Rspondin-1 contributes to the progression and stemness of gastric cancer by LGR5.

Gastric cancer is a one of the most common malignant tumors with poor prognosis worldwide. Leucine-rich G-protein-coupled receptor 5 (LGR5) is determined as a modulator of Wnt signaling cascade and R-spondins are a family of secretory agonists in the Wnt signaling and act as ligands to interact with LGR5. However, the function of Rspondin-1 in GC remains obscure. Here, we identified the effect of Rspondin-1 on GC progression. Rspondin-1 and LGR5 were upregulated in clinical gastric cancer tissues. CCK-8 assay revealed that the viability of GC cells was reduced by Rspondin-1 depletion and enhanced by Rspondin-1 overexpression. The depletion of Rspondin-1 decreased while the overexpression of Rspondin-1 increased the numbers of colony formation and Edu-positive GC cells. The depletion of Rspondin-1 attenuated the invasion and migration ability of GC cells. Moreover, sphere formation assays revealed that the knockdown of Rspondin-1 reduced the stemness of GC cells. The expression of cancer stem cell markers, including Nanog, OCT3/4, and SOX2 were suppressed by Rspondin-1 depletion in GC cells. Rspondin-1 induced tumor growth of gastric cancer cells in vivo. Mechanically, the cell viability and invasion suppressed by the depletion of Rspondin-1 in GC cells were rescued by LGR5 overexpression. Besides, the overexpression of LGR5 reversed Rspondin-1 knockdown-inhibited Nanog and OCT3/4 expression. Consequently, we concluded that Rspondin-1 contributes to the progression and stemness of gastric cancer by LGR5.

Defining the impact of platelet-to-lymphocyte ratio on patient survival with gastric neuroendocrine neoplasm: a retrospective cohort analysis.

BACKGROUND: Gastric neuroendocrine neoplasm (g-NEN) is a rare but heterogeneous neoplasm, with an increasing incidence yearly. Conventional prognostic markers of g-NEN remain limited which could only be detected after surgery. There is an urgent need to explore new prognostic markers for g-NEN patients. This study aimed to investigate the prognostic value of platelet-to-lymphocyte, ratio (PLR) and the association between PLR and body mass index (BMI) in patients with gastric neuroendocrine neoplasms (g-NEN). METHODS: A retrospective cohort of patients with g-NEN from January 2001 through June 2016 was examined. The prognostic significance of PLR was determined by multiple regression analysis in different models. Stratified analysis was performed to examine the prognostic value of PLR at different BMI levels. RESULTS: In total, 238 patients were enrolled. Those with higher PLRs tended to undergo open surgery, had larger tumor sizes, were diagnosed more frequently with neuroendocrine carcinoma, and had higher tumor grades. PLR was significantly associated with the survival of patients with g-NEN. With PLR increased per standard deviation, the all-cause mortality risk of patients with g-NEN increased by 67%, 63%, and 54% in the crude (HR = 1.67, 95% CI 1.32-2.12, P < 0.001), minimally adjusted (HR = 1.63, 95% CI 1.28-2.08, P < 0.001), and fully adjusted (HR = 1.54, 95% CI 1.202-1.98, P = 0.001) models, respectively. Patients with higher PLR (quartile 4, ≥ 187) had a 1.8-fold increase in all-cause mortality risk compared with those with lower PLR (quartile 1-3, < 187). Furthermore, there was a significant interaction effect between BMI subgroups and PLR in predicting the survival of patients with g-NEN (PLR regarded as a continuous variable: all P for interaction < 0.05 in the crude, minimally adjusted, and fully adjusted models; PLR regarded as a categorical variable: P for interaction < 0.05 in the fully adjusted model). Patients with g-NEN with the characteristics of higher PLR (quartile 4, ≥ 187) and non-obesity (BMI < 25 kg/m2) had worse survival than others (P < 0.05). CONCLUSION: The inflammation marker PLR has an independent prognostic value for patients with g-NENs, and high PLR combined with non-obesity increases the mortality risk of these patients.

Stearoyl-CoA-desaturase-1 regulates gastric cancer stem-like properties and promotes tumour metastasis via Hippo/YAP pathway.

