Cox model risk score to predict survival of intrahepatic cholangiocarcinoma after ultrasound-guided ablation.

PURPOSE: To explore factors associated with overall survival (OS) and progression-free survival (PFS) of intrahepatic cholangiocarcinoma (iCCA) after ultrasound-guided ablation and establish a model for survival risk evaluation. METHODS: Data from 54 patients with 86 iCCAs between August 2008 and October 2022 were retrospectively analyzed. Cox regression were used to analyze the effects of clinical features on OS and PFS. Based on the variables screened by multivariable analysis, a model was established to predict the survival of the patients. Time-dependent receiver operating characteristic (timeROC) curve was constructed to evaluate the performance of this model. The model was further verified by bootstrap validation. The clinical usefulness of the model was evaluated by the decision curve analysis (DCA). RESULTS: During follow up, 39 patients died and 49 patients developed recurrence. Pre-ablation CA199 level > 140 U/ml was the only independent predictor of poor PFS. Age > 70 years, early recurrence, maximal diameter of tumor size > 1.5 cm and pre-ablation CA199 level > 140 U/ml were significantly associated with poor OS. Then a model was established based on the above four variables. The areas under the timeROC curve (AUC) for 1-year, 2-year, 3-year, 5-year were 0.767, 0.854, 0.791 and 0.848, respectively. After bootstrapping for 1000 repetitions, the AUCs were similar to the initial model. DCA also demonstrated that the model had good positive net benefits. CONCLUSION: The established model in this study could predict the survival outcomes of the patients with iCCA after thermal ablation, but further research was needed to validate the results.

Three-dimensional ultrasound fusion imaging in precise needle placement for thermal ablation of hepatocellular carcinoma.

PURPOSE: To investigate the value of three-dimensional ultrasound fusion imaging (3DUS FI) technique for guiding needle placement in hepatocellular carcinoma (HCC) thermal ablation. METHODS: A total of 57 patients with 60 HCCs with 3DUS FI-guided thermal ablation were retrospectively included in the study. 3DUS volume data of liver were acquired preoperatively by freehand scanning with the tumor and predetermined 5 mm ablative margin automatically segmented. Plan of needle placement was made through a predetermined simulated ablation zone to ensure a 5 mm ablative margin with the coverage rate toward tumor and ablative margin. With real-time ultrasound and 3DUS fusion imaging, ablation needles were placed according to the plan. After ablation, the ablative margin was immediately evaluated by contrast-enhanced ultrasound and 3DUS fusion imaging. The rate of adequate ablative margin, complete response (CR), local tumor progression (LTP), disease-free survival (DFS), and overall survival (OS) was evaluated. RESULTS: According to postoperative contrast-enhanced CT or MR imaging, the complete response rate was 100% (60/60), and 83% of tumors (30/36) achieved adequate ablative margin (>5 mm) three-dimensionally. During the follow-up period of 6.0-42.6 months, LTP occurred in 5 lesions, with 1- and 2-year LTP rates being 7.0% and 9.4%. The 1- and 2-year DFS rates were 76.1% and 65.6%, and 1- and 2-year OS rates were 98.1% and 94.0%. No major complications or ablation-related deaths were observed in any patients. CONCLUSIONS: Three-dimensional ultrasound fusion imaging technique may improve the needle placement of thermal ablation for HCC and reduce the rate of LTP.

Quantitative Parameters of Contrast-Enhanced Ultrasound Predicting the Response to Combined Immune Checkpoint Inhibitor and Anti-angiogenesis Therapies for Unresectable Hepatocellular Carcinoma.

