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Autophagy May 2024Circular RNAs (circRNAs) are special non-coding RNA (ncRNA) molecules that play a significant role in many diseases. However, the biogenesis and regulation of circRNAs...
Circular RNAs (circRNAs) are special non-coding RNA (ncRNA) molecules that play a significant role in many diseases. However, the biogenesis and regulation of circRNAs in diabetic nephropathy (DN) are largely unknown. Here, we investigated the expression profile of circRNAs in kidney of DN mice through circular RNA sequencing (circRNA-seq). The renal biopsy samples of patients with DN had low expression, which was significantly associated with renal function. Furthermore, loss-of-function and gain-of-function experiments were carried out to prove the role of in DN. Podocyte conditional knockin (cKI) or systemic overexpression of alleviated albuminuria and restored macroautophagy/autophagy in kidney of diabetic mice. However, knockdown exacerbated albuminuria and podocyte injury. Mechanistically, we found directly binds to to perform its function. Silencing of or overexpression of recovered podocyte autophagy both and . To examine the cause of downregulation in DN, bioinformatics prediction found that sequence has a high possibility of containing an m6A methylation site. Additionally, METTL3 was proved to regulate the expression and methylation level of through YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). In conclusion, this study revealed that regulates podocyte autophagy by targeting in DN. Therefore, is a potential biomarker for prevention and cure of DN. CCL2/MCP-1: C-C motif chemokine ligand 2; ceRNA: competing endogenous RNA; circRNA: circular RNA; cKI: conditional knockin; cKO: conditional knockout; CRE: creatinine; DM: diabetes mellitus; DN: diabetic nephropathy; ESRD: end-stage renal disease; HG: high glucose; IF: immunofluorescence; MAP1LC3/LC3B: microtubule-associated protein 1 light chain 3 beta; MPC5: mouse podocyte clone 5; MTECs: mouse tubular epithelial cells; MTOR: mechanistic target of rapamycin kinase; NC: normal control; ncRNA: non-coding RNA; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PAS: periodic acid-Schiff; RELA/p65: v-rel reticuloendotheliosis viral oncogene homolog A (avian); SDs: slit diaphragm proteins; Seq: sequencing; STZ: streptozotocin; SV40: SV40-MES13-cells, mouse mesangial cell line; T1D: type 1 diabetes mellitus; T2D: type 2 diabetes mellitus; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor; VECs: vascular endothelial cells; WT1: WT1 transcription factor; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.
Topics: Diabetic Nephropathies; Animals; Podocytes; RNA, Circular; Autophagy; MicroRNAs; Autophagy-Related Proteins; Mice; Humans; Male; Mice, Inbred C57BL; Diabetes Mellitus, Experimental; Methyltransferases; Cysteine Endopeptidases
PubMed: 38050963
DOI: 10.1080/15548627.2023.2286128 -
Arthritis Research & Therapy Dec 2023To identify the role of gluconeogenesis in chondrocytes in osteoarthritis (OA).
PURPOSE
To identify the role of gluconeogenesis in chondrocytes in osteoarthritis (OA).
MATERIALS AND METHODS
Cartilage samples were collected from OA patients and C57 mice and were stained with Safranin O-Fast Green to determine the severity of OA. Periodic acid Schiff staining was used to characterize the contents of polysaccharides and SA-βGal staining was used to characterize the aging of chondrocytes. Immunohistochemistry and western blotting were used to detect fructose-bisphosphatase1 (FBP1), SOX9, MMP13, P21, and P16 in cartilage or chondrocyte. The mRNA levels of fbp1, mmp13, sox9, colX, and acan were analyzed by qPCR to evaluate the role of FBP1 in chondrocytes.
