Single-Exosome Profiling Identifies ITGB3+ and ITGAM+ Exosome Subpopulations as Promising Early Diagnostic Biomarkers and Therapeutic Targets for Colorectal Cancer.

Tumor metastasis is a hallmark of colorectal cancer (CRC), in which exosome plays a crucial role with its function in intercellular communication. Plasma exosomes were collected from healthy control (HC) donors, localized primary CRC and liver-metastatic CRC patients. We performed proximity barcoding assay (PBA) for single-exosome analysis, which enabled us to identify the alteration in exosome subpopulations associated with CRC progression. By in vitro and in vivo experiments, the biological impact of these subpopulations on cancer proliferation, migration, invasion, and metastasis was investigated. The potential application of exosomes as diagnostic biomarkers was evaluated in 2 independent validation cohorts by PBA. Twelve distinct exosome subpopulations were determined. We found 2 distinctly abundant subpopulations: one ITGB3-positive and the other ITGAM-positive. The ITGB3-positive cluster is rich in liver-metastatic CRC, compared to both HC group and primary CRC group. On the contrary, ITGAM-positive exosomes show a large-scale increase in plasma of HC group, compared to both primary CRC and metastatic CRC groups. Notably, both discovery cohort and validation cohort verified ITGB3+ exosomes as potential diagnostic biomarker. ITGB3+ exosomes promote proliferation, migration, and invasion capability of CRC. In contrast, ITGAM+ exosomes suppress CRC development. Moreover, we also provide evidence that one of the sources of ITGAM+ exosomes is macrophage. ITGB3+ exosomes and ITGAM+ exosomes are proven 2 potential diagnostic, prognostic, and therapeutic biomarkers for management of CRC.

m6A Regulator-Based Exosomal Gene Methylation Modification Patterns Identify Distinct Microenvironment Characterization and Predict Immunotherapeutic Responses in Colon Cancer.

Recent studies have highlighted the biological significance of exosomes and m6A modifications in immunity. Nonetheless, it remains unclear whether the m6A modification gene in exosomes of body fluid has potential roles in the tumor microenvironment (TME). Herein, we identified three different m6A-related exosomal gene modification patterns based on 59 m6A-related exosomal genes, which instructed distinguishing characteristics of TME in colon cancer (CC). We demonstrated that these patterns could predict the stage of tumor inflammation, subtypes, genetic variation, and patient prognosis. Furthermore, we developed a scoring mode-m6A-related exosomal gene score (MREGS)-by detecting the level of m6A modification in exosomes to classify immune phenotypes. Low MREGS, characterized by prominent survival and immune activation, was linked to a better response to anti-PDL1 immunotherapy. In contrast, the higher MREGS group displayed remarkable stromal activation, high activity of innate immunocytes, and a lower survival rate. Hence, this work provides a novel approach for evaluating TME cell infiltration in colon cancer and guiding more effective immunotherapy strategies.

Inhibition of GABAA receptors in intestinal stem cells prevents chemoradiotherapy-induced intestinal toxicity.

Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy. Chemoradiotherapy can trigger DNA damage and induce P53-dependent apoptosis in LGR5+ intestinal stem cells (ISCs). Gamma-aminobutyric acid (GABA) and its A receptors (GABAAR) are present in the gastrointestinal tract. However, the functioning of the GABAergic system in ISCs is poorly defined. We found that GABAAR α1 (GABRA1) levels increased in the murine intestine after chemoradiotherapy. GABRA1 depletion in LGR5+ ISCs protected the intestine from chemoradiotherapy-induced P53-dependent apoptosis and prolonged animal survival. The administration of bicuculline, a GABAAR antagonist, prevented chemoradiotherapy-induced ISC loss and intestinal damage without reducing the chemoradiosensitivity of tumors. Mechanistically, it was associated with the reduction of reactive oxygen species-induced DNA damage via the L-type voltage-dependent Ca2+ channels. Notably, flumazenil, a GABAAR antagonist approved by the U.S. Food and Drug Administration, rescued human colonic organoids from chemoradiotherapy-induced toxicity. Therefore, flumazenil may be a promising drug for reducing the gastrointestinal side effects of chemoradiotherapy.

Unraveling the Proteomic Landscape of Intestinal Epithelial Cell-Derived Exosomes in Mice.