OBJECTIVE: The aim of the work described here was to explore the value of contrast-enhanced ultrasound (CEUS) quantitative parameters in predicting the response of combined immune checkpoint inhibitor (ICI) and anti-angiogenesis therapies for unresectable hepatocellular carcinoma (HCC). METHODS: Sixty-six HCC patients who underwent combined ICI and anti-angiogenesis therapies were prospectively enrolled. A CEUS examination was performed at baseline, and tumor perfusion parameters were obtained with perfusion quantification software. The differences in CEUS quantitative parameters between the responder and non-responder groups were compared, and the correlations between CEUS parameters and progression-free survival (PFS) was evaluated. RESULTS: The objective response rate (ORR) was 21.2%. The values of rising time (RT) ratio, time to peak ratio, fall time ratio, peak enhancement ratio, wash-in rate ratio, wash-in perfusion index ratio and wash-out rate ratio differed significantly differed between the responder and non-responder groups (all p values < 0.05). Multivariable logistic regression analysis revealed that the RT ratio was the only independent factor associated with the ORR (odds ratio = 0.007, 95% confidence interval: 0.000-0.307, p = 0.010). The median RT ratios of the responder and non-responder groups were 36.9 and 58.9, respectively (p = 0.006). The appropriate cutoff point of the RT ratio was 80.1, determined with the X-tile program. Survival analysis indicated high PFS for the patients with a lower RT ratio (high RT ratio vs. low RT ratio = 4.4 mo vs. not reached, p = 0.001). CONCLUSION: CEUS quantitative parameters may predict the efficacy of ICI and anti-angiogenesis combined therapies for HCC.

Development and Assessment of Nomogram Based on AFP Response for Patients with Unresectable Hepatocellular Carcinoma Treated with Immune Checkpoint Inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have been increasingly used to treat hepatocellular carcinoma (HCC). Prognostic biomarkers are an unmet need. We aimed to develop a prognostic nomogram for patients with unresectable HCC receiving ICIs therapy. METHODS: A total of 120 patients with unresectable HCC receiving ICIs treatment were enrolled in this study. Patients were randomly divided into a training set (n = 84) and a validation set (n = 36) in a 7:3 ratio. Clinical characteristics were retrospectively analyzed. Serum α-fetoprotein protein (AFP) response was defined as a decline of ≥20% in AFP levels within the initial eight weeks of treatment. Univariable and multivariable Cox analyses were used to select relevant variables and construct the nomogram. The areas under the receiver operating characteristic curves (AUCs) were used to determine the performance of the model. Kaplan-Meier analysis with the log-rank test was used to compare different risk groups. RESULTS: The median progression-free survival (PFS) was 7.7 months. In the multivariate Cox analysis, the presence of extrahepatic metastasis (hazard ratio [HR] = 2.08, 95% confidence interval [CI]: 1.02-4.27, p < 0.05), white blood cell count (HR = 3.48, 95% CI: 1.02-11.88, p < 0.05) and AFP response (HR = 0.41, 95% CI: 0.18-0.95, p < 0.05) independently predicted PFS. A nomogram for PFS was established with AUCs of 0.79 and 0.70 in the training and validation sets, respectively. The median PFS of the high- and low-risk subgroups was 3.5 and 11.7 months, respectively (p < 0.05). CONCLUSION: The nomogram could predict PFS in patients with unresectable HCC receiving ICIs treatment and further help decision making in daily clinical practice.

Liquids with High Compressibility.

Compressibility is a fundamental property of all materials. For fluids, that is, gases and liquids, compressibility forms the basis of technologies such as pneumatics and hydraulics and determines basic phenomena such as the propagation of sound and shock waves. In contrast to gases, liquids are almost incompressible. If the compressibility of liquids could be increased and controlled, new applications in hydraulics and shock absorption could result. Here, it is shown that dispersing hydrophobic porous particles into water gives aqueous suspensions with much greater compressibilities than any normal liquids such as water (specifically, up to 20 times greater over certain pressure ranges). The increased compressibility results from water molecules being forced into the hydrophobic pores of the particles under applied pressure. The degree of compression can be controlled by varying the amount of porous particles added. Also, the pressure range of compression can be reduced by adding methanol or increased by adding salt. In all cases, the liquids expand back to their original volume when the applied pressure is released. The approach shown here is simple and economical and could potentially be scaled up to give large amounts of highly compressible liquids.

Student perceptions toward virtual reality training in dental implant education.