RESULTS
The level of polysaccharides in cartilage was reduced in OA and the expression of FBP1 was also reduced. We treated the chondrocytes with IL-1β to cause OA in vitro, and then made chondrocytes overexpress FBP1 with plasma. It shows that FBP1 alleviated the degeneration and senescence of chondrocytes in vitro and that it also showed the same effects in vivo experiments. To further understand the mechanism of FBP1, we screened the downstream protein of FBP1 and found that CRB3 was significantly downregulated. And we confirmed that CRB3 suppressed the degeneration and delayed senescence of chondrocytes.
CONCLUSIONS
FBP1 promoted the polysaccharide synthesis in cartilage and alleviated the degeneration of cartilage by regulating CRB3, so FBP1 is a potential target in treating OA.
Topics: Animals; Humans; Mice; Cartilage, Articular; Chondrocytes; Interleukin-1beta; Matrix Metalloproteinase 13; Osteoarthritis; Polysaccharides; Fructose-Bisphosphatase; Membrane Glycoproteins
PubMed: 38049890
DOI: 10.1186/s13075-023-03221-5 -
Virus Research Oct 2023Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal...
Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal disease outbreaks worldwide. Among the eight proteins encoded by the CeMV genome, the haemagglutinin (H) glycoprotein is responsible for the virus attachment to host cell receptors. CeMV H represents an attractive target for antiviral and diagnostic research, yet the elucidation of the molecular mechanisms underlying its role in infection and inter-species transmission was hampered thus far due to the unavailability of recombinant versions of the protein. Here we present the cloning, expression and purification of a recombinant CeMV H ectodomain (rH-ecto), providing an initial characterization of its biophysical and structural properties. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) combined to Western blot analysis and periodic acid Schiff assay showed that CeMV rH-ecto is purifiable at homogeneity from insect cells as a secreted, soluble and glycosylated protein. Miniaturized differential scanning fluorimetry, Blue Native PAGE and size exclusion chromatography coupled to multiangle light scattering revealed that CeMV rH-ecto is globularly folded, thermally stable and exists in solution in the oligomeric states of dimer and multiple of dimers. Furthermore, negative stain electron microscopy single particle analysis allowed us to delineate a low-resolution molecular architecture of the CeMV rH-ecto dimer, which recapitulates native assemblies from other morbilliviral H proteins, such as those from measles virus and canine distemper virus. This set of experiments by orthogonal techniques validates the CeMV rH-ecto as an experimental model for future biochemical studies on its structure and functions.
PubMed: 37769814
DOI: 10.1016/j.virusres.2023.199231 -
Liver International : Official Journal... Feb 2024Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important...
BACKGROUND AND AIMS
Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated.
METHODS
The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444-464 aa) were used for in vitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes.
RESULTS
Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions.
CONCLUSION
Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues.
Topics: Animals; Mice; Glucose; Glycogen; Lactates; Liver; Mitochondria; NAD; Oxidative Stress; Perilipin-5
PubMed: 37933091
DOI: 10.1111/liv.15775 -
Bio Systems Oct 2023The gene encoding the spike glycoprotein of the SARS-CoV-2 virus that causes COVID-19 disease, was analyzed through two types of periodic tables (standard and cube) of...
The gene encoding the spike glycoprotein of the SARS-CoV-2 virus that causes COVID-19 disease, was analyzed through two types of periodic tables (standard and cube) of the genetic code to discover the internal fine structure of the spike (S) protein. The analysis was performed on the Wuhan-Hu-1 SARS-CoV-2 sequence (GenBank accession number NC_045512.2). A partition was detected between codon numbers (three-letter code numbers) 47 and 48 that code amino acids in the S-protein. The population distribution of organized codes and amino acid replacements in the S-protein showed large differences between two regions of the cube-type periodic table. The genetic codes of codon numbers 48-63 (4th plane of the cube table) had a higher frequency than the genetic codes of each of the other three planes (1st-3rd planes). Planes-linkage structures involved in the partition were also analyzed and a simplified model for the S-protein gene was obtained where a planes-linkage of the 4th plane and another planes-linkage of the 1st-3rd planes were linked together in alternate shifts. Most of the code population in the 4th plane and their planes-linkage multiformity gave additional support to the partition between codon numbers 47 and 48 in the S-protein gene. Analysis of real lineages of the SARS-CoV-2 virus through the cube-type periodic table identified distinguishing features of the Omicron lineage that included not only a large code population within the receptor-binding domain of the S-protein, but also large percentage rises in the population of amino acid replacements in the 1st and 2nd planes.
