FAM83B promotes cell proliferation via regulating the expression of CDK4/CDK6/CCND1 complex in laryngeal squamous cell carcinoma.

FAM83B, as one of the FAM83 family members, has been closely involved in cell transformation, and a growing number of scholars have been studied its role in tumours over the years. Whereas the effect and potential mechanism of FAM83B in laryngeal squamous cell carcinoma (LSCC) have not been investigated. In this research, we discovered that the expression quantity of FAM83B was remarkably higher in LSCC tissues (79.65 ± 35.98) than in matched adjacent tissues (59.34 ± 32.59) by tissue microarrays and immunohistochemistry. Furthermore, expression of FAM83B was knocked down in HEP-2 and TU177 cell lines via lentivirus, and in the course of intracorporal and extracorporeal experiments, FAM83B knockdown showed the inhibition of tumour growth, migration, and invasion ability. Moreover, cell cycle assay showed that FAM83B knockdown leads to an apparent accumulation of cells in the G1 phase, indicating that FAM83B knockdown can inhibit cell proliferation. Meanwhile, western blotting (WB) demonstrated that FAM83B knockdown led to a significant reduction in CDK4/CDK6/CCND1 protein expression, which may have decelerated cell cycle progression. Collectively, this study demonstrates that FAM83B serves as an oncogene in LSCC, promoting cell proliferation by controlling the protein expression of CDK4, CDK6, and CCND1, thus inducing a transference of the G1 stage to S stage in cell-cycle of LSCC cells. These results provide an academic foundation for elucidating the mechanism of LSCC occurrence and evolution and for developing treatment strategies for LSCC.

Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes.

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

Effect of the ß-glucan from Lentinus edodes on colitis-associated colorectal cancer and gut microbiota.

Colorectal cancer is the third most common cancer in the world, and therapies with safety are in great need. In this study, the ß-glucan isolated from Lentinus edodes was successfully fractionated into three fractions with different weight-average molecular weight (Mw) by ultrasonic degradation and used for the treatment of colorectal cancer. In our findings, the ß-glucan was successfully degraded with the Mw decreased from 2.56 × 106 Da to 1.41 × 106 Da, exhibiting the triple helix structure without conformation disruption. The in vitro results indicate that ß-glucan fractions inhibited colon cancer cell proliferation, induced colon cancer cell apoptosis, and reduced inflammation. The in vivo results based on Azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model demonstrate that the lower-molecular weight ß-glucan fraction showed stronger anti-inflammatory and anti-colon cancer activities by reconstructing intestinal mucosal barrier, increasing short chain fatty acids (SCFAs) content, regulating metabolism of gut microbiota, and rebuilding the gut microbiota structure with the increased Bacteroides and the decreased Proteobacteria at the phylum level, as well as with the decreased Helicobacter and the increased Muribaculum at the genus level. These findings provide scientific basis for using the ß-glucan to regulate gut microbiota as an alternative strategy in the clinical treatment of colon cancer.

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Global Insights Into Lysine Acylomes Reveal Crosstalk Between Lysine Acetylation and Succinylation in Streptomyces coelicolor Metabolic Pathways.

Lysine acylations are reversible and ubiquitous post-translational modifications that play critical roles in regulating multiple cellular processes. In the current study, highly abundant and dynamic acetylation, besides succinylation, was uncovered in a soil bacterium, Streptomyces coelicolor. By affinity enrichment using anti-acetyl-lysine antibody and the following LC-MS/MS analysis, a total of 1298 acetylation sites among 601 proteins were identified. Bioinformatics analyses suggested that these acetylated proteins have diverse subcellular localization and were enriched in a wide range of biological functions. Specifically, a majority of the acetylated proteins were also succinylated in the tricarboxylic acid cycle and protein translation pathways, and the bimodification occurred at the same sites in some proteins. The acetylation and succinylation sites were quantified by knocking out either the deacetylase ScCobB1 or the desuccinylase ScCobB2, demonstrating a possible competitive relationship between the two acylations. Moreover, in vitro experiments using synthetically modified peptides confirmed the regulatory crosstalk between the two sirtuins, which may be involved in the collaborative regulation of cell physiology. Collectively, these results provided global insights into the S. coelicolor acylomes and laid a foundation for characterizing the regulatory roles of the crosstalk between lysine acetylation and succinylation in the future.