Objectives: Both the shortage of professional teaching resources and the expensive dental implant supplies impede the effective training of dental undergraduate in implantology. Virtual reality (VR) technology may provide solutions to solve these problems. This pilot study was implemented to explore the usability and acceptance of a VR application in the training of dental implant among dental students at the Jinan University School of Stomatology. Methods: We designed and developed a VR system with head-mounted displays (HMDs) to assist dental implant training. Undergraduate dental students were invited to experience a 30-minute "Introduction to dental implants" VR-HMDs training module. A total of 119 dental students participated the training. Firstly, the VR interactive training on dental implant was described, illustrated and practiced. Next, a system usability scale (SUS) survey was used to verify the usability and feasibility of the VR application on training dental students. Finally, the participants were given a questionnaire to provide their perceptions and feedback of the usefulness of the VR application for training dental implant skills. Results: The SUS score was 82.00 ± 10.79, indicating a top 10 percentage ranking of the system's usabilitys. The participants' answers to the questionnaire reflected most of them exhibited strong interests in the VR system, with a tendency that the female students were more confident than the male in manipulating the VR system. The participants generally acknowledged the usefulness of VR dental implants, ranking VR value above the traditional laboratory operations, and a preference for using the VR system on learning other skills. They also gave valuable suggestions on VR dental implants for substantial improvement. However, some students were not strongly positive about the VR training in this study, the reason might lie in a more theoretical module was selected for testing, which impacted the students' ratings. Conclusions: In this study we revealed the feasibility and usability of VR applications on training dental implant among undergraduate dental students. This pilot study showed that the participants benefited from the dental implant VR training by practicing the skills repeatedly. The feedback from student participants affirmed the advantages and their acceptance of the VR application in dental education. Especially, the VR-based technology is highly conducive to clinical operating skills and surgical procedures-focused training in medical education, indicating that the VR system should be combined with the traditional practice approach in improving dental students' practical abilities.

Dynamic chromatin conformation and accessibility changes mediate the spatial-specific gene regulatory network in Bombyx mori.

Silk gland genes of Bombyx mori can have strict spatial expression patterns, which impact their functions and silk quality. However, our understanding of their regulation mechanisms is currently insufficient. To address this, the middle silk gland (MSG) and posterior silk gland (PSG) of the silkworm were investigated. Gene ontology annotation showed that spatially specific expressed genes were involved in the formation of H3k9me and chromatin topology. Chromatin conformation data generated by Hi-C showed that the topologically associated domain boundaries around FibL and Sericin1 genes were significantly different between MSG and PSG. Changes in chromatin conformation led to changes in chromatin activity, which significantly affected the expression of nearby genes in silkworm. Chromatin accessibility regions of MSG and PSG were analyzed using FAIRE-seq, and 1006 transcription factor motifs were identified in open chromatin regions. Furthermore, the spatial-specific expression patterns of silk gland genes were mainly associated with homeobox-contained transcription factors, such as POU-M2, which was specifically bound and relatively highly expressed in the MSG. The regulatory network mediated by POU-M2 regulated most of the spatial-specific expressed genes in MSG, such as ADH1. These results can aid in improving silk performance, optimizing silkworm breeding, and improving the gene spatial regulatory model research for insects.

Two B-box proteins, PavBBX6/9, positively regulate light-induced anthocyanin accumulation in sweet cherry.

