Damaged DNA Is an Early Event of Neurodegeneration in Induced Pluripotent Stem Cell-Derived Motoneurons with UBQLN2P497H Mutation.

Ubiquilin-2 (UBQLN2) mutations lead to familial amyotrophic lateral sclerosis (FALS)/and frontotemporal dementia (FTLD) through unknown mechanisms. The combination of iPSC technology and CRISPR-mediated genome editing technology can generate an iPSC-derived motor neuron (iPSC-MN) model with disease-relevant mutations, which results in increased opportunities for disease mechanism research and drug screening. In this study, we introduced a UBQLN2-P497H mutation into a healthy control iPSC line using CRISPR/Cas9, and differentiated into MNs to study the pathology of UBQLN2-related ALS. Our in vitro MN model faithfully recapitulated specific aspects of the disease, including MN apoptosis. Under sodium arsenite (SA) treatment, we found differences in the number and the size of UBQLN2+ inclusions in UBQLN2P497H MNs and wild-type (WT) MNs. We also observed cytoplasmic TAR DNA-binding protein (TARDBP, also known as TDP-43) aggregates in UBQLN2P497H MNs, but not in WT MNs, as well as the recruitment of TDP-43 into stress granules (SGs) upon SA treatment. We noted that UBQLN2-P497H mutation induced MNs DNA damage, which is an early event in UBQLN2-ALS. Additionally, DNA damage led to an increase in compensation for FUS, whereas UBQLN2-P497H mutation impaired this function. Therefore, FUS may be involved in DNA damage repair signaling.

Curcumol and FTY720 synergistically induce apoptosis and differentiation in chronic myelomonocytic leukemia via multiple signaling pathways.

Chronic myelomonocytic leukemia (CML) is a myeloid tumor characterized by MDS (myelodysplastic syndrome) and MPN (myeloproliferative neoplasms). Allogeneic hematopoietic stem cell transplantation, chemotherapy, interferon, and targeted therapy are the main treatment methods for CML. Tyrosine kinase inhibitors (TKIs) are also a treatment option, and patients are currently recommended to take these drugs throughout their lives to prevent CML recurrence. Therefore, there is a need to investigate and identify other potential chemotherapy drugs. Currently, research on CML treatment with a single drug has shown little progress. Fingolimod (FTY720), an FDA-approved drug used to treat relapsing multiple sclerosis, has also shown great potential in the treatment of lymphocytic leukemia. In our study, we find that FTY720 and curcumol have a significant inhibitory effect on K562 cells, K562/ADR cells, and CD34+ cells from CML patients. RNAseq data analysis shows that regulation of apoptosis and differentiation pathways are key pathways in this process. Besides, BCR/ABL-Jak2/STAT3 signaling, PI3K/Akt-Jnk signaling, and activation of BH3-only genes are involved in CML inhibition. In a K562 xenograft mouse model, therapy with curcumol and FTY720 led to significant inhibition of tumor growth and induction of apoptosis. To summarize, curcumol and FTY720 synergistically inhibit proliferation involved in differentiation and induce apoptosis in CML cells. Therefore, synergistic treatment with two drugs could be the next choice of treatment for CML.

Fusion with ARRDC1 or CD63: A Strategy to Enhance p53 Loading into Extracellular Vesicles for Tumor Suppression.

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.

Identification and verification of genes associated with hypoxia microenvironment in Alzheimer's disease.

As the incidence of Alzheimer's disease (AD) increases year by year, more people begin to study this disease. In recent years, many studies on reactive oxygen species (ROS), neuroinflammation, autophagy, and other fields have confirmed that hypoxia is closely related to AD. However, no researchers have used bioinformatics methods to study the relationship between AD and hypoxia. Therefore, our study aimed to screen the role of hypoxia-related genes in AD and clarify their diagnostic significance. A total of 7681 differentially expressed genes (DEGs) were identified in GSE33000 by differential expression analysis and cluster analysis. Weighted gene co-expression network analysis (WGCNA) was used to detect 9 modules and 205 hub genes with high correlation coefficients. Next, machine learning algorithms were applied to 205 hub genes and four key genes were selected. Through the verification of external dataset and quantitative real-time PCR (qRT-PCR), the AD diagnostic model was established by ANTXR2, BDNF and NFKBIA. The bioinformatics analysis results suggest that hypoxia-related genes may increase the risk of AD. However, more in-depth studies are still needed to investigate their association, this article would guide the insights and directions for further research.

Selection and identification of a novel ssDNA aptamer targeting human skeletal muscle.

Skeletal muscle disorders have posed great threats to health. Selective delivery of drugs and oligonucleotides to skeletal muscle is challenging. Aptamers can improve targeting efficacy. In this study, for the first time, the human skeletal muscle-specific ssDNA aptamers (HSM01, etc.) were selected and identified with Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The HSM01 ssDNA aptamer preferentially interacted with human skeletal muscle cells in vitro. The in vivo study using tree shrews showed that the HSM01 ssDNA aptamer specifically targeted human skeletal muscle cells. Furthermore, the ability of HSM01 ssDNA aptamer to target skeletal muscle cells was not affected by the formation of a disulfide bond with nanoliposomes in vitro or in vivo, suggesting a potential new approach for targeted drug delivery to skeletal muscles via liposomes. Therefore, this newly identified ssDNA aptamer and nanoliposome modification could be used for the treatment of human skeletal muscle diseases.

Characterization and microRNA Expression Analysis of Serum-Derived Extracellular Vesicles in Severe Liver Injury from Chronic HBV Infection.

