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1.
Talanta ; 206: 120200, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514845

RESUMO

Highly-sensitive and contamination-free droplet digital PCR (ddPCR) is an enabling technology and widely needed for accurate quantification of nucleic acid in clinical applications. In this paper, a novel droplet reader was developed by combining a "quasi" confocal laser-induced fluorescence (LIF) cytometry with a delicate microfluidic chip design. The droplets with a size of 90 µm was illuminated at an out-of-focus position by two aligned laser beams to generate maximum fluorescent signal. Additionally, the lateral offset position of the microfluidic chip should be precisely tuned so that the bandwidth of the FAM and VIC channels were configured at the matching sizes. Then, PMT gain voltages and pneumatic pressures were optimized for better droplet detection efficiencies. An aerosol adsorption experiment was performed to demonstrate that there was no aerosol contamination, and detected copy numbers of both mutants and wild types scaled linearly with the expected input copy numbers (r2>0.998) with a LoB of 0.0 copies and LoD of 3.0 copies. The results demonstrated that this droplet reader with the delicate chip is a convenient, highly-sensitive and contamination-free to detect fluorescence signals inside droplets after ddPCR, which is highly promising for broad applications of ddPCR in clinical diagnosis.

2.
J Med Virol ; 92(1): 34-44, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31429942

RESUMO

Mounting evidence indicates that serum soluble E-cadherin (sE-cadherin) serves as an important player in various physiological and pathological processes. However, the crosstalk between serum sE-cadherin and oxidative stress in chronic hepatitis B (CHB) remains to be illustrated. The main purpose of this study is to explore the molecular mechanisms underlying the function of sE-cadherin in CHB virus infection. Levels of serum sE-cadherin, total antioxidant capacity (TAC), glutathione (GSH), superoxide dismutase (SOD), total oxidant activity (TOA), NADPH oxidase 2 (NOX2), and malondialdehyde (MDA), from 51 patients with hepatitis B envelope antigen (HBeAg)-negative CHB, 54 patients with HBeAg-positive CHB, and 109 healthy individuals were detected by enzyme-linked immunosorbent assay. In our study, patients with CHB showed significantly higher serum sE-cadherin levels than healthy individuals (P < .01). Furthermore, we also found that the serum sE-cadherin levels were significantly negatively correlated with TAC, antioxidant enzymes (GSH and SOD) in patients with CHB, and that serum sE-cadherin concentrations were significantly positively correlated with liver enzyme markers (alanine transaminase and aspartate aminotransferase) and oxidative markers (TOA, NOX2, and MDA) in patients with CHB. Therefore, serum sE-cadherin may act as a new candidate biomarker for reflecting inflammation and oxidative stress status in the development and progression of hepatitis B virus infection.

3.
Nanotechnology ; 31(7): 075708, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31665701

RESUMO

Perovskite nanocrystal embedded polymer nanofibers with polarized emissions are interesting materials for down-shifting applications. By using a folded aluminum foil as a collector, we fabricated inch-size aligned polymer nanofiber films with embedded CH3NH3PbBr3 nanocrystals by adapting an electrospinning technique. It was found that the addition of an appropriate amount of cyanoethyl cellulose (CEC) makes the dispersion of MAPbBr3 in the nanofibers more uniform. Using a precursor solution with MAPbBr3 of 10% and CEC of 1 wt%, the resulting nanofiber films show strong polarized emission with quantum yields up to 51%. The emission dichroic ratio and emission polarization ratio can reach 5.21 and 0.43, respectively. These polarized emissive films can be potentially applied as down converters for liquid crystal display backlights and other polarization selective photonic devices.

4.
Meat Sci ; 159: 107919, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31472934

RESUMO

The effects of edible chitosan (CTS) coatings (0, 1, 2, and 3%) on Harbin red sausage storage stability at room temperature compared to vacuum packaging are discussed. The pH, moisture and L* of sausages decreased gradually, while the a*, TBARS, total aerobic bacteria (TAB) and lactic acid bacteria (LAB) increased significantly (P < 0.05) with the prolongation of storage time. In the same storage time, the storage stability was improved with the increase in the concentration of edible CTS coating, but there was no significant difference between 2% and 3% (P > 0.05). Although vacuum packaging can effectively maintain colour, moisture and reduce lipid oxidation, 2% CTS is outstanding in sustaining storage stability (pH and water distribution) and inhibition of microbial growth (TAB and LAB). In addition, no significant difference was observed between CTS coating and vacuum packaging on aroma and flavour (P > 0.05). In conclusion, CTS coating as edible packaging material can be used in meat products preservation.