Anthocyanin production in bicolored sweet cherry (Prunus avium cv. Rainier) fruit is induced by light exposure, leading to red coloration. The phytohormone abscisic acid (ABA) is essential for this process, but the regulatory relationships that link light and ABA with anthocyanin-associated coloration are currently unclear. In this study, we determined that light treatment of bicolored sweet cherry fruit increased anthocyanin accumulation and induced ABA production and that ABA participates in light-modulated anthocyanin accumulation in bicolored sweet cherry. Two B-box (BBX) genes, PavBBX6/9, were highly induced by light and ABA treatments, as was anthocyanin accumulation. The ectopic expression of PavBBX6 or PavBBX9 in Arabidopsis (Arabidopsis thaliana) increased anthocyanin biosynthesis and ABA accumulation. Overexpressing PavBBX6 or PavBBX9 in sweet cherry calli also enhanced light-induced anthocyanin biosynthesis and ABA accumulation. Additionally, transient overexpression of PavBBX6 or PavBBX9 in sweet cherry peel increased anthocyanin and ABA contents, whereas silencing either gene had the opposite effects. PavBBX6 and PavBBX9 directly bound to the G-box elements in the promoter of UDP glucose-flavonoid-3-O-glycosyltransferase (PavUFGT), a key gene for anthocyanin biosynthesis, and 9-cis-epoxycarotenoid dioxygenase 1 (PavNCED1), a key gene for ABA biosynthesis, and enhanced their activities. These results suggest that PavBBX6 and PavBBX9 positively regulate light-induced anthocyanin and ABA biosynthesis by promoting PavUFGT and PavNCED1 expression, respectively. Our study provides insights into the relationship between the light-induced ABA biosynthetic pathway and anthocyanin accumulation in bicolored sweet cherry fruit.

Genome-Wide Identification of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Sweet Cherry Fruit.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play important regulatory roles during plant growth and development, fruit ripening, inflorescence branching, and biotic and abiotic stresses. However, there have been no identification or systematic studies of the SPL gene family in the sweet cherry. In this study, 12 SPL genes were identified in the sweet cherry reference genome, which were distributed over 6 chromosomes and classified into six groups according to phylogenetic relationships with other SPL gene families. Nine PavSPLs were highly expressed at green fruit stages and dramatically decreased at the onset of fruit ripening, which implied that they were important regulators during fruit development and ripening. The expression patterns of PavSPL genes under ABA, GA, and MeJA treatments showed that the PavSPLs were involved in the process of fruit ripening. A subcellular localization experiment proved that PavSPL4 and PavSPL7 proteins were localized in the nucleus. The genome-wide identification of the SPL gene family provided new insights while establishing an important foundation for sweet cherry studies.

MicroRNA156ab regulates apple plant growth and drought tolerance by targeting transcription factor MsSPL13.

Drought stress substantially reduces the productivity of apple plants and severely restricts the development of apple industry. Malus sieversii, wild apples with excellent drought resistance, is a valuable wild resource for a rootstock improvement of cultivated apple (Malus domestica). miRNAs and their targets play essential roles in plant growth and stress responses, but their roles in drought stress responses in apple are unknown. Here, we demonstrate that microRNA156ab is upregulated in M. sieversii in response to drought stress. Overexpressing msi-miR156ab promoted auxin accumulation, maintained the growth of apple plants, and increased plant resistance to osmotic stress. Antioxidant enzyme activities and proline contents were also increased in miR156ab-OE transgenic apple lines, which improved drought resistance. The squamosa promoter binding protein-like transcription factor MsSPL13 is the target of msi-miR156ab, as demonstrated by 5'-RACE and dual luciferase assays. Heterologous expression of MsSPL13 decreased auxin contents and inhibited growth in Arabidopsis (Arabidopsis thaliana) under normal and stress conditions. The activities of antioxidant enzymes were also suppressed in MsSPL13-OE transgenic Arabidopsis, reducing drought resistance. We showed that MsSPL13 regulates the expression of the auxin-related genes MsYUCCA5, PIN-FORMED7 (MsPIN7), and Gretchen Hagen3-5 (MsGH3-5) by binding to the GTAC cis-elements in their promoters, thereby regulating auxin metabolism. Finally, we demonstrated that the miR156ab-SPL13 module is involved in mediating the difference in auxin metabolism and stress responses between M. sieversii and M26 (M. domestica) rootstocks. Overall, these findings reveal that the miR156ab-SPL13 module enhances drought stress tolerance in apples by regulating auxin metabolism and antioxidant enzyme activities.

Hemodynamic effects of intravenous bolus injection of iopromide 370 twice in abdominal contrast-enhanced CT and coronary CTA dual-site sequential examinations.

