO2@PDA@Zr-MOF. Then they were packed in capillary columns for selective enrichment of phosphopeptides via interaction between Zr-O clusters and phosphate groups. The pre-concentration resulted in a better detection of phosphopeptides by mass spectrometry. Tris: Tris(hydroxymethyl)aminomethane; DMF: Dimethyl Formamide; Zr-MOF: Zirconium(IV)-organic framework; MOAC: Metal oxide affinity chromatography.

Design of five-layer gold nanoparticles self-assembled in a liquid open tubular column for ultrasensitive nano-LC-MS/MS proteomic analysis of 80 living cells.

In this work, for the first time, a liquid open tubular column modified by five-layer gold nanoparticles and linked with C18 (GNPs@C18 ) was designed and fabricated for nano-LC-MS/MS analysis of 80 living cells. Sixty nanometer gold nanoparticles were self-assembled layer by layer on the inner wall of a 20 µm id fused-silica capillary. C18 was then linked on the gold nanoparticles to make the liquid open tubular column show hydrophobic character. Enough loading capacities for analysis of 80 living cells, ∼100 fmol for pk-10 and ∼30 fmol for insulin, were obtained with the 2 m × 20 µm id five-layer GNPs@C18 open tubular column. The open tubular column was used in an online pretreatment and direct nano-LC-MS/MS analysis system to analyze 80 living HepG2 cells. In total, 650 proteins were identified in triplicate runs. The subcellular localization of the identified proteins showed that our system had no bias toward different cellular compartments. Protein copy number per cell of the identified proteins showed that the detection limit could reach 50 zmol and the abundance of the identified proteins could cover a dynamic range of 6 orders.

Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.

BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.

Quantitative method for analysis of tobacco-specific N-nitrosamines in mainstream cigarette smoke by using heart-cutting two-dimensional liquid chromatography with tandem mass spectrometry.

A heart-cutting two dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for the analysis of tobacco-specific N-nitrosamines (TSNAs) at low concentration level in Virginia-type cigarette smoke. A strong cation exchange column was utilized for the first dimensional separation, which effectively removed acidic and neutral components in the smoke, followed by a reversed phase liquid chromatography coupled with tandem mass spectrometric analysis. To capture components of the TSNAs in the effluent on the trapping column, a compensating pump was applied for online dilution and pH adjustment during the period of the TSNAs fraction transferring and enrichment. Highly sensitive determination of the TSNAs in mainstream cigarette smoke was achieved by isotope deuterated internal standards under the multiple reaction monitoring mode. Compared with traditional methodologies, the method was almost no matrix interference. Limits of quantity for the TSNAs were within 0.027-0.094 ng/mL, and the results showed good reproducibility and accuracy. Finally, the new method was applied for analysis of the Kentucky reference cigarettes and the results agreed well with joint experiments of Cooperation Centre for Scientific Research Relative to Tobacco.

Differential intensity-dependent effects of pulsed electromagnetic fields on RANKL-induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells.

Pulsed electromagnetic fields (PEMF) have been proven to be effective for promoting bone mass and regulating bone turnover both experimentally and clinically. However, the exact mechanisms for the regulation of PEMF on osteoclastogenesis as well as optical exposure parameters of PEMF on inhibiting osteoclastic activities and functions remain unclear, representing significant limitations for extensive scientific application of PEMF in clinics. In this study, RAW264.7 cells incubated with RANKL were exposed to 15 Hz PEMF (2 h/day) at various intensities (0.5, 1, 2, and 3 mT) for 7 days. We demonstrate that bone resorbing capacity was significantly decreased by 0.5 mT PEMF mainly by inhibiting osteoclast formation and maturation, but enhanced at 3 mT by promoting osteoclast apoptosis. Moreover, gene expression of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 was significantly decreased by 0.5 mT PEMF, but increased by 3 mT. Our findings reveal a significant intensity window for low-intensity PEMF in regulating bone resorption with diverse nature for modulating osteoclastogenesis and apoptosis. This study not only enriches our basic knowledge for the regulation of PEMF in osteoclastogenesis, but also may lead to more efficient and scientific clinical application of PEMF in regulating bone turnover and inhibiting osteopenia/osteoporosis. Bioelectromagnetics. 38:602-612, 2017. © 2017 Wiley Periodicals, Inc.