5.
J Nanosci Nanotechnol ; 20(2): 1232-1237, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31383123

RESUMO

CO atmosphere plasma and first H2 then CO plasma were attempted to substitute the traditional "calcination-reduction-carburization" processes for the preparation of metallic cobalt phase with hcp structure. CoRu/SiO2 catalyst precursor was prepared via incipient wetness impregnation. Characterization and catalytic results showed that CO could be decomposed in glow discharge plasma field, a large fraction of carbon species was deposited as elementary substance on the catalyst surface, while some of the carbon reacted with cobalt and formed cobalt carbide, which could transform into Co0 with hcp structure after reduction. The hcp structured cobalt containing catalysts showed higher initial Fischer-Tropsch synthesis activity than both calcined and air plasma treated samples.

6.
J Affect Disord ; 260: 334-341, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31521871

RESUMO

BACKGROUND: To investigate mental and physical health comorbidity with chronic back or neck pain in the Chinese population, and assess the level of disability associated with chronic back or neck pain. METHODS: Data were derived from a large-scale and nationally representative community survey of adult respondents on mental health disorders in China (n = 28,140). Chronic back or neck pain, other chronic pain conditions and chronic physical conditions were assessed by self-report. Mental disorders were assessed by the Composite International Diagnostic Interview (CIDI). Role disability during the past 30 days was assessed with the World Health Organization Disability Assessment Schedule (WHO-DAS-II). RESULTS: The 12-month prevalence of chronic back or neck pain was 10.8%. Most of respondents with chronic back or neck pain (71.2%) reported at least one other comorbid condition, including other chronic pain conditions (53.4%), chronic physical conditions (37.9%), and mental disorders (23.9%). It was found by logistic regression that mood disorders (OR = 3.7, 95%CI:2.8-4.8) showed stronger association with chronic back or neck pain than anxiety disorders and substance disorders. Most common chronic pains and physical conditions were significantly associated with chronic back or neck pain. Chronic back or neck pain was associated with role disability after controlling for demographics and for comorbidities. Physical and mental comorbidities explained 0.7% of the association between chronic back or neck pain and role disability. CONCLUSIONS: Chronic back or neck pain and physical-mental comorbidity is very common in China and chronic back or neck pain may increase the likelihood of other physical and mental diseases. This presents a great challenge for both clinical treatment and public health education. We believe that further study needs to be conducted to improve the diagnostic and management skills for comorbidity conditions.

7.
Sensors (Basel) ; 19(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671560

RESUMO

This work presents a cost-effective shadow mask printing approach to fabricate flexible sensors. The liquid-state sensing material can be directly brushed on a flexible substrate through a shadow mask. The ink leakage issue which often occurs in printed electronics is addressed with a custom taping scheme. A simple thermal compression bonding approach is also proposed to package the functional area of the sensor. To verify the feasibility and robustness of the proposed fabrication approach, a prototyped strain gauge displacement sensor is fabricated using carbon ink as the sensing material and a flexible polyimide (PI) film as the substrate. Once the substrate is deformed, cracks in the solidified ink layer can cause an increased resistance in the conductive path, thus achieving function of stable displacement/strain sensing. As a demonstration for displacement sensing application, this sensor is evaluated by studying its real-time resistance response under both static and dynamic mechanical loading. The fabricated sensor shows a comparable performance (with a gauge factor of ~17.6) to those fabricated using costly lithography or inkjet printing schemes, while with a significantly lower production cost.

8.
ACS Appl Mater Interfaces ; 11(47): 44700-44707, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31670938

RESUMO

With the minimization and higher power of electronic devices, materials with effective heat dissipation and high electrical insulation have attracted relentless interest. Especially, highly thermally conductive, highly electrically insulating but low filler content of polymer-based composites are desirable. Herein, a facile and eco-friendly cotton candy-templated method (CTM) to construct three-dimensional heat transport pathways inside epoxy resin is reported. The fabricated Al2O3/epoxy composites with enhanced heat transport capability feature a 15-fold increase in thermal conductivity at a filler content of 36.2 vol % compared to pristine epoxy. Moreover, the remarkable thermal conductive property has excellent stability over a wide range of temperature before and after heating and cooling cycles. Meanwhile, the CTM composite still retain highly electrical insulation. The cotton candy-templated method proposed in this work is a new avenue for the preparation of three-dimensional heat transport pathways within polymer-based composites for microelectronic packaging and electrical engineering systems.