Little information is available about how intravenous bolus injection of iopromide 370 twice in a short time will affect hemodynamics and whether the changes reach clinically relevant levels. In the present study, 31 healthy adult volunteers received abdominal contrast-enhanced CT and coronary CTA sequential examinations. The same dose and rate of normal saline was injected 30 min in advance as self-control. Hemodynamic data were noninvasively collected at selected time points from 1 min prior to injection to 30 min post-injection. The results showed that after iopromide 370 injection, except for stroke volume, all other indicators changed immediately during the first injection, changed most significantly during the second injection (P < 0.05), and returned to baseline within 10 min. Heart rate and cardiac output exhibited the most pronounced changes, with an increasing rate of 33.5% and 33.8%, respectively. For indicators with a change range of > 15% during the second injection, except for mean arterial pressure and total peripheral resistance, the proportions of subjects for the other indicators between the two groups were statistically different (P < 0.05). In conclusion, intravenous bolus injection of iopromide 370 twice in dual-site sequential examinations induced dose-cumulative and time-dependent hemodynamic effects, which all fluctuated within the normal ranges.

Recoil attenuation for deepwater drilling riser systems via delayed H∞ control.

This paper deals with the recoil suppression problem of a deepwater drilling riser system via active H∞ control using both current and delayed states. First, based on the three degrees of freedom spring-mass-damping model of the riser system, an incremental dynamic equation of the system subject to the platform heave motion and the friction force induced by drilling discharge mud and seawater is established. Then, to reject recoil movements of the riser, a delayed state feedback H∞ controller with delayed states as well as current states is designed. The existence conditions and the design method of the delayed H∞ recoil controllers are presented. Third, the effects of the introduced time-delays on the recoil control of the riser are analyzed, and the design of optimal artificial time-delays is formulated as the minimum value problem of a series of quintic algebraic polynomials, which are related to the weights of average response amplitudes, steady-state errors, and the control force. Lastly, simulation results are provided to demonstrate the effectiveness of delay-free and delayed H∞ recoil control schemes for the riser. It is shown that (i) under the delayed H∞ controllers, the recoil responses of the riser can be controlled significantly; (ii) the decay rate of the recoil response under the delay-free H∞ controller is slightly faster than the one under the delayed H∞ controllers. However, the former requires more control cost than the latter; (iii) compared with the delayed H∞ controller with the existing linear quadratic optimal controller, the control cost by the former is larger than that by the latter. However, the steady-state errors of the riser under the latter are slightly smaller than that under the former; (iv) the introduced time-delays with proper size play positive role of suppressing recoil response of the system, and the corresponding delayed H∞ controller series provide more options for recoil control of the riser.

miR164g-MsNAC022 acts as a novel module mediating drought response by transcriptional regulation of reactive oxygen species scavenging systems in apple.

Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species Malus sieversii and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor MsNAC022 in M. sieversii. The heterologous accumulation of miR164g in Arabidopsis thaliana results in enhanced sensitivity to drought stress, while overexpression of MsNAC022 in Arabidopsis and the cultivated apple line 'GL-3' (Malus domestica Borkh.) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly PEROXIDASE (MsPOD). Furthermore, we showed that expression of MsPOD is activated by MsNAC022 in transient assays. Interestingly, Part1 (P1) region is the key region for the positive regulation of MsPOD promoter by MsNAC022, and the different POD expression patterns in M. sieversii and M. domestica is attributed to the specific fragments inserted in P1 region of M. sieversii. Our findings reveal the function of the miR164g-MsNAC022 module in mediating the drought response of M. sieversii and lay a foundation for breeding drought-tolerant apple cultivars.

Abscisic acid-responsive transcription factors PavDof2/6/15 mediate fruit softening in sweet cherry.