Purpose: This study aimed to identify the biological functions of small intestine intestinal epithelial cell derived exosomes (IEC-Exos) and further distinguished the difference proteins in IEC-Exos between ileum and jejunum related to function of the digestive system and occurrence of several diseases. Materials and Methods: IECs of Male C57BL/6J mice were isolated. IEC-Exos were extracted from jejunum and ileum epithelial cell culture fluid by ultracentrifugation. In addition, isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to detect IEC-Exo proteins and conduct biological information analysis. Results: The results showed that compared with jejunum IEC-Exos from ileum IEC-Exos, there were 393 up-regulated proteins and 346 down-regulated proteins. IECs-Exos, especially derived from jejunum, were rich in angiotensin-converting enzyme 2 (ACE2). The highly expressed proteins from ileum IEC-Exos were mostly enriched in genetic information processing pathways, which mainly mediate the processes of bile acid transport, protein synthesis and processing modification. In contrast, the highly expressed proteins from jejunum IEC-Exos were mainly enriched in metabolic pathways involved in sugar, fatty acid, amino acid, drug, and bone metabolism, etc. The differentially expressed proteins between ileum and jejunum IEC-Exos were not only related to the function of the digestive system but also closely related to the occurrence of infectious diseases, endocrine diseases and osteoarthritis, etc. Conclusion: IEC-Exos there were many differentially expressed proteins between ileum and jejunum, which played different roles in regulating intestinal biological functions. ACE2, the main host cell receptor of SARS-CoV-2, was highly expressed in IEC-Exos, which indicated that IEC-Exos may be a potential route of SARS-CoV-2 infection.

Correlation of myopia onset and progression with corneal biomechanical parameters in children.

BACKGROUND: Recent epidemiological studies have shown that general eye measurement parameters and corneal biomechanical properties can predict the speed of myopic progression in children. AIM: To investigate the correlation between the onset and progression of myopia and corneal biomechanical parameters in children. METHODS: The study included 102 cases in the emmetropia group, 207 cases in the myopic group, and 109 cases in the hyperopic group. The correlation between the change in corneal biomechanical indexes and the change in general ocular measurement parameters was analyzed. A one-way ANOVA test compared general ocular measurement and corneal biomechanical parameters. Pearson's correlation coefficient was analyzed to correlate corneal biomechanical and general ocular measurement parameters. RESULTS: The general ophthalmometric parameters: Spherical equivalent (SE), intraocular pressure (IOP), and axial length (AL), differed significantly among subjects in myopia, emmetropia, and hyperopic groups. Children's SE positively correlated with corneal biomechanical parameters: Second velocity of applanation (A2V), peak distance (PD), and deformation amplitude (DA) (P < 0.05), and second applanation length (A2L) (P < 0.05). But it was negatively correlated with PD, DA and integral radius (IR) (P < 0.05). Also, IOP was negatively correlated with A2L and IR (P < 0.05). AL positively correlated with A2V and negatively correlated with second applanation time (A2T), highest concavity, and PD. Central corneal thickness positively correlated with first applanation length, first applanation time, first applanation deformation amplitude, A2V, A2L, A2T, second applanation deformation amplitude, central curvature radius at highest concavity (HCR), PD, DA, IR, ambrosia relational thickness-horizontal, first applanation stiffness parameter, corvis biomechanical index, topographic and biomechanics index and the first velocity of applanation. The general ocular Km in children positively correlated with corneal biomechanical parameters DA and IR and negatively correlated with A2L, HCR, and PD. There was a positive correlation between the general ocular measurement parameters ΔSE and corneal biomechanical parameters ΔA2V and ΔA2L, and a negative correlation with ΔIR. The increase in general ocular measurement parameter ΔKm positively correlated with changes in corneal biomechanical parameters, ΔDA and ΔIR, and negatively correlated with ΔHCR and ΔPD. CONCLUSION: Myopia development in children was associated with multiple corneal biomechanical parameters.

Resolving the difference between left-sided and right-sided colorectal cancer by single-cell sequencing.

Colorectal cancers (CRCs) exhibit differences in incidence, pathogenesis, molecular pathways, and outcome depending on the location of the tumor. The transcriptomes of 27,927 single human CRC cells from 3 left-sided and 3 right-sided CRC patients were profiled by single-cell RNA-Seq (scRNA-Seq). Right-sided CRC harbors a significant proportion of exhausted CD8+ T cells of a highly migratory nature. One cluster of cells from left-sided CRC exhibiting states preceding exhaustion and a high ratio of preexhausted/exhausted T cells were favorable prognostic markers. Notably, we identified a potentially novel RBP4+NTS+ subpopulation of cancer cells that exclusively expands in left-sided CRC. Tregs from left-sided CRC showed higher levels of immunotherapy-related genes than those from right-sided CRC, indicating that left-sided CRC may have increased responsiveness to immunotherapy. Antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) induced by M2-like macrophages were more pronounced in left-sided CRC and correlated with a good prognosis in CRC.

Plasma Proteomics Characteristics of Subclinical Vitamin E Deficiency of Dairy Cows During Early Lactation.

