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Yen-Ho Lai, Chih-Sheng Chiang, Tzu-Hsun Kao, San-Yuan Chen Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu, Taiwan, Republic of China Introduction: Deep penetration of large-sized drug nanocarriers into tumors is important to improve the efficacy of tumor therapy. Methods: In this study, we developed a size-changeable “Trojan Horse” nanocarrier (THNC) composed of paclitaxel (PTX)-loaded Greek soldiers (GSs; ~20 nm) assembled in an amphiphilic gelatin matrix with hydrophilic losartan (LST) added. Results: With amphiphilic gelatin matrix cleavage by matrix metalloproteinase-2, LST showed fast release of up to 60% accumulated drug at 6 h, but a slow release kinetic (~20%) was detected in the PTX from the GSs, indicating that THNCs enable controllable release of LST and PTX drugs for penetration into the tumor tissue. The in vitro cell viability in a 3D tumor spheroid model indicated that the PTX-loaded GSs liberated from THNCs showed deeper penetration as well as higher cytotoxicity, reducing a tumor spheroid to half its original size and collapsing the structure of the tumor microenvironment. Conclusion: The results demonstrate that the THNCs with controlled drug release and deep penetration of magnetic GSs show great potential for cancer therapy. Keywords: amphiphilic gelatin, nanocarriers, controlled release, deep tumor penetration

K Suresh Babu,1,† M Anandkumar,1,† TY Tsai,2 TH Kao,2 B Stephen Inbaraj,2 BH Chen2,31Centre for Nano Sciences and Technology, Madanjeet School of Green Energy Technologies, Pondicherry University, Kalapet, India; 2Department of Food Science, 3Graduate Institute of Medicine, Fu Jen University, Taipei, Taiwan†These authors contributed equally to this workBackground: Cerium oxide nanoparticles (CeO2) have been shown to be a novel therapeutic in many biomedical applications. Gold (Au) nanoparticles have also attracted widespread interest due to their chemical stability and unique optical properties. Thus, decorating Au on CeO2 nanoparticles would have potential for exploitation in the biomedical field. Methods: In the present work, CeO2 nanoparticles synthesized by a chemical combustion method were supported with 3.5% Au (Au/CeO2) by a deposition-precipitation method. The as-synthesized Au, CeO2, and Au/CeO2 nanoparticles were evaluated for antibacterial activity and cytotoxicity in RAW 264.7 normal cells and A549 lung cancer cells. Results: The as-synthesized nanoparticles were characterized by X-ray diffraction, scanning and transmission electron microscopy, and ultraviolet-visible measurements. The X-ray diffraction study confirmed the formation of cubic fluorite-structured CeO2 nanoparticles with a size of 10 nm. All synthesized nanoparticles were nontoxic towards RAW 264.7 cells at doses of 0–1,000 µM except for Au at >100 µM. For A549 cancer cells, Au/CeO2 had the highest inhibitory effect, followed by both Au and CeO2 which showed a similar effect at 500 and 1,000 µM. Initial binding of nanoparticles occurred through localized positively charged sites in A549 cells as shown by a shift in zeta potential from positive to negative after 24 hours of incubation. A dose-dependent elevation in reactive oxygen species indicated that the pro-oxidant activity of the nanoparticles was responsible for their cytotoxicity towards A549 cells. In addition, cellular uptake seen on transmission electron microscopic images indicated predominant localization of nanoparticles in the cytoplasmic matrix and mitochondrial damage due to oxidative stress. With regard to antibacterial activity, both types of nanoparticles had the strongest inhibitory effect on Bacillus subtilis in monoculture systems, followed by Salmonella enteritidis, Escherichia coli, and Staphylococcus aureus, while, in coculture tests with Lactobacillus plantarum, S. aureus was inhibited to a greater extent than the other bacteria. Conclusion: Gold-supported CeO2 nanoparticles may be a potential nanomaterial for in vivo application owing to their biocompatible and antibacterial properties.Keywords: cerium oxide nanoparticles, gold supported cerium oxide, cytotoxicity, antibacterial activity, cellular uptake, reactive oxygen species

Recent evidence that luteinizing hormone (LH) stimulation of ovulatory follicles causes transactivation of the epidermal growth factor receptor (EGFR) has provided insights into the mechanisms of ovulation. However, the complete array of signals that promote oocyte reentry into the meiotic cell cycle in the follicle are still incompletely understood. To elucidate the signaling downstream of EGFR involved in oocyte maturation, we have investigated the LH responses in granulosa cells with targeted ablation of EGFR. Oocyte maturation and ovulation is disrupted when EGFR expression is progressively reduced. In granulosa cells from mice with either global or granulosa cell-specific disruption of EGFR signaling, LH-induced phosphorylation of MAPK3/1, p38MAPK, and connexin-43 is impaired. Although the LH-induced decrease in cGMP is EGFR-dependent in wild type follicles, LH still induces a decrease in cGMP in Egfr(delta/f) Cyp19-Cre follicles. Thus compensatory mechanisms appear activated in the mutant. Spatial propagation of the LH signal in the follicle also is dependent on the EGF network, and likely is important for the control of signaling to the oocyte. Thus, multiple signals and redundant pathways contribute to regulating oocyte reentry into the cell cycle.