BACKGROUND: Stearoyl-CoA desaturase-1 (SCD1) is reported to play essential roles in cancer stemness among several cancers. Our previous research revealed significant overexpression of SCD1 in primary gastric cancer stem cells (GCSCs), with its functional role still unknown. METHODS: We stably established three primary GCSCs by sphere-forming assays and flow cytometry. Protein quantification and bioinformatics analysis were performed to reveal the differential protein pattern. Lentivirus-based small-interfering RNA (siRNA) knockdown and pharmacological inhibition approaches were used to characterise the function and molecular mechanism role of SCD1 in the regulation of GC stemness and tumour metastasis capacity both in vitro and in vivo. RESULTS: SCD1 was found to increase the population of GCSCs, whereas its suppression by an SCD1 inhibitor or knockdown by siRNA attenuated the stemness of GCSCs, including chemotherapy resistance and sphere-forming ability. Furthermore, SCD1 suppression reversed epithelial-to-mesenchymal transition and reduced the GC metastasis probability both in vitro and in vivo. Downregulation of SCD1 in GCSCs was associated with the expression of Yes-associated protein (YAP), a key protein in the Hippo pathway, and nuclear YAP translocation was also blocked by the SCD1 decrease. CONCLUSIONS: SCD1 promotes GCSC stemness through the Hippo/YAP pathway. Targeting SCD1 might be a novel therapeutic strategy, especially to suppress GC metastasis and sensitise chemotherapy.

Peptide-Mediated Synthesis of Zeolitic Imidazolate Framework-8 with Controllable Morphology and Size.

Peptides with a sequence of Nap-Ix-GPLGLAG-R4-NH2 (x = 2, 4, and 6, shorted as I2R4, I4R4, and I6R4) were used as capping agents for the synthesis of zeolitic imidazolate framework-8 (ZIF-8) in water. Peptide addition can significantly inhibit the growth of ZIF-8 crystals. The shape and size of ZIF-8 crystals was related closely to the number of isoleucine (Ile, I) residues as well as concentration of the peptide. The shape of ZIF-8 crystals changes from rhomboid dodecahedron to truncated rhombic dodecahedron to cube with the decreasing number of isoleucine residues from six to two. At a peptide concentration of 1.0 mM, the morphology of ZIF-8 crystals was cubic, truncated rhombic dodecahedron, and typical rhombic dodecahedron in the cases of I2R4, I4R4, and I6R4, respectively. Also, the particle size can be regulated from ca. 1.7 µm to <100 nm by controlling the peptide concentration from 0 to 2.0 mM. This work develops a simple and green method for the synthesis of ZIF-8 crystals with controllable shape and size in water, which shows high potential for biomedical and biological applications.

In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.

Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4-5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths.

Measurement of 3-photon excitation and emission spectra and verification of Kasha's rule for selected fluorescent proteins excited at the 1700-nm window.

Fluorescent proteins are widely used to visualize structures and dynamics in various biological samples. Multiphoton microscopy is especially suitable for imaging structures expressing fluorescent proteins with subcellular resolution. 3-photon microscopy (3PM) excited at the 1700-nm window has proven to be promising for deep-tissue (such as brain) imaging expressing red fluorescent proteins. However, the 3-photon excitation and emission spectra of fluorescent proteins suitable at this window remain largely unknown, hampering protein selection and detection optimization. Here we demonstrate detailed measurement of 3-photon excitation and emission spectra for selected fluorescent proteins, suitable for 3-photon excitation at the 1700-nm window. The measured 3-photon excitation spectra will provide guidelines for protein and excitation wavelength selection. The measured 3-photon emission spectra and comparison with the 1-photon emission spectra, on one hand proves that the fundamental Kasha's rule is still valid for 3-photon fluorescence in these fluorescent proteins, on the other hand will be helpful for efficient fluorescence signal detection.

High-energy polarized soliton synthesis and its application to deep-brain 3-photon microscopy in vivo.