Softening is a key step during fruit ripening that is modulated by the interplay between multiple phytohormones. The antagonistic action of abscisic acid (ABA) and auxin determines the rate of fruit ripening and softening. However, the transcription factors that integrate ABA and auxin signals to regulate fruit softening remain to be determined. In this study, we identified several DNA-binding with One Finger (Dof) transcription factors essential for ABA-promoted fruit softening, based on transcriptome analysis of two sweet cherry (Prunus avium L.) varieties with different fruit firmness. We show that PavDof6 directly binds to the promoters of genes encoding cell wall-modifying enzymes to activate their transcription, while PavDof2/15 directly repress their transcription. Transient overexpression of PavDof6 and PavDof2/15 in sweet cherry fruits resulted in precocious and delayed softening, respectively. In addition, we show that the auxin response factor PavARF8, the expression of whose encoding gene is repressed by ABA, activates PavDof2/15 transcription. Furthermore, PavDof2/6/15 and PavARF8 directly bind to the 9-cis-epoxycarotenoid dioxygenase 1 (PavNCED1) promoter and regulate its expression, forming a feedback mechanism for ABA-mediated fruit softening. These findings unveil the physiological framework of fruit softening and establish a direct functional link between the ABA-PavARF8-PavDofs module and cell-wall-modifying genes in mediating fruit softening.

Virtual reality approach for orthodontic education at School of Stomatology, Jinan University.

OBJECTIVES: The development of virtual reality (VR) has exerted an enormous impact on higher education. However, the VR application in dental education is still at an early stage in China. This study explored the usability of a VR application in training the practical skills of dental students. METHODS: We first utilized the "system usability scale" (SUS) to verify the validity of the VR application in this study. Next, the VR training on orthodontic bracket bonding was experienced by the participants. The subsequent survey was delivered to collect the participants' perception and evaluation of the VR system application in training the practical skills of prospective dentists. RESULTS: The SUS score was 76.17 ± 9.89, suggesting an above-average evaluation of the system's usability and maturity. The response from the questionnaire supplied relatively positive responses to the VR system application on "user experiences," "perceived usefulness of VR application on orthodontics," and "perceived ease of manipulation." The male students exhibited strong interest in manipulating the VR system and were more capable of manipulating the VR system than the female students. VR experience did not affect the participants' responses to the questions. CONCLUSIONS: Overall, the use of VR in dental education achieved the expected outcomes. Most students identified the VR training as an enjoyable learning process, and it could be repetitively experienced without further costs, which might have potential positive effects on long-term learning outcomes, although there is room to further improve the effectiveness evaluation of VR.

Identification of immunophenotypes in esophageal squamous cell carcinoma based on immune gene sets.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is a malignant tumor with high heterogeneity. Research on molecular mechanisms involved in the process of tumor origination and progression is extremely limited to investigating mechanisms of molecular typing for ESCC. METHODS: After comprehensively analyzing the gene expression profiles in The Cancer Genome Atlas and Gene Expression Omnibus databases, we identified four immunotypes of ESCC (referred to as C1-C4) based on the gene sets of 28 immune cell subpopulations. The discrepancies in prognostic value, clinical features, drug sensitivity, and tumor components between the immunotypes were individually analyzed. RESULTS: The ranking of immune infiltration is C1 > C4 > C3 > C2. These subtypes are characterized by high and low expression of immune checkpoint proteins, enrichment and insufficiency of immune-related pathways, and differential distribution of immune cell subgroups. Poorer survival was observed in the C1 subtype, which we hypothesized could be caused by an immunosuppressive cell population. Fortunately, C1's susceptibility to anti-PD-1 therapy offers hope for patients with poor prognosis in advanced stages. On the other hand, C4 is sensitive to docetaxel, which may offer novel treatment strategies for ESCC in the future. It is worth noting that immunophenotyping is tightly bound to the abundance of stromal components and stem cells, which could explain the tumor immune escape to some extent. Ultimately, determination of hub genes based on the C1 subtypes provides a reference for the discovery of immunotarget drugs against ESCC. CONCLUSION: The identification of immunophenotypes in our study provides new therapeutic strategies for patients with ESCC.

Multi-Omics Integration to Reveal the Mechanism of Sericin Inhibiting LPS-Induced Inflammation.