Disease-specific oligomers Tau assay system is anticipated in Alzheimer disease (AD) to elucidate their etiological roles. We developed a highly sensitive and selective ELISA for high-molecular-weight oligomer tau (HMWoTau) with LLOQ of 0.3 pg/well for the first time, using a novel mouse monoclonal antibody APNmAb005. The target molecule was identified as HMWoTau with circa 2000 kD as a minimum size and the more oligomerized species (>5000 kD), in combination analysis with Size-Exclusion-Chromatography and Sucrose-Density-Gradient-Centrifugation for both recombinant human (rh) Tau-derived aggregates and AD brain-lysates in PBS(-). HMWoTau was labeled by Thioflavin S and visualized as a homogeneous globular particle (about 30 nm in diameter) by two different technologies of atomic force microscopy and dSTORM-Nanoimager. Specific quantitation was also confirmed by immune-absorption, rhHMWoTau-spiked, and cross-reactivity studies. APNmAb005 failed to detect the HMWoTau signal by treatment with DTT/SDS under no influence on the pan-tau antibody, indicating its conformation-specific recognition. APNmAb005-ELISA showed AD-specific and statistically significant ELISA signals from 1 ng brain lysate protein/well. Analysis of the frontal neocortex (N = 40, Braak stage I-VI) by ELISA revealed the detection-limit levels of HMWoTau species at stage I-III, and drastic and statistically significant increases at stage V/VI (AD). By contrast, total Tau and p181 Tau showed 1/4-1/5 levels of AD even at Stage I, while both tau species also showed a statistically significant increase in AD. In sum, our novel APNmAb005-ELISA clarified the disease-specific increase in HMWoTau species and will be useful for not only further etiological elucidation but also the potential diagnostics in AD and relevant tauopathy., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

In recent years, liquid crystal materials have drawn great interest because of their wide range of applications. Among various thermochromic materials, cholesteric liquid crystalline (CLC) materials have been well studied and reported. CLC materials have the advantages of ready manipulation and multiple color transitions. For the further development of smart clothing and wearable electronics, however, the incorporation of CLC materials into polymers still remains challenging. The difficulties lie in the prevention of leakage of CLC and retention of the cholesteric liquid crystalline phase. In this work, we demonstrate a versatile nonsolvent and phase separation method using polar solvents to incorporate CLC microspheres into polymer matrix. Poly(vinyl alcohol) (PVA), a water-soluble polymer, is chosen as the polymer because of its high transparency and ease to handle. Using spin-coating and wet spinning techniques, PVA/CLC films and fibers can be fabricated. The formation of CLC microspheres in the polymer matrix is characterized through optical and polarized microscopy. Compared with the CLC films, the PVA/CLC composites demonstrate superior thermal stability. Moreover, both PVA/CLC films and fibers exhibit good color stability from the electrical tests. This work provides an effective strategy to prepare polymer/CLC composites, paving a wide avenue toward applications in smart textiles, display technologies, and medical devices.

Concurrent chemoradiotherapy (CCRT) is the standard treatment for patients with locally advanced squamous cell carcinoma of the head and neck (HNSCC). Pneumonia is a significant complication in these patients. This study aims to identify pneumonia risk factors and their impact on survival in HNSCC patients undergoing CCRT. Data from the Chang Gung Research Database (CGRD) were retrospectively reviewed for patients treated between January 2007 and December 2019. Of 6959 patients, 1601 (23.01%) developed pneumonia, resulting in a median overall survival (OS) of 1.2 years compared to 4.9 years in the non-pneumonia group ( p < 0.001). The pneumonia group included older patients with advanced tumors, more patients with diabetes mellitus (DM), more patients with invasive procedures, longer chemotherapy and radiotherapy durations, and lower body weight. The 2-year, 5-year, and 10-year OS rates were significantly lower in the pneumonia group. Multivariate analysis identified alcohol consumption, DM, gastrostomy, nasogastric tube use, longer chemotherapy, and a 2-week radiotherapy delay as independent risk factors. Understanding these risks can lead to early interventions to prevent severe pneumonia-related complications. A better understanding of the risks of pneumonia enables early and aggressive interventions to prevent severe complications.

Objectives : Cervical disc herniation (CDH) leads to pain, numbness, and potential disability. Percutaneous endoscopic cervical discectomy (PECD) offers an anterior or posterior approach. This study aims to compare postoperative disc height and angle changes one year after PECD, considering both approaches. Methods : We retrospectively reviewed the data from patients with CDH who underwent PECD from October 2017 to July 2022. Cervical disc height was measured using the preoperative and one-year postoperative magnetic resonance imaging (MRI) examinations. Lordotic angle (LA), global alignment angle (GAA), segmental alignment angle (SAA), and slippage distance (SD) at the surgical level were measured on radiographs in the neutral, flexion, and extension positions. Results : Thirty-eight patients who underwent posterior PECD (PPECD) and five patients who underwent anterior PECD (APECD) were included in the evaluation. The mean age of the patients was 47.4 years (range: 29-69 years). There was a significant difference in the preoperative and one-year postoperative GAA and SAA in extension in the PPECD group ( p = 0.003 and 0.031, respectively). The mean decreased disc height one-year postoperative was 1.30 mm in the APECD group and 0.3 mm in the PPECD group by MRI. A significant disc height decrease was observed in the APECD group ( p < 0.001). Conclusions : Treating CDH with PPECD or APECD is feasible, as it can relieve symptoms and reduce disability. Stability remained unaffected during the first year after surgery, even though there was an increase in angulation during extension. Despite a significant decrease in disc space following APECD, patients reported significant symptom improvement and no new symptoms.

Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1β. We previously showed that IL-1β release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1β release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1β release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1β release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1β release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1β, but resulted in the retention of mature IL-1β within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1β was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1β release from viable cells. This study expands on the molecular pathways that promote IL-1β in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1β release during infection., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Objective: This retrospective study was designed to evaluate the incidence and predisposing factors of heterotopic ossification (HO) after cervical disc arthroplasty (CDA) with a specific implant at 1 and 2 levels, and to investigate the biomechanical effects related to HO. The study goal was to identify ways to reduce the likelihood of HO formation after surgery., Methods: The study included patients who underwent only 1- or 2-level CDA with the Baguera C disc between November 2014 and December 2021 at a single medical center. All patients were operated on by the same neurosurgeon. The surgical indication included 1-level or 2-level disc herniation between C3 and C7 with radiculopathy, myelopathy, or both, with minimal spondylosis. The various factors were assessed by evaluating plain radiographs and cervical CT scans. The presence of HO was evaluated at different intervals postsurgery, and HO severity was graded using the McAfee classification., Results: Of 107 patients who underwent CDA, 47 (43.9%) had HO at 63 of 171 levels (36.8%). Most cases with HO were grade 1, and no grade 4 was observed. Statistically significant risk factors for HO were the length of endplate coverage ratio and inferior anterior residual exposed endplate (AREE); sex, age, implant height and width, shell angle, and pre- and postoperative functional spinal unit (FSU) angle were not significant. More AREE and greater kyphotic postoperative FSU angle in the flexion position were significant factors differentiating HO grades 0 and 1 from grades 2 and 3. Furthermore, the non-HO group showed a trend of higher range of motion at any postoperative time compared to the HO group, especially at 1 month after surgery., Conclusions: The HO incidence after CDA was correlated with the residual length of endplate coverage and inferior AREE. Additionally, the AREE and kyphotic postoperative FSU angle in the flexion position were associated with HO grade progression. Patients with HO also showed a trend of lower range of motion at 1 month after surgery. Using an adequately sized implant and encouraging neck motion may help prevent HO development and progression.

Background : Cervical disc arthroplasty (CDA) is currently used instead of fusion to preserve cervical spine motion. Cervical implant subsidence is a potential complication after CDA. Methods : Radiological measurements were recorded via patient anteroposterior and lateral radiographs in the neutral position. Subsidence was defined as a decrease of 3 mm or more in functional spinal unit height (FSUH) from which was measured on a post-operative (OP) radiograph. Results : This study included 104 patients who underwent 153 CDA levels with the Bryan Disc. Approximately one-quarter of the implants (22.9%) showed subsidence. Binary logistic regression analysis indicated that pre-OP mean disc height (DH) was identified as an independent risk factor for subsidence in multivariate analysis (0.151, 95% Confidence Interval 0-0.073, p = 0.018). Receiver operating characteristic curve analysis (area under the curve = 0.852, sensitivity 84.7%, specificity 77.1%) revealed a cut-off value of 4.48 mm for pre-OP Mean-DH in the risk for implant subsidence. Conclusions : In this study, the subsidence rate significantly increased when the implants were oversized beyond a pre-OP Mean-DH of approximately >4 mm. Moreover, the implant subsidence incidence was higher than that reported in previous studies. This is possibly due to endplate over-preparation or disc space over-distraction during placement at the same height as the Bryan Disc (8.5 mm).

Introduction: X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder that may result in severe speech impairment. The literature suggests that there are differences in the speech of individuals with XDP and healthy controls. This study aims to examine the motor speech characteristics of the mixed dystonia-parkinsonism phase of XDP., Method: We extracted acoustic features representing coordination, consistency, speed, precision, and rate from 26 individuals with XDP and 26 controls using Praat, MATLAB, and R software. Group demographics were compared using descriptive statistics. A one-way analysis of variance (ANOVA) with Tukey's post hoc test was used to test for acoustic differences between the two groups., Results: The XDP group had significantly lower consistency, speed, precision, and rate than controls (p < 0.05). For coordination, the XDP group had a smaller ratio of pause duration during transitions when compared to controls., Discussion: To our knowledge, this study is the first to describe the motor speech characteristics of the mixed dystonia-parkinsonism phase of XDP. The motor speech of mixed dystonia-parkinsonism XDP is similar to prior characterizations of mixed hyperkinetic-hypokinetic dysarthria with noted differences in articulatory coordination, consistency, speed, precision, and rate from healthy controls. Identifying the motor speech components of all three phenotypes of XDP (i.e., dystonia-dominant phase, parkinsonism-dominant phase, and mixed dystonia-parkinsonism phase) is needed to establish markers of speech impairment to track disease progression., Competing Interests: Declaration of competing interest This work was supported by the Massachusetts General Hospital Collaborative Center for X-linked Dystonia-Parkinsonism, Boston, MA; the National Institutes of Health (NIH)National Institute on Deafness and Other Communication Disorders [grant number K24DC0016312]; and the NIHNational Institute of Neurological Disorders and Stroke [grant number K23NS123369]. The funding sources did not participate in the study design; data collection, analysis, and interpretation; report writing; or decision to submit the article for publication. The authors declare that there are no conflicts of interest relevant to this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aim: An estimated 88% of rheumatoid arthritis (RA) patients experience various degrees of cervical spine involvement. The excessive movement of the atlantoaxial joint, which connects the occiput to the upper cervical spine, results in atlantoaxial instability (AAI). AAI stabilization is usually achieved by C1 lateral mass-to-C2 pedicle screw-rod fixation (LC1-PC2 fixation), which is technically challenging in RA patients who often show destructive changes in anatomical structures. This study aimed to analyze the clinical results and operative experiences of C1-C2 surgery, with emphasis on the advancement of image-guided surgery and augmented reality (AR) assisted navigation., Methods: We presented our two decades of experience in the surgical management of AAI from April 2004 to November 2022., Results: We have performed surgery on 67 patients with AAI, including 21 traumatic odontoid fractures, 20 degenerative osteoarthritis, 11 inflammatory diseases of RA, 5 congenital anomalies of the os odontoideum, 2 unknown etiologies, 2 movement disorders, 2 previous implant failures, 2 osteomyelitis, 1 ankylosing spondylitis, and 1 tumor. Beginning in 2007, we performed LC1-PC2 fixation under C-arm fluoroscopy. As part of the progress in spinal surgery, since 2011 we used surgical navigation from presurgical planning to intraoperative navigation, using the preoperative computed tomography (CT) -based image-guided BrainLab navigation system. In 2021, we began using intraoperative CT scan and microscope-based AR navigation., Conclusion: The technical complexities of C1-C2 surgery can be mitigated by CT-based image-guided surgery and microscope-based AR navigation, to improve accuracy in screw placement and overall clinical outcomes, particularly in RA patients with AAI., (© 2023 The Authors. International Journal of Rheumatic Diseases published by Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Vascular injury is one of the potential complications of percutaneous transpedicular screws fixation. Carefully review the vascular anatomy preoperatively is important. Once unexpected low blood pressure or anemia occurs, vascular injury should be taken into consideration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In Petunia (Solanaceae family), self-incompatibility (SI) is regulated by the polymorphic S-locus, which contains the pistil-specific S-RNase and multiple pollen-specific S-Locus F-box (SLF) genes. SLFs assemble into E3 ubiquitin ligase complexes known as Skp1-Cullin1-F-box complexes (SCFSLF). In pollen tubes, these complexes collectively mediate ubiquitination and degradation of all nonself S-RNases, but not self S-RNase, resulting in cross-compatible, but self-incompatible, pollination. Using Petunia inflata, we show that two pollen-expressed Cullin1 (CUL1) proteins, PiCUL1-P and PiCUL1-B, function redundantly in SI. This redundancy is lost in Petunia hybrida, not because of the inability of PhCUL1-B to interact with SSK1, but due to a reduction in the PhCUL1-B transcript level. This is possibly caused by the presence of a DNA transposon in the PhCUL1-B promoter region, which was inherited from Petunia axillaris, one of the parental species of Pe. hybrida. Phylogenetic and syntenic analyses of Cullin genes in various eudicots show that three Solanaceae-specific CUL1 genes share a common origin, with CUL1-P dedicated to S-RNase-related reproductive processes. However, CUL1-B is a dispersed duplicate of CUL1-P present only in Petunia, and not in the other species of the Solanaceae family examined. We suggest that the CUL1s involved (or potentially involved) in the SI response in eudicots share a common origin., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The objective of this study was to develop a simultaneous analysis method of furan and its 10 derivatives in different food commodities. The results indicated that furan and its 10 derivatives could be separated within 9.5 min by using a HP-5MS column and gas chromatography-tandem mass spectrometry (GC-MS/MS) with multiple reaction monitoring mode for detection. Furthermore, this method could resolve several furan isomers, such as 2-methyl furan and 3-methyl furan, as well as 2,3-dimethyl furan and 2,5-dimethyl furan. The most optimal extraction conditions were: 5 g of the fruit or juice sample mixed with 5 mL of the saturated NaCl solution, separately, or 1 g of the canned oily fish sample mixed with 9 mL of the saturated NaCl solution, followed by the equilibration of each sample at 35 °C for 15 min, using a carboxen-polydimethylsiloxane SPME arrow to adsorb the analytes for 15 min at 35 °C for subsequent analysis by GC-MS/MS. For method validation of all the analytes in the different food matrices, the recovery was 76-117% and the limit of the quantitation was 0.003-0.675 ng/g, while the relative standard deviation (RSD%) of the intra-day variability range from 1-16%, and that of the inter-day variability was from 4-20%. The method validation data further demonstrated that a reliable method was established for the analysis of furan and its 10 derivatives in commercial foods., Competing Interests: The authors read and approved the manuscript and declare that they have no competing interest.