Here, we demonstrate a polarized high-energy soliton synthesis technique for deep-brain 3-photon microscopy (3PM) excited at the 1700-nm window. Through coherent combining, we generate linearly polarized high-energy solitons whose energy is twice as high than those of each linearly polarized solitons. Due to the nonlinear origin of signals, both measured 3-photon fluorescence signal and third-harmonic signals are thus boosted by ~8 times in a tissue phantom. Using this technique, we further demonstrate 3PM of sulforhodamine 101 labeled vasculature 1600 µm in the mouse brain in vivo, which cannot be achieved by single-polarized soliton excitation.

Peptide-mediated green synthesis of the MnO2@ZIF-8 core-shell nanoparticles for efficient removal of pollutant dyes from wastewater via a synergistic process.

The MnO2@ZIF-8 core-shell nanoparticles for highly efficient dye degradation have been synthesized with a green method. ZIF-8 crystals with controlled morphology and size are first synthesized by using peptide to modulate the crystal growth. MnO2 is then coated on ZIF-8 via in situ reaction. The surface MnO2 density can be controlled by the dosage of KMnO4. The MnO2@ZIF-8 nanoparticles work as photocatalyst to degrade rhodamine B in a Fenton-like process, giving a degradation ratio of > 96.0%. The degradation kinetics comply well with the Pseudo-second-order model and the experimental equilibrium data meet the Langmuir model best. The specific hierarchical structure of MnO2@ZIF-8 assures a synergistic enhancement of the catalytic degradation performance from several aspects. First, anchoring of the MnO2 nanoparticles on ZIF-8 allows their well disperse to provide more active surface area. Second, highly porous ZIF-8 can adsorb dye molecules to accumulate them at the surface reactive sites. Third, the MnO2/ZIF-8 nano-heterojunctions enhance charge carrier transfer and accelerate the production of free oxidative radicals. The study demonstrates a green method for fabrication of hierarchical hybrid structures, paving the way for designing novel photocatalysts with potential applications for wastewater treatment.

In vivo deep-brain blood flow speed measurement through third-harmonic generation imaging excited at the 1700-nm window.

Measurement of the hemodynamic physical parameter blood flow speed in the brain in vivo is key to understanding brain physiology and pathology. 2-photon fluorescence microscopy with single blood vessel resolution is typically used, which necessitates injection of toxic fluorescent dyes. Here we demonstrate a label-free nonlinear optical technique, third-harmonic generation microscopy excited at the 1700-nm window, that is promising for such measurement. Using a simple femtosecond laser system based on soliton self-frequency shift, we can measure blood flow speed through the whole cortical grey matter, even down to the white matter layer. Together with 3-photon fluorescence microscopy, we further demonstrate that the blood vessel walls generate strong THG signals, and that plasma and circulating blood cells are mutually exclusive in space. This technique can be readily applied to brain research.

Air-core fiber or photonic-crystal rod, which is more suitable for energetic femtosecond pulse generation and three-photon microscopy at the 1700-nm window?

Energetic femtosecond pulses at the 1700-nm window are a prerequisite for deep-tissue three-photon microscopy (3PM). Soliton self-frequency shift (SSFS) in photonic-crystal (PC) rod has been the only technique to generate such pulses suitable for 3PM. Here we demonstrate through SSFS in an air-core fiber, we can generate most energetic femtosecond soliton pulses at the 1700-nm window, 5.2 times higher than that from PC rod. However, the air-core soliton pulse width is 5.9 times longer than that of PC rod soliton. Based on comparative 3PM excited with both air-core and PC rod solitons, we propose the more suitable source for 3PM. We further elucidate the challenge of generating shorter soliton pulses from air-core fibers through numerical simulation.

Visualizing astrocytes in the deep mouse brain in vivo.

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3-photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength-dependent 3-photon action cross section (ησ 3 ) measurement of SR101. 3-photon fluorescence imaging of the SR101-labeled vasculature enabled an imaging depth of 1340-µm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal-to-background ratio limit encountered in 2-photon fluorescence imaging. Visualization of astrocytes 910 µm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2-photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101-labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.

Deep-brain three-photon microscopy excited at 1600 nm with silicone oil immersion.