9.
Chem Commun (Camb) ; 55(95): 14287-14290, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712798

RESUMO

A novel bright near-infrared II (NIR-II, 1000-1700 nm) fluorescent probe with excellent water-solubility, superior photostability, and excellent in vitro and in vivo biocompatibility was facilely synthesized for in vivo biomedical imaging of xenograft breast tumor and chemically induced spontaneous breast carcinoma. To the best of our knowledge, it is the first time that the superior practical applications of this NIR-II probe in dimethylbenzanthracene (DMBA)-induced rat mammary carcinoma imaging and image-guided rat carcinoma surgery were demonstrated.

10.
Chemosphere ; 243: 125356, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31743867

RESUMO

1-nitropyrene (1-NP) is widespread in the environment, as a typical nitrated polycyclic aromatic hydrocarbon. The purpose of this research was to explore the effects of gestational 1-NP exposure on susceptibility of allergic asthma in offspring. Maternal mice were exposed to 1-NP (100 µg kg-1) by gavage throughout the whole pregnancy. Pups were sensitized by injecting with ovalbumin (OVA) on postnatal day (PND)23, 29, and 36, respectively. At 7 days following the last injection, sensitized mice were exposed to aerosol OVA. As expected, there were quite a few inflammatory cells in the lungs of OVA-sensitized pups, accompanied by bronchial wall thickening and hyperemia. Elevated goblet cells and overproduced mucus were observed in the airways of OVA-sensitized pups. Interestingly, gestational 1-NP exposure aggravated infiltration of inflammatory cells, mainly eosinophils, in OVA-sensitized offspring. Although it had little effect on airway smooth muscle layer thickening and basement membrane fibrosis, gestational 1-NP exposure aggravated goblet cell hyperplasia, Muc5ac mRNA upregulation, and mucus secretion in the airways of OVA-sensitized and challenged offspring. Mechanistically, gestational 1-NP exposure aggravated elevation of pulmonary IL-5 in OVA-sensitized pups. These findings suggest that gestational 1-NP exposure increases susceptibility of allergic asthma in offspring.

11.
Environ Pollut ; : 113593, 2019 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-31771930

RESUMO

Di (2-ethyl-hexyl)phthalate (DEHP) is an environmental endocrine disruptor and commonly used as plasticizer. Maternal DEHP exposure during pregnancy reduces placental size and destroys placental structure. However, the underlying mechanisms were unclear. In this study, we supposed that DEHP disturbs endocrine function of placenta to inhibit the proliferation of placental cell. Using radioimmunoassay and ELISA, we found that DEHP and its active metabolite mono (2-ethyl-hexyl) phthalate (MEHP) promoted progesterone secretion in pregnant mouse and in JEG-3 cells, respectively. Therefore, placental endocrine function was altered by DEHP. The mRNA and protein level of progesterone synthetase 3ß-HSD1 was elevated by DEHP, which is conducive to the synthesis of progesterone. The level of progesterone receptor was down-regulated by DEHP and MEHP in mouse placenta and in JEG-3 cells, respectively. PR deficiency further promoted the level of 3ß-HSD1, which leads to a higher progesterone level. In turn, higher concentration of progesterone further inhibited the expression of PGR (PR gene). Therefore, higher progesterone down-regulated the level of its receptor PR. Meanwhile, decreased PR induced more progesterone secretion. There is a feedback loop between progesterone and PR. PR deficiency down-regulated the protein level of Cyclin D1 which plays an important role in cell proliferation. Accordingly, DEHP and its active metabolite MEHP can restrain proliferation of placental cell and disturb the progesterone secretion via decreasing the level of PR.

12.
Integr Zool ; 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31773881

RESUMO

Post-dispersal seed predation plays an important role in plant demography and biodiversity maintenance. However, the effects of seed density on seed predation from previous studies have been inconsistent. We dissected the effects of density on the two-step processes of seed predation by using 101,520 seeds from 62 plant species in an alpine pine forest for three consecutive years. We present a clear manner to explain the current controversy surrounding the effects of density on seed predation. Seed encounter frequency (at least one seed being predated from an experiment depot) showed positive density dependence, while seed exploitation (the proportion of seeds being predated of the encountered depots) showed negative density dependence. Both density effects showed a consistent trend but with different effect magnitudes among years. Final seed predation is the combination of seed encounter and seed exploitation. Final seed predation could be either positively or negatively density-dependent and was contingent on the magnitude of the difference between positive density-dependent seed encounter and negative density-dependent seed exploitation. Furthermore, our results also indicated that studies including only a few species would produce biased results, because the density effect on seed predation differed greatly among plant species. Future studies should include a large number of plant species that possess a wide range of diverse seed traits to avoid potential bias and produce more comprehensive and accurate results. This article is protected by copyright. All rights reserved.