Objectives: The objectives of this study were to identify the risk factors and incidence of anterior bone loss (ABL) after Baguera C cervical disc arthroplasty (CDA) and identify whether design differences in artificial discs affect ABL., Methods: In this retrospective radiological review of patients who underwent single-level Baguera C CDA in a medical center, the extent of ABL and the following radiological parameters were recorded: global and segmental alignment angle, lordotic angle (or functional spinal unit angle), shell angle, global range of motion (ROM), and ROM of the index level. ABL at the index level was grade 0-2. Grade 0 was defined as no remodeling, grade 1 as spur disappearance or mild change in body contour, and grade 2 as obvious bone regression with Baguera C Disc exposure., Results: Combining grade 1 and grade 2, ABL was found in 56 upper adjacent vertebrae and 52 lower adjacent vertebrae of the 77 patients. Only 18 patients (23.4%) had no ABL. Shell angle differed significantly between ABL grades of both the upper and lower adjacent level: 0.0° in grade 0 and 1 ABL vs. 2.0° in grade 2 ABL of the upper adjacent level ( p < 0.05); and 0.0° in grade 0 and 1 ABL vs. 3.5° in grade 2 ABL of the lower adjacent level ( p < 0.05). A female predominance of ABL was found. Hybrid surgery and artificial disc size were also related to ABL., Conclusions: ABL is more common in Baguera C Disc arthroplasty than Bryan Disc arthroplasty. Larger shell angle was related to ABL after CDA with Baguera C Discs, which may indicate that shell angle is pivotal in determining the incidence of ABL after CDA. Females had more ABL with Baguera C Disc arthroplasty; this might be related to shorter endplate lengths as well as a smaller endplate-implant mismatch., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Chih-Chang Yang et al.)