Three-photon microscopy excited at the 1700-nm window (roughly covering 1600-1840 nm) is especially suitable for deep-brain imaging in living animals. To match the brain refractive index, D2 O has been exclusively used as the immersion medium. However, the hygroscopic property of D2 O leads to a decrease of transmittance of the excitation light and as a result a decrease in three-photon signals over time. Solutions such as replacing D2 O from time to time, wrapping both the objective lens and the immersion D2 O, and sealing D2 O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D2 O for deep-brain imaging. Excited at 1600 nm, our comparative deep-brain imaging using both D2 O and silicone oil immersion show that silicone oil immersion yields 17% higher three-photon signal in third-harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three-photon fluorescence imaging of vasculature up to 1460 µm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long-span three-photon brain imaging excited at the 1700-nm window.

Visualizing the "sandwich" structure of osteocytes in their native environment deep in bone in vivo.

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3-photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700-nm window enables the highest optical transmittance through the skull. Using label-free third-harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140-µm mouse skull and 155 µm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the "sandwich" structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.

Rapid effect of bisphenol A on glutamate-induced Ca2+ influx in hippocampal neurons of rats.

Intracellular Ca2+ signaling plays an essential role in synaptic plasticity. This study examined the effect of BPA on concentration of intracellular Ca2+ ([Ca2+]i) by measuring fluorescence intensity of Ca2+ in hippocampal neurons in vitro. The results showed that BPA for 30 min exerted dose-dependently dual effects on glutamate-elevated [Ca2+]i: BPA at 1-10 µM suppressed but at 1-100 nM enhanced glutamate-raised [Ca2+]i. BPA-potentiated [Ca2+]i was blocked by the antagonist of NMDA receptor and was eliminated by an estrogen-related receptor gamma (ERRγ) antagonist rather than an AR antagonist. Both inhibitors of MAPK/ERKs and MAPK/p38 blocked BPA-enhanced [Ca2+]i. Co-treatment of BPA with 17ß-E2 or DHT eliminated the enhancement of 17ß-E2, DHT, and BPA in glutamate-elevated [Ca2+]i. These results suggest that BPA at nanomole level rapidly enhances Ca2+ influx through NMDA receptor by ERRγ-mediated MAPK/ERKs and MAPK/p38 signaling pathways. However, BPA antagonizes both estrogen and androgen enhancing NMDA receptor-mediated Ca2+ influx in hippocampal neurons.

Refractive index and pulse broadening characterization using oil immersion and its influence on three-photon microscopy excited at the 1700-nm window.

Three-photon microscopy excited at the 1700-nm window enables deep-tissue penetration. However, the refractive indices of commonly used immersion oils, and the resultant pulse broadening are not known, preventing imaging optimization. Here, we demonstrate detailed characterization of the refractive index, pulse broadening and distortion for excitation pulses at this window for commonly used immersion oils. On the physical side, we uncover that absorption, rather than material dispersion, is the main cause of pulse broadening and distortion. On the application side, comparative three-photon imaging results indicate that 1600-nm excitation yields 5 times higher three-photon signal than 1690-nm excitation.

Ex and in vivo characterization of the wavelength-dependent 3-photon action cross-sections of red fluorescent proteins covering the 1700-nm window.

Multiphoton action cross-sections are the prerequisite for excitation light selection. At the 1700-nm window suitable for deep-tissue imaging, wavelength-dependent 3-photon action cross-sections ησ3 for RFPs are unknown, preventing wavelength selection. Here we demonstrate: (1) ex vivo measurement of wavelength-dependent ησ3 for purified RFPs; (2) a multiphoton imaging guided measurement system for in vivo measurement; and (3) in vivo measurement of wavelength-dependent ησ3 in RFP labeled cells. These fundamental results will provide guidelines for excitation wavelength selection for 3-photon fluorescence imaging of RFPs at the 1700-nm window, and augment the existing database of multiphoton action cross-sections for fluorophores.

Self-referenced axial chromatic dispersion measurement in multiphoton microscopy through 2-color third-harmonic generation imaging.

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2-color third-harmonic generation imaging excited by a 2-color soliton source with tunable wavelength separation. Our technique is self-referenced, eliminating potential measurement error when 1-color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2-color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.