Vitamin E (VE) is an essential fat-soluble nutrient for dairy cows. Vitamin E deficiency leads to immune suppression and oxidative stress and increases the susceptibility of cows to reproductive disorders in the early post-partum period. However, studies on plasma proteomics of VE deficiency have not been reported so far. Therefore, the purpose of this study was to understand the changes of blood protein profile in cows with subclinical VE deficiency in the early post-partum period. In this study, plasma protein levels of 14 healthy cows (>4 µg/ml α-tocopherol) and 13 subclinical VE-deficient cows (2-3 µg/ml α-tocopherol) were analyzed by tandem mass tag (TMT). The results showed that there were 26 differentially expressed proteins (DEPs) in the plasma of cows with subclinical VE deficiency compared with healthy controls. Twenty-one kinds of proteins were downregulated, and five kinds were upregulated, among which eight proteins in protein-protein interactions (PPI) network had direct interaction. These proteins are mainly involved in the MAPK signaling pathway, pantothenic acid and coenzyme A (CoA) biosynthesis, PPAR signaling pathway, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. The top four DEPs in PPI (APOC3, APOC4, SAA4, PHLD) and one important protein (VNN1) by literature review were further verified by ELISA and Western blot. The expression levels of APOC3, VNN1, and SAA4 were significantly lower than those of healthy controls by ELISA. VNN1 was significantly lower than those of healthy controls by Western blot. VNN1 is closely related to dairy cow subclinical VE deficiency and can be a potential biomarker. It lays a foundation for further research on the lack of pathological mechanism and antioxidative stress of VE.

M6A methylation of DEGS2, a key ceramide-synthesizing enzyme, is involved in colorectal cancer progression through ceramide synthesis.

N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulator in cancer. However, the understanding of m6A modification on lipid metabolism regulation in colorectal cancer (CRC) is very limited. Here, we observed that human CRCs exhibited increased m6A mRNA methylation mediated by dysregulation of m6A erasers and readers. By performing methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and transcriptomic sequencing (RNA-seq), we identified DEGS2 as a downstream target of m6A dysregulation. Overexpression or knockdown of DEGS2 confirmed the role of DEGS2 in proliferation, invasion and metastasis of CRC both in vitro and in vivo. Mechanistic studies identified the specific m6A modification site within DEGS2 mRNA, and mutation of this target site was found to drastically enhance the proliferative and invasive ability of CRC cells in vitro and promote tumorigenicity in vivo. Lipidome analysis showed that lipid metabolism was dysregulated in CRC. Moreover, ceramide synthesis was suppressed due to DEGS2 upregulation mediated by m6A modification in CRC tissues. Our findings highlight that the function of DEGS2 m6A methylation in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

Polarization-Modulated Bent-Core Liquid Crystal Thin Films without Layer Undulation.

Spatial confinement is known to affect molecular organizations of soft matter. We present an important manifestation of this statement for thin films of bent-core smectic liquid crystals. Prior freeze-fracture transmission electron microscopy (FFTEM) studies carried out on nitro-substituted bent-core mesogens (n-OPIMB-NO_{2}) revealed an undulated smectic layer structure with an undulation periodicity of ∼8 nm. We report cryogenic TEM measurements on ∼100 nm thick 8-OPIMB-NO_{2} films. In contrast to FFTEM results, our studies show only density modulation with periodicity b=16.2 nm, and no smectic layer undulation. We show that the discrepancy between the FFTEM and cryogenic transmission electron microscopy (cryo-TEM) results can be attributed to the different sample thicknesses used in the experiments. FFTEM monitors cracked surfaces of a relatively thick (5-10 µm) frozen sample, whereas cryo-TEM visualizes the volume of a thin (0.1 µm) film that was quenched from its partially fluid phase. These results have importance in possible photovoltaics and organic electronics applications where submicron thin films are used.

An Optically Isotropic Antiferroelectric Liquid Crystal (OI-AFLC) Display Mode Operating over a Wide Temperature Range using Ternary Bent-Core Liquid Crystal Mixtures.

We report on the synthesis and characterization of bent-core liquid crystal (LC) compounds and the preparation of mixtures that provide an optically isotropic antiferroelectric (OI-AFLC) liquid crystal display mode over a very wide temperature interval and well below room temperature. From the collection of compounds synthesized during this study, we recognized that several ternary mixtures displayed a modulated SmCaPA phase down to below -40 °C and up to about 100 °C on both heating and cooling, as well as optical tilt angles in the transformed state of approximately 45° (optically isotropic state). The materials were fully characterized and their liquid crystal as well as electro-optical properties analyzed by polarized optical microscopy, differential scanning calorimetry, synchrotron X-ray diffraction, dielectric spectroscopy, and electro-optical tests.