13.
Cell Oncol (Dordr) ; 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776938

RESUMO

PURPOSE: BRCA1-associated protein (BRAP) was first identified by its ability to bind to the nuclear localization signalling motif of BRCA1 and other proteins. Subsequently, human BRAP has been found to exert multiple functions, many of which are related to cancer development. Up till now, however, the role of BRAP in glioma development has remained obscure. Here, we report a role for BRAP in mediating the proliferation and migration of glioma cells both in vitro and in vivo. METHODS: The expression of BRAP in 98 glioma patient samples was determined by immunohistochemistry, after which associations between BRAP expression and patient prognosis were assessed. A short hairpin RNA (shRNA) was used to knock down BRAP and an expression vector was used to exogenously overexpress BRAP in glioma cells. The effects of BRAP expression on tumour cell behaviour in vitro and in an in vivo xenograft mouse model were examined. RESULTS: We found that in glioma patients BRAP expression was associated with a favourable prognosis. We also found that shRNA-mediated knockdown of BRAP facilitated the proliferation and migration of glioma cells and the self-renewal of glioma stem cells. In parallel, we found that BRAP knockdown increased tumour growth and invasion and decreased survival in an in vivo glioma xenograft mouse model. Mechanistically, we found that BRAP inhibited glioma cell proliferation and migration, as well as glioma stem cell self-renewal via the TGF-ß/PI3K/AKT/mTOR signalling pathway. CONCLUSIONS: Together, our findings identify BRAP as a mediator of glioma cell proliferation, migration and glioma stem cell self-renewal.

14.
Chem Commun (Camb) ; 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31777862

RESUMO

Correction for 'A bright NIR-II fluorescent probe for breast carcinoma imaging and image-guided surgery' by Xiaodong Zeng et al., Chem. Commun., 2019, DOI: 10.1039/c9cc07694h.

15.
J Am Chem Soc ; 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31769979

RESUMO

(S)-2-hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its non-heme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves ~ 95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE•FeII•1 and HppE•ZnII•2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues - Leu120, Leu144, Phe182, and Leu193 - close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans ~ 99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (~ 82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (~ 13:87), trifluorination of C3 (~ 23:77), or both (~ 1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (~ 56:44) and Leu144Phe/Leu193Phe (~ 90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical - as proposed in a recent computational study - to direct formation of the more potently antibacterial cis epoxide by radicaloid C-O coupling.

16.
J Exp Clin Cancer Res ; 38(1): 466, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31718694

RESUMO

BACKGROUND: Circular RNAs (circRNAs) have recently been shown to play important roles in different tumors. However, their detailed roles and regulatory mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not well understood. This study aimed to identify enriched circRNAs and detect their functions and mechanisms in PDAC cells and tissues. METHODS: circRNA-ASH2L (circ-ASH2L) was identified by circRNA microarray studies based on previous studies, and further detected in PDAC cells and samples by qRT-PCR. The functions of circ-ASH2L were identified by transwell, EdU, cell cycle or Tube formation assays. The regulatory mechanisms of circ-ASH2L were explored by WB, RIP, FISH, dual-luciferase assays, RNA pulldown or other assays. RESULTS: We identified a circRNA (circ-ASH2L) based on our previous studies, detected its expression in different malignant cells and found that circ-ASH2L was highly expressed in pancreatic cells or tumor tissues and correlated with tumor malignancy. Further studies revealed that circ-ASH2L promoted tumor invasion, proliferation and angiogenesis by regulating miR-34a, thus regulate Notch 1 expression. Circ-ASH2L served as a miRNA sponge for miR-34a and promoted tumor progression in vivo. Finally, we analyzed circ-ASH2L expression in clinical tissues and found that high circ-ASH2L expression was correlated with lymphatic invasion and TNM stage and was an independent risk factor for pancreatic patient survival. CONCLUSIONS: circ-ASH2L play an important role in tumor invasion, and high circ-ASH2L may be a useful marker of PDAC diagnosis or progression.