Furan has been described as a carcinogen, being present in numerous food products available to the everyday consumer. The objectives of this study were to determine the formation of furan and its derivatives in 3 model systems during heating, with their contents being determined in selected commercial food products by HS-SPME Arrow coupled with GC/MS/MS. A high accuracy and precision was attained for the method developed in this study. The model system of 0.5 M glucose/alanine + 0.1 M ascorbic acid generated the highest furan level (5603.63 ng/g) when heated at 121 °C for 2 h. The addition of ascorbic acid favored formation of furfural and furfuryl alcohol, while linoleic acid favored formation of furfuryl alcohol. In addition, the total furan contents in various commercial foods ranged from 21.14 to 3183.39 ng/g. Most importantly, the outcome of model systems may be used to predict the presence of furan and its derivatives in commercial foods., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study explored the effects of sterilization conditions on the formation of furan and its 10 derivatives in canned foods with a sterilizing value (F0) at 4. The contents of furans were determined by SPME arrow-GC-MS/MS, along with the furan precursors analyzed for elucidating the possible mechanism of furan formation. Results revealed that the total furan contents rose substantially in canned meat paste, tomato mackerel, chicken puree, tomato paste, pineapple slice, pineapple juice and carrot juice following sterilization. However, the total furan content did not change significantly ( p > 0.05) in canned oily mackerel, but decreased significantly ( p < 0.05) in canned apple puree after sterilization. With the exception of apple puree and pineapple slice, all the other canned foods showed a higher total furan content under low-temperature-long-time condition than that under high-temperature-short-time condition. Following heating, only the furan level showed a large increase in chicken puree, meat paste and tomato mackerel, whereas in canned fruit- and vegetable-based foods, the contents of furan and furfural showed a pronounced increase. The levels of alkylated furans were higher in sterilized samples containing high level of amino acid, while that of oxygenated furans were higher in sterilized samples containing high level of reducing sugar.

Owing to the presence of significant levels of toxic furan compounds reported globally in commercial foods by various food authorities, the objectives of this study were to develop an analytical method for determination of furan and its 10 derivatives in commercial foods using headspace-solid phase microextraction (HS-SPME)-Arrow coupled with gas chromatography-tandem mass spectrometry. Furan and its 10 derivatives were separated within 10 min by employing an HP-5MS capillary column with d 4 -furan as the internal standard for quantitation. The most optimal sample weight and extraction time for various commercial food samples, respectively, ranged from 1 to 5 g and 10-15 min depending on the sample variety. For extraction, carboxen/poly(dimethylsiloxane) (CAR/PDMS) cellulose was used with the temperature at 30 °C, equilibration time of 15 min, and desorption time of 3 min. The limit of detection ranged from 0.001 to 1.071 ng/g, while the limit of quantitation ranged from 0.003 to 3.571 ng/g. A high precision and accuracy were obtained for this method. The total furan content in commercial foods ranged from nd to 40 725.85 ng/g, in which the mean contents were the highest for brewed coffee (35 082.26 ng/g) and canned coffee (25 152.22 ng/g), while the lowest were for potato chip and cookies (0.57-1.48 ng/g), donut (1.50 ng/g), milk (0.34-30.38 ng/g), and oat (6.56 ng/g).

Surface-enhanced Raman scattering (SERS) has been a useful sensing technique, in which inelastic light scattering can be significantly enhanced by absorbing molecules onto rough metal surfaces or nanoparticles. Although many methods have been developed to prepare SERS substrates, it is still highly desirable and challenging to design SERS substrates, especially with highly ordered and controlled three-dimensional (3D) structures. In this work, we develop novel SERS substrates with regular volcano-shaped polymer structures using the versatile solvent on-film annealing method. Polystyrene (PS) nanospheres are first synthesized by surfactant-free emulsion polymerization and assembled on poly(methyl methacrylate) (PMMA) films. After annealing in acetic acid vapors, PMMA chains are selectively swollen and wet the surfaces of the PS nanospheres. By selectively removing the PS nanospheres using cyclohexane, volcano-shaped PMMA films can be obtained. Compared with flat PMMA films with water contact angles of ∼74°, volcano-shaped PMMA films exhibit higher water contact angles of ∼110° due to the sharp features and rough surfaces. The volcano-shaped PMMA films are then coated with gold nanoparticles (AuNPs) as SERS substrates. Using rhodamine 6G as the probe molecules, the SERS results show that the Raman signals of the volcano-shaped PMMA/AuNP hybrid substrates are much higher than those of the pristine PMMA films and PMMA films with AuNPs. For the volcano-shaped PMMA/AuNP hybrid substrates using 400 nm PS nanospheres, a high enhancement factor (EF) value of ∼1.12 × 10 5 with a detection limit of 10 -8 M is obtained in a short integration time of 1 s. A linear calibration line with an R 2 value of 0.918 is also established, demonstrating the ability to determine the concentrations of the analytes. This work offers significant insight into developing novel SERS substrates, which is crucial for improving the detection limits of analytes.

This paper describes a low-temperature metallization and laser trimming process for microwave dielectric ceramic filters. The ceramic was metalized by electroless copper plating at a temperature lower than those of conventional low-temperature co-fired ceramic (LTCC) and direct bond copper (DBC) methods. Compared with filters made via traditional silver paste sintering, the metal in the holes of the microwave dielectric filters is uniform, smooth, and does not cause clogging nor become detached. Further, the batches of fabricated filters do not require individual inspection, reducing energy, labor, cost, and time requirements. A microwave dielectric filter was then manufactured from the prepared ceramic using a laser trimming machine with a line width and position error within ±50 μm; this demonstrates a more accurately controlled line width than that offered by screen printing. After using HFSS software simulations for preliminary experiments, the microwave dielectric filter was tuned to a target Wi-Fi band of 5.15-5.33 GHz; the return loss was <-10 dB, and the insertion loss was >-3 dB. To implement the real-world process, the laser parameters were optimized. Laser trimming has a higher success rate than traditional manual trimming, and the microwave dielectric filter manufactured here verified the feasibility of this process.