Topics: Humans; SARS-CoV-2; COVID-19; Spike Glycoprotein, Coronavirus; Amino Acids; Glycoproteins
PubMed: 37611861
DOI: 10.1016/j.biosystems.2023.104990 -
Advanced Science (Weinheim,... Aug 2023DNA has been used as a robust material for the building of a variety of nanoscale structures and devices owing to its unique properties. Structural DNA nanotechnology... (Review)
Review
DNA has been used as a robust material for the building of a variety of nanoscale structures and devices owing to its unique properties. Structural DNA nanotechnology has reported a wide range of applications including computing, photonics, synthetic biology, biosensing, bioimaging, and therapeutic delivery, among others. Nevertheless, the foundational goal of structural DNA nanotechnology is exploiting DNA molecules to build three-dimensional crystals as periodic molecular scaffolds to precisely align, obtain, or collect desired guest molecules. Over the past 30 years, a series of 3D DNA crystals have been rationally designed and developed. This review aims to showcase various 3D DNA crystals, their design, optimization, applications, and the crystallization conditions utilized. Additionally, the history of nucleic acid crystallography and potential future directions for 3D DNA crystals in the era of nanotechnology are discussed.
Topics: Nucleic Acid Conformation; DNA; Nanotechnology
PubMed: 37327311
DOI: 10.1002/advs.202302021 -
Nature Communications Jul 2023Enzymes fold into unique three-dimensional structures to distribute their reactive amino acid residues, but environmental changes can disrupt their essential folding and...
Enzymes fold into unique three-dimensional structures to distribute their reactive amino acid residues, but environmental changes can disrupt their essential folding and lead to irreversible activity loss. The de novo synthesis of enzyme-like active sites is challenging due to the difficulty of replicating the spatial arrangement of functional groups. Here, we present a supramolecular mimetic enzyme formed by self-assembling nucleotides with fluorenylmethyloxycarbonyl (Fmoc)-modified amino acids and copper. This catalyst exhibits catalytic functions akin those of copper cluster-dependent oxidases, and catalytic performance surpasses to date-reported artificial complexes. Our experimental and theoretical results reveal the crucial role of periodic arrangement of amino acid components, enabled by fluorenyl stacking, in forming oxidase-mimetic copper clusters. Nucleotides provide coordination atoms that enhance copper activity by facilitating the formation of a copper-peroxide intermediate. The catalyst shows thermophilic behavior, remaining active up to 95 °C in an aqueous environment. These findings may aid the design of advanced biomimetic catalysts and offer insights into primordial redox enzymes.
Topics: Copper; Biomimetics; Oxidoreductases; Metalloproteins; Amino Acids; Nucleotides
PubMed: 37419896
DOI: 10.1038/s41467-023-39779-6 -
International Journal of Molecular... Dec 2023Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid‑β (Aβ) in the brain. The gut/brain axis may serve a role in AD...