Fabrication of tumor-targeting composites based on the triple helical ß-glucan through conjugation of aptamer.

Herein the nucleic acid aptamers were attached to the polydeoxyadenylic acid (poly(dA)) tail for improving the tumor-targetability and cellular internalization of s-LNT/poly(dA) composite composed of two single chains of triple helical ß-glucan lentinan (s-LNT) and one poly(dA) chain. The in vitro results demonstrate that the cellular uptake of s-LNT/poly(dA) composites in MCF-7 cancer cells was enhanced effectively after attaching the aptamer. The as-prepared fluorescin isothiocyanate (FITC)-labelled LNT (LNT-FITC) through grafting was used for tracing the enhanced tumor-targetability of the composites. As a result, the cellular internalization of the LNT-FITC into MCF-7 and 4T1 cancer cells was further increased by the aptamer conjugated to poly(dA). Meanwhile, the in vivo experiments further demonstrate more s-LNT/poly(dA)-aptamer composites were effectively accumulated at the tumor site compared with s-LNT alone. This work provides a novel strategy for fabricating triplex ß-glucan as delivery vectors with active tumor-targetability.

Inhibition of tumor growth by ß-glucans through promoting CD4+ T cell immunomodulation and neutrophil-killing in mice.

ß-glucans are polysaccharides comprising ß-D-glucoses with various bioactivities. Herein, we extracted three ß-glucans from Lentinus edodes with different sources and assessed their antitumor activities on a mice model with intragastric, intraperitoneal and intratumoral injection. Three polysaccharides were shown to have the same chemical structure of ß-(1,3)-glucan with ß-(1,6) branches, and exhibited S-180 tumor-suppressing ability with good safety. It was found that ß-glucans up-regulated CD4+ T cell level in lymphoid organs decreased by tumor-burden, indicating promotion of immunomodulation. ß-glucans targeted tumors in vivo even after oral or intraperitoneal injection. Furthermore, ß-glucans not only targeted to lymphoid organs and increased CD4+ T cells number, but also enhanced CD4+ T cells and neutrophils populations in tumors. It was proposed that ß-glucans promoted CD4+ T cell immunomodulation and neutrophils infiltration into tumors, leading to tumor growth inhibition. These findings reveal that ß-glucans can be used as an effective agent for cancer immunotherapy.

Nanoplatform Constructed from a ß-Glucan and Polydeoxyadenylic Acid for Cancer Chemotherapy and Imaging.

A nanoplatform carrying doxorubicin (Dox) for cancer therapy and a dye for imaging was developed based on a natural triple helix ß-glucan (t-LNT) and polydeoxyadenylic acid (poly(dA)). The t-LNT-Dox conjugates were prepared through Schiff-base reaction between the aldehyde group in the oxidized t-LNT and the amino group of Dox, the single chains (s-LNT-Dox) of which interacted with the poly(dA)-dye to form a composite s-LNT-Dox/poly(dA)-dye through hydrogen bonding between s-LNT and poly(dA). t-LNT-Dox was confirmed to acid-responsively release Dox in vitro, showing enhanced cytotoxicity against HeLa cancer cells with time. It was confirmed that Dox and the dye could be simultaneously delivered into HeLa cells or the tumors with a prolonged duration time. Furthermore, LNT-Dox conjugates effectively inhibited tumor growth and decreased adverse effects of the free Dox in vivo. Hence, this work develops a new strategy to fabricate the nanoplatform for therapy and imaging using a natural polysaccharide.

Orally Delivered Antisense Oligodeoxyribonucleotides of TNF-α via Polysaccharide-Based Nanocomposites Targeting Intestinal Inflammation.