Ciliated muconodular papillary tumor (CMPT) is an uncommon tumor of the lung, usually detected incidentally in the peripheral lung fields on computed tomography. It is most commonly diagnosed in patients between the sixth and last decades of life. The canonical microscopic feature is a tripartite mixture of ciliated columnar, mucous, and basal cells with papillary architecture. However, studies have shown wide morphological variations in this tumor, such as a lack of ciliated columnar or mucous cells and flat instead of papillary architecture. Hence, a new umbrella term, bronchiolar adenoma (BA), has been proposed to encompass all tumors comprising a uniform layer of basal cells underneath the luminal epithelium displaying bronchiolar differentiation. Recent molecular studies on CMPT/BA discovered a high frequency of driver gene alterations, including mutations in the BRAF, EGFR, AKT1, KRAS, and HRAS genes, as well as ALK gene rearrangements. The tumor follows an indolent clinical course, as no recurrences or metastases have been reported in patients who underwent surgical resection. In this article we review the clinical, radiological, and pathological features, and molecular characteristics of CMPTs/BAs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

More and more information on tourist spots is being represented as pictures rather than text. Consequently, tourists who are interested in a specific attraction shown in pictures may have no idea how to perform a text search to get more information about the interesting tourist spots. In the view of this problem and to enhance the competitiveness of the tourism market, this research proposes an innovative tourist spot identification mechanism, which is based on deep learning-based object detection technology, for real-time detection and identification of tourist spots by taking pictures on location or retrieving images from the Internet. This research establishes a tourist spot recognition system, which is a You Only Look Once version 3 model built in Tensorflow AI framework, and is used to identify tourist attractions by taking pictures with a smartphone's camera. To verify the possibility, a set of tourist spots in Hsinchu City, Taiwan is taken as an example. Currently, the tourist spot recognition system of this research can identify 28 tourist spots in Hsinchu. In addition to the attraction recognition feature, tourists can further use this tourist spot recognition system to obtain more information about 77 tourist spots from the Hsinchu City Government Information Open Data Platform, and then make dynamic travel itinerary planning and Google MAP navigation. Compared with other deep learning models using Faster region-convolutional neural networks or Single-Shot Multibox Detector algorithms for the same data set, the recognition time by the models using You Only Look Once version 3, Faster region-convolutional neural networks, and Single-Shot Multibox Detector algorithms are respectively 4.5, 5, and 9 s, and the mean average precision for each when IoU = 0.6 is 88.63%, 85%, and 43.19%, respectively. The performance experimental results of this research show the model using the You Only Look Once version 3 algorithm is more efficient and precise than the models using the Faster region-convolutional neural networks or the Single-Shot Multibox Detector algorithms, where You Only Look Once version 3 and Single-Shot Multibox Detector are one-stage learning architectures with efficient features, and Faster region-convolutional neural networks is a two-stage learning architecture with precise features.

Self-incompatibility in Petunia is controlled by the polymorphic S-locus, which contains S-RNase encoding the pistil determinant and 16-20 S-locus F-box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S 2 -haplotype of the S-locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S-RNase. Two SLF pseudogenes and 28 potential protein-coding genes were identified, 20 of which were also found at the S-loci of both the S 6a -haplotype of P. inflata and the S N -haplotype of self-compatible Petunia axillaris, but not in the S-locus remnants of self-compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S-locus sequences of these three S-haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S-haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S-loci of all three S-haplotypes and in the flanking regions of all S-locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F-box genes as putative SLF parent genes. Our results shed light on the importance of non-coding sequences in the evolution of the S-locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Global heterochromatin reduction, which is one of the hallmarks of senescent cells, is associated with reduced transposable element repression and increased risk of chromatin instability. To ensure genomic integrity, the irreparable cells in a population exit permanently from the cell cycle, and this process is termed "senescence." However, senescence only blocks the expansion of unwanted cells, and the aberrant chromatin of senescent cells remains unstable. Serendipitously, we found that the transient ectopic expression of a repressive epigenetic modulator, DNA methyltransferase 3-like (DNMT3L) was sufficient to delay the premature senescence progression of late-passage mouse embryonic fibroblasts (MEFs) associated with a tightened global chromatin structure. DNMT3L induces more repressive H3K9 methylation on endogenous retroviruses and downregulates the derepressed transposons in aging MEFs. In addition, we found that a pulse of ectopic DNMT3L resulted in the reestablishment of H3K27me3 on polycomb repressive complex 2 (PRC2)-target genes that were derepressed in old MEFs. We demonstrated that ectopic DNMT3L interacted with PRC2 in MEFs. Our data also suggested that ectopic DNMT3L might guide PRC2 to redress deregulated chromatin regions in cells undergoing senescence. This study might lead to an epigenetic reinforcement strategy for overcoming aging-associated epimutation and senescence., (Copyright © 2020 Yu, Hui, Kao, Liao, Yang, Hou, Hsieh, Chang, Tsai, Pinskaya, Yang, Chen, Morillon, Tsai and Lin.)

The transient receptor potential (TRP) channel TRPV4 is a calcium-permeable cation channel protein which plays a mechanosensory and osmosensory role in several musculoskeletal tissues. Previous studies have shown that some specific mutations in the ankyrin repeat domain (ARD) of TRPV4 can reduce channel activity and further cause the osteoarthropathy related disease. Mutations in this region probably influence the constitutive activity of the channel, which mainly regulated by the binding of a small ligand such as adenosine triphosphate (ATP). These findings suggest that it is crucial to understand the fundamental mechanisms regulated by chemical ligands such as ATP binding with the ankyrin repeat domain (ARD) of TRPV4. However, how these mutations at the molecular level resulting in the related diseases are still unclear. Here we use full atomistic simulations to investigate the mutation induced conformational changes and ATP binding ability differences of TRPV4-ARD. Conformation characteristics of different mutations of TRPV4-ARD are explored. Optimal communication paths are studied to explain how a point mutation away from aim region (Finger 3) can cause a significant alteration on the conformation. We identify two molecular mechanisms through the conformation of Finger 3 and through alter the ATP binding mechanism correspondently to explain these unknowns. Our study provides fundamental insights into the mutation induced structural changes of the TRPV4-ARD and helps to explain how the mutations alter the ATP binding ability of the TRPV4-ARD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA) is an endocrine disruptor. To evaluate the effect of canned food consumption on internal BPA dose, urinary BPA concentrations were measured before and after intake of canned foods. This study applied a randomized crossover design, recruited 20 healthy volunteers, and divided them into two groups. One group consumed canned food; the other group consumed fresh food. After a 1-day washout, the dietary interventions were reversed. In each period, urine samples were collected immediately before meals and then 2 h, 4 h, and 6 h after meals. A mixed-effects model was used to assess BPA changes over time. Our results showed urinary BPA concentrations increased after consumption of canned food. Specifically, urinary BPA concentrations significantly differed between consumption of canned food and fresh food at 2 h, 4 h, and 6 h after intake (p values of 0.001, < 0.001, and < 0.001, respectively). Mean BPA concentrations at 2 h, 4 h, and 6 h after meals were 152%, 206%, and 79% higher, respectively, than mean BPA concentrations before meals. Urine concentration profiles of canned food intake showed that peaks were at 4 h, the increase diminished at 6 h, and returned to baseline levels at 24 h after intake. Therefore, dietary intervention and a 1-day washout period are effective for limiting internal BPA burden. This study provides convincing evidence of a human exposure route to BPA and a basis for designing interventions to mitigate exposure.