Sericin is a natural protein with high application potential, but the research on its efficacy is very limited. In this study, the anti-inflammatory mechanism of sericin protein was investigated. Firstly, the protein composition of sericin extracts was determined by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). This was then combined with Enzyme-linked Immunosorbent Assay (ELISA) and Quantitative Real-time PCR (qRT-PCR), and it was confirmed that the anti-inflammation ability of sericin was positively correlated with the purity of sericin 1 protein. Finally, RNA-seq was performed to quantify the inhibitory capacity of sericin sample SS2 in LPS-stimulated macrophages. The gene functional annotation showed that SS2 suppressed almost all PRRs signaling pathways activated by lipopolysaccharides (LPS), such as the Toll-like receptors (TLRs) and NOD-like receptors (NLRs) signaling pathways. The expression level of adaptor gene MyD88 and receptor gene NOD1 was significantly down-regulated after SS2 treatment. SS2 also reduced the phosphorylation levels of NF-κB P65, P38, and JNK, thereby reducing the expressions of IL-1ß, IL-6, INOS, and other inflammatory cytokines. It was confirmed that sericin inhibited LPS-induced inflammation through MyD88/NF-κB pathway. This finding provides necessary theoretical support for sericin development and application.

Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.).

Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis-elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.

Genome-Wide Identification of the Xyloglucan endotransglucosylase/Hydrolase (XTH) and Polygalacturonase (PG) Genes and Characterization of Their Role in Fruit Softening of Sweet Cherry.

Fruit firmness is an important economical trait in sweet cherry (Prunus avium L.) where the change of this trait is related to cell wall degradation. Xyloglucan endotransglycosylase/hydrolase (XTH) and polygalacturonases (PGs) are critical cell-wall-modifying enzymes that occupy a crucial position in fruit ripening and softening. Herein, we identified 18 XTHs and 45 PGs designated PavXTH1-18 and PavPG1-45 based on their locations in the genome of sweet cherry. We provided a systematical overview of PavXTHs and PavPGs, including phylogenetic relationships, conserved motifs, and expression profiling of these genes. The results showed that PavXTH14, PavXTH15 and PavPG38 were most likely to participated in fruit softening owing to the substantial increment in expression during fruit development and ripening. Furthermore, the phytohormone ABA, MeJA, and ethephon significantly elevated the expression of PavPG38 and PavXTH15, and thus promoted fruit softening. Importantly, transient expression PavXTH14, PavXTH15 and PavPG38 in cherry fruits significantly reduced the fruit firmness, and the content of various cell wall components including hemicellulose and pectin significantly changed correspondingly in the transgenic fruit. Taken together, these results present an extensive analysis of XTHs and PGs in sweet cherry and provide potential targets for breeding softening-resistant sweet cherry cultivars via manipulating cell wall-associated genes.

Expression of the Malus sieversii NF-YB21 Encoded Gene Confers Tolerance to Osmotic Stresses in Arabidopsis thaliana.

Drought is the main environmental factor that limits the yield and quality of apples (Malus × domestica) grown in arid and semi-arid regions. Nuclear factor Ys (NF-Ys) are important transcription factors involved in the regulation of plant growth, development, and various stress responses. However, the function of NF-Y genes is poorly understood in apples. Here, we identified 43 NF-Y genes in the genome of apples and conducted an initial functional characterization of the apple NF-Y. Expression analysis of NF-Y members in M. sieversii revealed that a large number of NF-Ys were highly expressed in the roots compared with the leaves, and a large proportion of NF-Y genes responded to drought treatment. Furthermore, heterologous expression of MsNF-YB21, which was significantly upregulated by drought, led to a longer root length and, thus, conferred improved osmotic and salt tolerance in Arabidopsis. Moreover, the physiological analysis of MsNF-YB21 overexpression revealed enhanced antioxidant systems, including antioxidant enzymes and compatible solutes. In addition, genes encoding catalase (AtCAT2, AtCAT3), superoxide dismutase (AtFSD1, AtFSD3, AtCSD1), and peroxidase (AtPER12, AtPER42, AtPER47, AtPER51) showed upregulated expression in the MsNF-YB21 overexpression lines. These results for the MsNF-Y gene family provide useful information for future studies on NF-Ys in apples, and the functional analysis of MsNF-YB21 supports it as a potential target in the improvement of apple drought tolerance via biotechnological strategies.