17.
Nanotechnology ; 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747652

RESUMO

Bismuth telluride (Bi2Te3) as a typical of topological insulator, which possesses narrow band gap and exhibited fascinating performance on photodetector filed. In this work, we have fabricated Bi2Te3/graphene heterostructure via a facile one-pot hydrothermal method. The as-prepared composites used as electrode materials of photoelectrochemical (PEC) type photodetector. From the results of photoelectrochemical tests, we can obviously find Bi2Te3/graphene heterostructure have a remarkable improvement on photoresponse compared to sole Bi2Te3, which effectively demonstrates effective photocarrier generation and transfer at the interface between graphene and Bi2Te3 can enhance the properties of photoresponse. Moreover, owing to the self-powered ability of PEC-type photodetector, it can work under the bias potential of 0 V and exhibited prominent photoresponse which can reach to 2.2 mA/W. Meanwhile, the photocurrent density of the prepared Bi2Te3/graphene heterostructure based photodetector can almost linearly rise with the increased irradiation power density. Even if the light intensity reduced to 40 mW/cm2, the photocurrent density can also reach 67 µA/cm2, which ensure photodetection ability of the as-prepared Bi2Te3/graphene under low light intensity. The excellent performance of Bi2Te3/graphene heterostructure for PEC-type photodetector hold the great promising in the field of photoelectric detection.

18.
Sci Rep ; 9(1): 16726, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723206

RESUMO

The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) recently issued updated guidelines on human epidermal growth factor receptor 2 (HER2) testing by fluorescence in situ hybridization (FISH) in invasive breast cancers. In this study, we aimed to investigate the impact of the new recommendations on HER2 FISH interpretation in invasive breast cancers with immunohistochemically (IHC) equivocal results. 1810 breast cancer cases with IHC equivocal results were enrolled in this study between January 2012 and May 2019. Concomitant IHC was performed on the same tissue blocks detected by FISH testing. According to the 2018 guidelines, all the cases in ISH group 2 were categorized as HER2 negative; three of four cases in ISH group 3 were considered as HER2 positive, while the one scored IHC 1+ was reclassified as HER2 negative; Fifty-three previously ISH equivocal cases were redistributed into ten HER2-positive cases and forty-three HER2-negative cases. In conclusion, the utility of 2018 ASCO/CAP guidelines resulted in a slight decrease in HER2 positive rate, due to the reclassification of cases in ISH group 2 and group 4. The implementation of the new guidelines can reduce reflex FISH test and make the diagnosis of HER2 gene status more definitive.

19.
Histol Histopathol ; : 18186, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31745968

RESUMO

Gallbladder cancer (GBC) is a malignant tumor of the biliary tract. The main problem affecting the treatment of gallbladder cancer is late diagnosis and poor prognosis. EIF5A2 is one of two isoforms of the EIF5A family and is reported to be a new oncogenic protein in many human cancers. In this study, our results showed for the first time that EIF5A2 was overexpressed in GBC samples compared with non-tumor tissue. Overexpression of EIF5A2 was associated with lymph node metastasis, tumor differentiation, UICC (Union for International Cancer Control) staging, histological type, metastasis, and tumor size. Overexpression of EIF5A2 in gallbladder carcinoma tissues is also associated with poor prognosis in patients. The interference of EIF5A2 significantly inhibited the proliferation, cell cycle, migration and colony formation of GBC-SD cells in vitro. Our results suggest that EIF5A2 is a target oncogene and may be an important prognostic biomarker in the pathogenesis of gallbladder cancer.

20.
Artigo em Inglês | MEDLINE | ID: mdl-31712419

RESUMO

Insect pollination of flowering plants (angiosperms) is responsible for the majority of the world's flowering plant diversity and is key to the Cretaceous radiation of angiosperms. Although both insects and angiosperms were common by the mid-Cretaceous, direct fossil evidence of insect pollination is lacking. Direct evidence of Cretaceous insect pollination is associated with insect-gymnosperm pollination. Here, we report a specialized beetle-angiosperm pollination mode from mid-Cretaceous Burmese amber (99 mega-annum [Ma]) in which a tumbling flower beetle (Mordellidae), Angimordella burmitina gen. et sp. nov., has many tricolpate pollen grains attached. A. burmitina exhibits several specialized body structures for flower-visiting behavior including its body shape and pollen-feeding mouthparts revealed by X-ray microcomputed tomography (micro-CT). The tricolpate pollen in the amber belongs to the eudicots that comprise the majority of extant angiosperm species. These pollen grains exhibit zoophilous pollination attributes including their ornamentation, size, and clumping characteristics. Tricolpate pollen grains attached to the beetle's hairs are revealed by confocal laser scanning microscopy, which is a powerful tool for investigating pollen in amber. Our findings provide direct evidence of insect pollination of Cretaceous angiosperms, extending the range insect-angiosperm pollination association by at least 50 million years. Our results support the hypothesis that specialized insect pollination modes were present in eudicots 99 million years ago.

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