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection., Competing Interests: The authors have declared that no competing interests exist.

This study developed a high performance liquid chromatography with diode array detection (HPLC-DAD) and tandem mass spectrometry (MS-MS) method for determination of prenylflavonoids and hop bitter acids in surplus yeast, a byproduct from beer brewing process. This method enabled the simultaneous separation of 4 prenylflavonoids and 20 hop bitter acids within 30 min by employing a Hypersil-Keystone HyPURITY C18 column and a gradient mobile phase composed of phosphoric acid aqueous solution at pH 1.6 and acetonitrile. For HPLC-DAD analysis, the limits of detection and limits of quantitation ranged from 0.04 to 0.15 µg/mL and from 0.12 to 0.45 µg/mL, respectively, and the recoveries ranged from 82.6 to 99.7%. The intra-day variability and inter-day variability ranged from 1.37 to 8.82% and from 0.68 to 9.74%, respectively. For qualitation by MS-MS, the positive mode was discovered to possess satisfactory collision capacity and high sensitivity for prenylflavonoids, while the negative mode was more suitable for the ionization of hop bitter acids. The content of hop bitter acids in surplus yeast were higher than that of prenylflavonoids, and isomers and oxidation products of hop bitter acids were found. This study has advantages in identifying more components, short separation time, satisfactory resolution, high accuracy and high precision.

Self-incompatibility (SI) in Petunia is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistil-specific S-RNase gene and multiple pollen-specific S-locus F-box ( SLF ) genes. Both gain-of-function and loss-of-function experiments have shown that S-RNase alone regulates pistil specificity in SI. Gain-of-function experiments on SLF genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-of-function experiments must be performed to determine if SLF proteins are essential for SI of pollen. Here, we used CRISPR/Cas9 to generate two frame-shift indel alleles of S 2 - SLF1 ( SLF1 of S 2 -haplotype) in S 2 S 3 plants of P. inflata and examined the effect on the SI behavior of S 2 pollen. In the absence of a functional S 2 -SLF1, S 2 pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible S -haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of S 2 -haplotype and these eight S-RNases that had been determined by gain-of-function experiments performed previously or in this work. Our loss-of-function results provide definitive evidence that SLF proteins are solely responsible for SI of pollen, and they reveal their diverse and complex interaction relationships with S-RNases to maintain SI while ensuring cross-compatibility., (© 2018 American Society of Plant Biologists. All rights reserved.)

Key Message: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2 -haplotype and S 3 -haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCF SLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3 ) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2 ) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S 3 -RNase was completely suppressed by an antisense S 3 -RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

Cholesterol is widely present in animal fats and meat products and can undergo oxidation to form cholesterol-oxidation products (COPs) during heating. The objective of this study was to develop a QuEChERS method for the determination of COPs in edible animal fats and meat products via gas chromatography-mass spectrometry in which the required solvent volume and extraction time were reduced. By employing a DB-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) and a temperature-programming method, seven COPs, cholesterol, and the internal standard 5α-cholestane could be separated within 19 min. The limits of detection and limits of quantitation based on the COP standards ranged from 0.16 to 180 ng/mL and from 0.32 to 400 ng/mL, respectively, and the recoveries ranged from 89.1 to 107.6% for boiled pork and from 80.5 to 105.6% for lard. The intraday variabilities for boiled pork and lard ranged from 2.27 to 6.87% and from 1.52 to 9.78%, respectively, whereas the interday variabilities ranged from 1.81 to 7.89% and from 3.57 to 9.26%, respectively. Among the various meat samples, fish showed the highest level of COPs (31.84 μg/g). For the edible fats, the COP contents in tallow (22.79-60.15 μg/g) were much higher than those in lard (0.152-2.55 μg/g) and butter (0.526-1.36 μg/g). Collectively, this method can be applied to determine COPs in cholesterol-containing foodstuffs.

Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Petunia inflata possesses a self-incompatibility (SI) mechanism, which involves S-RNase and multiple S-locus F-box (SLF) genes at the polymorphic S-locus. For a given S-haplotype, each SLF is thought to interact with some of its non-self S-RNases, but not with its self S-RNase. In this work, we studied an allelic pair of SLF1, S2-SLF1 and S3-SLF1, which differ in 44 amino acids and show differential interactions with S3-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of S2-SLF1 and S3-SLF1, each with one of the three functional domains swapped, interact with S3-RNase. The results narrowed the candidate amino acids for specific interaction of S2-SLF1 with S3-RNase to the 16 in domain FD3. We then examined seven additional chimeric proteins by dividing FD3 into two subdomains and four mini-domains (A, B, C and D). The results further narrowed the candidate amino acids to four in mini-domain A and four in mini-domain D. Molecular modeling of interactions between S3-RNase and S2-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in S1-SLF1 and S6a-SLF1, both of which interact with S3-RNase based on the in vivo transgenic assay. Three of the chimeric proteins were used for the in vivo transgenic assay to determine whether FD3 alone contains the amino acids required for S2-SLF1 to interact with S7-RNase and S13-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.