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid‑β (Aβ) in the brain. The gut/brain axis may serve a role in AD pathogenesis. The present study investigated deposition of Aβ in the intestinal epithelium and its potential effects on intestinal barrier function in a transgenic mouse model of AD. To investigate alterations in the structure and functionality of the intestinal mucosal barrier in AD model mice, hematoxylin and eosin staining for Paneth cell count, Alcian blue‑periodic acid Schiff staining for goblet cells, immunohistochemistry and immunofluorescence for mucin (MUC)2 and wheat germ agglutin expression, transmission electron microscopy for mucosal ultrastructure, FITC‑labeled dextran assay for intestinal permeability, quantitative PCR for goblet cell precursor expression and western blot analysis for tight junction proteins, MUC2 and inflammatory cytokine detection were performed. The results showed that AD model mice exhibited excessive Aβ deposition in the intestinal epithelium, which was accompanied by increased intestinal permeability, inflammatory changes and decreased expression of tight junction proteins. These alterations in the intestinal barrier led to an increased proliferation of goblet and Paneth cells and increased mucus synthesis. Dysfunction of gut barrier occurs in AD and may contribute to its etiology. Future therapeutic strategies to reverse AD pathology may involve early manipulation of gut physiology and its microbiota.
Topics: Mice; Animals; Mucins; Alzheimer Disease; Tight Junctions; Intestinal Mucosa; Mice, Transgenic; Permeability; Tight Junction Proteins
PubMed: 37830152
DOI: 10.3892/ijmm.2023.5316 -
Anatomia, Histologia, Embryologia Sep 2023Avian ceca play an important role in liquid absorption, cellulose digestion, and defensive mechanism. This study aims to demonstrate histological and histochemical...
Avian ceca play an important role in liquid absorption, cellulose digestion, and defensive mechanism. This study aims to demonstrate histological and histochemical characteristics of developing chicken cecum. For this purpose, 10 embryos on the 18th day of incubation, 10 chicks on hatching day and 10 chicks on the seventh day post-hatching were used. The histological sections prepared from proximal, middle, and distal parts of cecum were stained with Crossmon's triple stain, periodic-acid Schiff, Alcian blue (pH 2.5), Masson-Fontana's argentaffin silver stain and Gordon and Sweets's silver stain. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were also demonstrated in the sections. In the proximal part, although the villi were rudimentary on the 18th day of incubation, well-developed villi were seen at seventh day post-hatching. In middle and distal parts, while it was seen that rudimentary folds appeared on the 18th day of incubation, mucosal folds were prominent and short villi were formed on the hatching day and seventh day post-hatching. Goblet cells and enteroendocrine cells were detected from the 18th day of incubation. The lymphoid follicles supported with reticular fibres were seen on the seventh day post-hatching in proximal cecum wall. While ACP-ase (+) lymphocytes were rarely observed, more ANAE (+) lymphocytes were in lymphoid follicles. As a result, development of cecum in chickens has been demonstrated by histological techniques in this study.
Topics: Animals; Chickens; Naphthol AS D Esterase; Cecum; Lymphocytes
PubMed: 37271871
DOI: 10.1111/ahe.12936 -
Journal of Molecular Biology Jul 2023Metal-binding proteins are essential for the vital activities and engage in their roles by acting in concert with metal cations. MbPA (The Metal-binding Protein Atlas)...
Metal-binding proteins are essential for the vital activities and engage in their roles by acting in concert with metal cations. MbPA (The Metal-binding Protein Atlas) is the most comprehensive resource up to now dedicated to curating metal-binding proteins. Currently, it contains 106,373 entries and 440,187 sites related to 54 metals and 8169 species. Users can view all metal-binding proteins and species-specific proteins in MbPA. There are also metal-proteomics data that quantitatively describes protein expression in different tissues and organs. By analyzing the data of the amino acid residues at the metal-binding site, it is found that about 80% of the metal ions tend to bind to cysteine, aspartic acid, glutamic acid, and histidine. Moreover, we use Diversity Measure to confirm that the diversity of metal-binding is specific in different area of periodic table, and further elucidate the binding modes of 19 transition metals on 20 amino acids. In addition, MbPA also embraces 6855 potential pathogenic mutations related to metalloprotein. The resource is freely available at http://bioinfor.imu.edu.cn/mbpa.
Topics: Amino Acids; Binding Sites; Cations; Metalloproteins; Metals
PubMed: 37086947
DOI: 10.1016/j.jmb.2023.168117