Tumor necrosis factor alpha (TNF-α) is usually regarded as a potential target for inflammatory bowel disease therapy. Herein, a promising strategy for effective delivery of phosphorothioated antisense oligodeoxyribonucleotide of TNF-α (PS-ATNF-α), targeting the intestinal inflammation based on the interaction of the single chain of triple helical ß-glucan (s-LNT) with poly-deoxyadenylic acid [poly(dA)], and the colon-specific degradation of chitosan-alginate (CA) hydrogel, is reported. The target gene of PS-ATNF-α, with a poly(dA) tail through a disulfide bond (-SS-), interacts with s-LNT to form a rod-like nanocomposite of s-LNT/poly(dA)-SS-PS-ATNF-α, which significantly inhibits lipopolysaccharide (LPS)-induced TNF-α at the protein level by 38.2% and mRNA level by 48.9% in RAW264.7 macrophages. The nanocomposites carried by the CA hydrogel with the loading amount of 83.5% are then orally administered and specifically released to the inflamed intestine, followed by internalization into intestinal cells such as macrophages, to reduce TNF-α production by 36.4% and dextran sulfate sodium-induced inflammation by decreasing myeloperoxidase and malondialdehyde. This study defines a new strategy for the oral delivery of antisense oligonucleotides to attenuate inflammatory response, demonstrating a notable potential for clinical applications in intestine-inflammation-targeted therapy.

Yeast ß-Glucan Suppresses the Chronic Inflammation and Improves the Microenvironment in Adipose Tissues of ob/ob Mice.

Inflammation in visceral adipose tissues (VATs) contributes to the pathology of diabetes. This study focused on the inflammatory regulation in VATs by a yeast ß-1,3-glucan (BYG) orally administered to ob/ob mice. BYG decreased pro-inflammatory modulators of TNF-α, IL-6, IL-1ß, CCL2, and SAA3, and increased anti-inflammatory factors of Azgp1 (2.53 ± 0.02-fold change) at protein and/or mRNA levels (p < 0.05). Remarkably, BYG decreased the degree of adipose tissue macrophages (ATMs) infiltration to 82.5 ± 8.3%, especially the newly recruited ATMs. Interestingly, BYG increased the protective Th2 cell regulator GATA3 (7.72 ± 0.04-fold change) and decreased immunosuppressors IL-10 and IL-1ra, suggesting that BYG elicited inflammation inhibition via stimulating immune responses. Additionally, BYG increased the gut microbiota proportion of Akkermansia from 0.07% to 4.85% and improved the microenvironment of VATs through decreasing fibrosis and angiogenesis. These findings suggest that BYG has anti-inflammatory effect in diabetic mice, which can be used as a food component and/or therapeutic agent for diabetes.

The ß-glucan from Lentinus edodes suppresses cell proliferation and promotes apoptosis in estrogen receptor positive breast cancers.

Breast cancer is now the most common cancer in worldwide women, and novel interventions are needed to overcome the resistance occurring in the estrogen-targeted endocrine therapy. Herein, we demonstrate that the ß-glucan from Lentinus edodes (LNT) exhibited a profound inhibition ratio of ∼53% against estrogen receptor positive (ER+) MCF-7 tumor growth in nude mice similar to the positive control of cisplatin. Immunohistochemistry images showed that LNT evidently suppressed cell proliferation and promoted apoptosis in MCF-7 tumor tissues. The Western blotting analysis indicated that LNT up-regulated the tumor suppressor p53, phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2), cleaved-Caspase 3 and poly [ADP (ribose)] polymerase 1 (PARP 1) protein levels, and reduced the expression of mouse double minute 2 (MDM2), telomerase reverse transcriptase (TERT), nuclear factor-kappa B (NF-κB) p65, B-cell lymphoma-2 (Bcl-2), estrogen receptor α (ERα), etc. in tumor tissues. Moreover, LNT significantly suppressed phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt) and mammalian target of rapamycin (mTOR) protein levels. It was thus proposed that LNT inhibited MCF-7 tumor growth through suppressing cell proliferation and enhancing apoptosis possibly via multiple pathways such as PI3K/Akt/mTOR, NF-κB-, ERK-, ERα-, caspase- and p53-dependent pathways. Interestingly, the cell viability assay, siRNA transfection, Western blotting and flow cytometric analysis suggested that LNT targeted p53/ERα to only suppress cell proliferation via cell cycle arrest at G2/M phase without apoptosis in vitro. The big difference between in vivo and in vitro data suggested that the immune responses triggered by the polysaccharide should mainly contribute to the apoptotic effect in vivo. Overall, this work provides a novel strategy to treat ER+ breast cancers by using a naturally occurring ß-glucan from mushrooms.