The objectives of this study were to determine the variety and amount of various functional components in Scutellaria barbata D. Don as well as study their anti-inflammatory activity on RAW 264.7 cells. Both ethanol and ethyl acetate extracts were shown to contain the functional components including phenolics, flavonoids, chlorophylls, and carotenoids, with the former mainly composed of phenolics and flavonoids, and the latter of carotenoids and chlorophylls. Both extracts could significantly inhibit (p < 0.05) the production of lipopolysaccharide-induced nitric oxide, prostaglandin E 2 , interlukin-6, and interlukin-1β, as well as the expressions of phosphor extracellular signal-regulated kinase and phosphor-c-Jun N-terminal kinase (p-JNK), but failed to retard tumor necrosis factor-α expression. Both ethanol and ethyl acetate extracts had a dose-dependent anti-inflammatory activity on RAW 264.7 cells. Furthermore, the anti-inflammatory efficiency can be varied for both ethanol and ethyl acetate extracts, which can be attributed to the presence of different varieties and amounts of functional components, as mentioned above. This finding suggested that S. Barbata extract may be used as an anti-inflammatory agent for possible future biomedical application., (Copyright © 2017. Published by Elsevier B.V.)

The traditional way to analyze heterocyclic amines (HAs) is time-consuming and uses large amounts of solvents. The objective of this study is to develop a quick and simultaneous analysis method for multiple types of HAs contained in meat products. Results showed that 20 HAs and 1 internal standard (4,7,8-TriMeIQx) can be separated within 30 min using an Inspire C18 column and a gradient solvent system containing 10 mM ammonium acetate (pH 2.9) and acetonitrile. This process resulted in a high degree of separation. Using acetonitrile with 1% acetic acid as an extraction solvent, followed by primary and secondary amine, MgSO 4 , and C18EC as purified reagent, is highly suitable for extracting HAs using the quick, easy, cheap, effective, rugged, and safe method (QuEChERS). Tandem mass spectrometry with selected reaction monitoring mode were used for analysis, which indicated reasonable recovery (58.9-117.4%) for all 20 types of HAs along with limits of detection and quantification in the range of 0.003-0.05 and 0.01-0.05 ng/g, respectively.

Lycium barbarum L., a traditional Chinese herb widely used in Asian countries, has been demonstrated to be protective against chronic diseases such as age-related macular degeneration. The objectives of this study were to determine the carotenoid content in L. barbarum by high-performance liquid chromatography-mass spectrometry, followed by preparation of a carotenoid nanoemulsion to evaluate the mechanism of inhibition on HT-29 colon cancer cells. The highest extraction yield of carotenoids was attained by employing a solvent system of hexane-ethanol-acetone (1:1:1, v/v/v). Nine carotenoids, including neoxanthin (4.47 μg g -1 ), all-trans-zeaxanthin and its cis-isomers (1666.3 μg g -1 ), all-trans-β-cryptoxanthin (51.69 μg g -1 ), all-trans-β-carotene and its cis-isomers (20.11 μg g -1 ), were separated within 45 min and quantified using a YMC C 30 column and a gradient mobile phase of methanol-water (9:1, v/v) (A) and methylene chloride (B). A highly stable carotenoid nanoemulsion composed of Capryol TM 90, Transcutol ® HP, Tween 80 and deionized water was prepared with a mean particle size of 15.1 nm. Characterization of zeaxanthin standard, blank nanoemulsion, carotenoid extract and carotenoid nanoemulsion by differential scanning calorimetry curves and Fourier transform infrared spectra revealed a good dispersion of zeaxanthin-dominated carotenoid extract with no significant chemical change after incorporation into nanoemulsion. The in vitro release kinetic study showed a higher release profile at pH 5.2 than at physiological pH 7.4, suggesting a rapid release of carotenoids in the acidic environment (pH 4.5-6.5) characteristic of tumors. Both the carotenoid nanoemulsion and the extract were effective at inhibiting growth of HT-29 colon cancer cells, with an IC 50 of 4.5 and 4.9 μg ml -1 , respectively. Also, both treatments could up-regulate p53 and p21 expression and down-regulate CDK2, CDK1, cyclin A and cyclin B expression and arrest the cell cycle at G 2 /M. The study may form a basis for further exploration of L. barbarum nanoemulsion in cancer treatment.

Study Design  Pilot study using the rabbit model. Objective  Low back pain is often associated with disk degeneration. Cell therapy for degenerating disks may promote tissue regeneration and repair. Human dermal fibroblasts, obtained from the patient's skin tissue or donated tissue, may be a promising cell therapy option for degenerating disks. The objective of these studies is to determine the effects of intradiscal transplantation of neonatal human dermal fibroblasts (nHDFs) on intervertebral disk (IVD) degeneration by measuring disk height, magnetic resonance imaging (MRI) signal intensity, gene expression, and collagen immunostaining. Methods  New Zealand white rabbits ( n  = 16) received an annular puncture to induce disk degeneration and were treated with nHDFs or saline 4 weeks later. At 2 and 8 weeks post-treatment, X-ray and MRI images were obtained. IVDs were isolated and examined for changes in collagen staining and gene expression. Results  In the nHDF-treated group, there was a 10% increase in the disk height index after 8 weeks of treatment ( p  ≤ 0.05), and there was no significant difference in the saline-treated group. When compared with the saline-treated disks, disks treated with nHDFs showed reduced expression of inflammatory markers, a higher ratio of collagen type II over collagen type I gene expression, and more intense immunohistochemical staining for both collagen types I and II. Conclusions  Human dermal fibroblast introduction into the disk reduced inflammation and promoted tissue rich in both type I and type II collagens. The results of this study suggest that nHDFs would be a feasible cell therapy option for disk degeneration.

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)