Direct observation of liquid crystals using cryo-TEM: specimen preparation and low-dose imaging.

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies.

Nanostructures of nematic materials of laterally branched molecules.

The synthesis and small-angle X-ray scattering (SAXS) characterization is reported for 20 laterally branched mesogenic molecules, which are derived from the common rod-shaped 2,5-bis([4-(octyloxy)phenyl]carbonyloxy) benzoic acid unit. These compounds have a varying degree of flexibility, in that their lateral branch is formed upon conversion of the acid to either an ester or an amide, and most laterally branched molecules exhibit relatively wide nematic liquid-crystal phases with a direct nematic-to-crystal transition at lower temperatures. SAXS studies reveal the presence of smectic-like nanostructures (clusters) with short-range order in the nematic phase, with characteristic correlation lengths from 3 to over 10 nm. The smectic layers that are contained in these clusters are tilted with respect to the nematic director by angles ranging from 0° (i.e. untilted) to 55°. In some compounds, the intensity of the SAXS peak corresponding to the smectic layer spacing depends strongly on temperature. The main features of the nanostructures can be understood based on the molecular structure; therefore, guiding future synthetic work towards more precisely controlled and technologically useful nanostructures in nematics.

Structural study of fucoidan from sea cucumber Acaudina molpadioides: a fucoidan containing novel tetrafucose repeating unit.

Structure of fucoidan from sea cucumber Acaudina molpadioides (Am-FUC) was investigated in this study. Low molecular weight polysaccharide of Am-FUC (Am-LMW) was prepared by enzymatic degradation and assayed by nuclear magnetic resonance. According to the structure of Am-LMW, and referring to previously reported information of Am-FUC oligosaccharides, the structure of Am-FUC was elucidated as [→3-α-l-Fucp-1→3-α-l-Fucp2,4(OSO3(-))-1→3-α-l-Fucp-1→3-α-l-Fucp2(OSO3(-))-1→]n. Compared to other investigated fucoidans, which also consist of tetrafucose repeating units with 1→3-linkages, Am-FUC contains a novel repeating unit distinctive in sulphation pattern.

Enzymatic preparation and structural determination of oligosaccharides derived from sea cucumber (Acaudina molpadioides) fucoidan.

Sea cucumber fucoidan is a major bioactive component of sea cucumber. Sea cucumber is widely consumed in East Asian countries as healthy food. Employing the degrading enzyme from the marine bacterium strain Flavobacteriaceae CZ1127, sea cucumber (Acaudina molpadioides) fucoidan oligosaccharides were prepared by enzymatic hydrolysis. The oligosaccharide profile of the hydrolysate was determined by liquid chromatography coupled with mass spectrometry (LC-MS). With the assistance of LC-MS, four major oligosaccharides in the hydrolysate were purified. By using tandem mass spectrometry and nuclear magnetic resonance, delicate structures of the oligosaccharides were verified as α-l-Fucp-1→3-α-l-Fucp(2,4OSO3(2-))-1→3-α-l-Fucp, α-l-Fucp-1→3-α-l-Fucp(2,4OSO3(2-))-1→3-α-l-Fucp-1→3-α-l-Fucp, α-l-Fucp-1→3-α-l-Fucp(2,4OSO3(2-))-1→3-α-l-Fucp-1→3-α-l-Fucp-1→3-α-l-Fucp-1→3-α-l-Fucp(2,4OSO3(2-))-1→3-α-l-Fucp and α-l-Fucp-1→3-α-l-Fucp(2,4OSO3(2-))-1→3-α-l-Fucp-1→3-α-l-Fucp-1→3-α-l-Fucp-1→3-α-l-Fucp(2,4OSO3(2-))-1→3-α-l-Fucp-1→3-α-l-Fucp.

Crystalline structure and thermal property characterization of chitin from Antarctic krill (Euphausia superba).

Antarctic krill (Euphausia superba) has been widely studied and extensively recognized as a target for commercial fishing. In this study, Antarctic krill chitin was extracted from defatted Antarctic krill shell, and its crystalline structure and thermal properties were characterized by employing Fourier transform infrared spectroscopy, X-ray diffractometry, scanning electron microscopy, thermogravimetry, and differential scanning calorimetry. Results showed that Antarctic krill chitin corresponded to the α-polymorph, and was composed of small, stable, and uniform microcrystals. The degree of N-deacetylation was 11.28 ± 0.86%. The d-spacings of Antarctic krill chitin were 9.78 Å and 4.63 Å at (020) and (110) planes. The crystalline sizes were 6.07 nm and 5.16 nm at (020) and (110) planes, respectively. The activation energy of the polysaccharide chain decomposition was 123.35 kJ/mol and the glass transition (T(g)) of Antarctic krill chitin was 164.96 °C.