Orally Administered Baker's Yeast ß-Glucan Promotes Glucose and Lipid Homeostasis in the Livers of Obesity and Diabetes Model Mice.

Baker's yeast glucan (BYG) has been reported to be an anti-diabetic agent. In the work described herein, further study on the effect of orally administered BYG on glucose and lipid homeostasis in the livers of ob/ob mice was performed. It was found that BYG decreased the blood glucose and the hepatic glucose and lipid disorders. Western blotting analysis revealed that BYG up-regulated p-AKT and p-AMPK, and down-regulated p-Acc in the liver. Furthermore, RNA-Seq analysis indicated that BYG down-regulated genes responsible for gluconeogenesis (G6pase and Got1), fatty acid biosynthesis (Acly, Acc, Fas, etc.), glycerolipid synthesis (Gpam and Lipin1/2), and cholesterol synthesis (Hmgcr, Fdps, etc.). Additionally, BYG decreased glucose transporters SGLT1 and GLUT2, fat emulsification, and adipogenic genes/proteins in the intestine to decrease glucose and lipid absorption. All these findings demonstrated that BYG is beneficial for regulating glucose and lipid homeostasis in diabetic mice, and thus has potential applications in anti-diabetic foods or drugs.

Dendritic nanotubes self-assembled from stiff polysaccharides as drug and probe carriers.

Dendritic nanotubes (DNTs) with hydrophobic cavities were constructed directly from rigid branched ß-1,3-d-glucan (AF1) in aqueous solution, and the AF1 sample was isolated from the fruiting bodies of Auricularia auricula-judae, a household nutritional food. The structure of AF1 dendritic nanotubes was demonstrated with a transmission electron microscope (TEM) and a scanning electron microscope (SEM), and a schematic diagram was proposed to describe the formation process, which was supported by the results of static/dynamic light scattering (SLS/DLS) and atomic force microscopy (AFM). In solution, a sequential self-assembly of the AF1 chains in a parallel manner occurred to form lamellas followed by self-curling into nanotubes with the mean diameters from 20 to 80 nm, depending on the concentration and molecular weight of AF1, through hydrogen bonding and hydrophilic/hydrophobic interaction. As a result of the dendritic structure, the AF1 aggregates exhibited highly condensed hydrophobic regions, which could be used as carriers to achieve a high concentration of the target molecules. In our findings, the anticancer drug DOX and the fluorescent probe TPA-BMO could be loaded into the hydrophobic region of DNTs. Interestingly, DOX-loaded DNTs of AF1 exhibited high drug loading capacity and pH-triggered sustained release behaviors (>23 days) with reduced cytotoxicity in vitro. Moreover, the bioimaging experiment demonstrated that TPA-BMO-loaded DNTs of AF1 induced stronger fluorescence intensity than TPA-BMO alone, and maintained a longer duration time (18 days) in vivo. Therefore, the DNTs of AF1 have promising applications as bioactive carriers, especially in the fields of drug delivery and bioimaging.

Hypoglycemic activity of the Baker's yeast ß-glucan in obese/type 2 diabetic mice and the underlying mechanism.

SCOPE: ß-Glucans have been shown to reduce the risk of obesity and diabetes. However, they often contain diverse polysaccharides and other ingredients, leading to elusive experimental data and mechanisms. In this study, a pure ß-glucan was obtained from the crude Baker's yeast polysaccharides for investigating its effect on the metabolic disorders in high-fat diet induced obese (DIO)/type 2 diabetic (T2D) mice and the underlying mechanism. METHODS AND RESULTS: The Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy data indicated that the pure ß-glucan (BYGlc) was a linear ß-(1→3)-glucan. It was first found that the oral administration of BYGlc into T2D and DIO mice significantly downregulated the blood glucose through suppressing sodium-glucose transporter-1 expression in intestinal mucosa. Meanwhile, BYGlc promoted glycogen synthesis and inhibited fat accumulation in liver, and depressed macrophage infiltration and pro-inflammatory cytokines production measured by histochemistry/immunohistochemistry and ELISA. Additionally, BYGlc remarkably decreased Firmicutes population and increased the proportion of Akkermansia by 16S rDNA analysis. CONCLUSION: BYGlc showed hypoglycemic activity accompanied by promotion of metabolism and inhibition of inflammation in T2D/DIO mice model. The hypoglycemic mechanisms were first declared to be through suppressing sodium-glucose transporter-1 expression and possibly associated with the altered gut microbiota.

Anti-tumor effect of ß-glucan from Lentinus edodes and the underlying mechanism.

ß-Glucans are well known for its various bioactivities, but the underlying mechanism has not been fully understood. This study focuses on the anti-tumor effect and the potential mechanism of a branched ß-(1, 3)-glucan (LNT) extracted from Lentinus edodes. The in vivo data indicated that LNT showed a profound inhibition ratio of ~75% against S-180 tumor growth, even significantly higher than the positive control of Cytoxan (~54%). Interestingly, LNT sharply promoted immune cells accumulation into tumors accompanied by cell apoptosis and inhibition of cell proliferation during tumor development. Furthermore, LNT not only up-regulated expressions of the tumor suppressor p53, cell cycle arrestin p21 and pro-apoptotic proteins of Bax and caspase 3/9, but also down-regulated PARP1 and anti-apoptotic protein Bcl-2 expressions in tumor tissues. It was first found that LNT initiated p53-dependent signaling pathway to suppress cell proliferation in vitro, and the caspase-dependent pathway to induce cell apoptosis in vivo. The underlying anti-tumor mechanism was proposed that LNT activated immune responses to induce cell apoptosis through caspase 3-dependent signaling pathway and to inhibit cell proliferation possibly via p53-dependent signaling pathway in vivo. Besides, LNT inhibited angiogenesis by suppressing VEGF expression, leading to slow progression of tumors.

The linear structure of ß-glucan from baker's yeast and its activation of macrophage-like RAW264.7 cells.

Yeast ß-glucan has many formulations with different chemical structures, water solubility and purity. In particular, the purity of ß-glucan in these formulations is variable and relatively low, contributing to different data on its biological activity. In this study, the major polysaccharide component in the crude Baker's yeast polysaccharides coded as BBG with high purity of 99% was obtained, and its chemical structure was determined to be a linear ß-(1,3)-glucan. It was found that BBG interacted with complement receptor 3 (CR3) and toll-like receptor 2 (TLR2) on the surface of macrophage-like RAW264.7 cells, and initiated activation of RAW264.7 cells characterized by significant production of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1). Additionally, activation of the nuclear factor kappaB p65 (NF-κB p65), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) induced by BBG, were also observed, further confirming the stimulation of RAW264.7 cells by BBG. All these findings provided important scientific evidences for better understanding the molecular mechanism of action for the linear ß-(1,3)-glucan in cells.

Construction of selenium nanoparticles/ß-glucan composites for enhancement of the antitumor activity.

We report on a green procedure for the stabilization of selenium nanoparticles (SeNPs) by a naturally occurring ß-glucan with triple helical conformation known as Lentinan (t-LNT) in water after denaturing into single chains (s-LNT) at 140 °C. The results demonstrated that the s-LNT can interact with SeNPs through Se-O-H interaction. Transmission electron microscopy (TEM), energy dispersive X-ray (EDX) spectra, UV/vis, X-ray diffraction (XRD) and dynamic light scattering (DLS) showed that s-LNT coated SeNPs to form a stable nano-composite Se/s-LNT, leading to good dispersion of SeNPs. Especially, the as-prepared Se/s-LNT composite in the solution could remain homogeneous and translucent for 30 days without any precipitates. Different size distribution of SeNPs was prepared by simply controlling the concentrations of selenite sodium and the corresponding reducing agent ascorbic acid. The size effect of SeNPs on anti-tumor activity was revealed that the SeNPs with more evenly particle size distribution show the higher anticancer activity.