BACKGROUND: Extracellular vesicle (EV) microRNAs have been documented in several studies to have significantly different expressions in hepatitis B virus (HBV)-related liver diseases, such as hepatocellular carcinoma (HCC). The current work aimed to observe the characteristics of EVs and EV miRNA expressions in patients with severe liver injury chronic hepatitis B (CHB) and patients with HBV-associated decompensated cirrhosis (DeCi). METHODS: The characterization of the EVs in the serum was carried out for three different groups, namely, patients with severe liver injury-CHB, patients with DeCi, and healthy controls. EV miRNAs were analyzed using miRNA-seq and RT-qPCR arrays. Additionally, we assessed the predictive and observational values of the miRNAs with significant differential expressions in serum EVs. RESULTS: Patients with severe liver injury-CHB had the highest EV concentrations when compared to the normal controls (NCs) and patients with DeCi (p < 0.001). The miRNA-seq of the NC and severe liver injury-CHB groups identified 268 differentially expressed miRNAs (|FC| > 2, p < 0.05). In this case, 15 miRNAs were verified using RT-qPCR, and it was found that novel-miR-172-5p and miR-1285-5p in the severe liver injury-CHB group showed marked downregulation in comparison to the NC group (p < 0.001). Furthermore, compared with the NC group, three EV miRNAs (novel-miR-172-5p, miR-1285-5p, and miR-335-5p) in the DeCi group showed various degrees of downregulated expression. However, when comparing the DeCi group with the severe liver injury-CHB group, only the expression of miR-335-5p in the DeCi group decreased significantly (p < 0.05). For the severe liver injury-CHB and DeCi groups, the addition of miR-335-5p improved the predictive accuracy of the serological levels, while miR-335-5p was significantly correlated with ALT, AST, AST/ALT, GGT, and AFP. Conclusions: The patients with severe liver injury-CHB had the highest number of EVs. The combination of novel-miR-172-5p and miR-1285-5p in serum EVs helped in predicting the progression of the NCs to severe liver injury-CHB, while the addition of EV miR-335-5p improved the serological accuracy of predicting the progression of severe liver injury-CHB to DeCi.

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica.

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A(565) to A(280) of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS-PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.

Phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates from phycobilisomes of the marine red alga Polysiphonia urceolata.

Phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates showing special spectral characteristics were prepared from the partly dissociated products of phycobilisomes from Polysiphonia urceolata. The absorption difference spectra between the aggregates and phycoerythrins showing normal spectral characteristics show peaks at 583 nm. The fluorescence emission difference spectra between the phycoerythrin aggregates and normal phycoerythrins show peaks at 602 nm. The special spectral characteristics of the PE aggregates disappeared when the PE aggregates were dissociated in deionized water. The intact phycobilisomes show similar characteristics as phycoerythrin-phycocyanin aggregates. When the peak values at 583 nm in absorption difference spectra were compared with those at 615 nm, it can be found that the amount of the 583 nm chromophores in the phycoerythrin-phycocyanin aggregates and intact phycobilisomes is similar to the amount of PCB chromophores in phycocyanins. It can be concluded that the 583 nm chromophores are at the interface between phycoerythrins and phycocyanins in phycobilisomes and transfer the light energy absorbed by other chromophores in phycoerythrins to the PCB chromophores in phycocyanins. A rod linker polypeptide with molecular weight of 40 kDa was found in the phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates, and it is believed to play roles in the spectral red shift of the aggregates.

Phycoerythrins in phycobilisomes from the marine red alga Polysiphonia urceolata.

Phycoerythrins (PE) in phycobilisomes from Polysiphonia urceolata were studied in this research. Dissociative products of phycobilisomes were analyzed by sucrose density gradient centrifugation and native-PAGE. At least three types of PEs were found in the dissociative products of phycobilisomes. According to their molecular weights, absorption spectra and subunit components, they should be PE hexamer containing γ1 subunit, PE hexamer containing γ2 subunit and PE monomer containing no γ subunit. PEs bigger than hexmer were also found in the dissociative products of phycobilisomes in 200mM phosphate buffer when dissociated phycobilisomes were analyzed by sucrose density gradient centrifugation. PE trimers containing no γ subunits were also found in products of dissociated phycobilisomes in deionized water when dissociated phycobilisomes were analyzed by native-PAGE. This is the first time that pure PE hexamers containing γ2 subunits were isolated from P. urceolata. The PE monomers containing no γ subunits should come from PE trimers or hexamers containing no γ subunits in the "rod" of phycobilisomes. It can be concluded that there are three types of PEs in "rod" of phycobilisomes from P. urceolata: PE containing γ1 subunit, PE containing γ2 subunit and PE containing no γ subunit.

Isolation, purification and properties of an R-phycocyanin from the phycobilisomes of a marine red macroalga Polysiphonia urceolata.

Phycobilisomes were prepared from a marine red macroalga Polysiphonia urceolata (P. urceolata) by sucrose step-gradient ultracentrifugation. From the prepared phycobilisomes, an R-phycocyanin was isolated by gel filtration on Sephadex G-150 and then purified by ion exchange chromatography on DEAE-Sepharose Fast Flow and native polyacrylamide gel electrophoresis (PAGE) performed in neutral buffer systems. The purified R-phycocyanins showed not only a homogeneous trimer of 136 kDa in gel filtration and a single band in native PAGE, but also exhibited one band at about pH 5.7 in native isoelectric focusing (IEF). By a gradient SDS-PAGE the purified R-phycocyanin was determined to contain one a subunit of 17.5 kDa (α17.5) and two b subunits of 21.3 kDa and 22.6 kDa (ß21.3 and ß22.6). The analysis from denaturing isoelectric focusing and two-dimension PAGE demonstrated that α17.5, ß21.3 and ß22.6 had their pIs of 6.4, 5.3 and 5.4, respectively. Furthermore, mass spectroscopy analysis of ß21.3 and ß22.6 by MALDI-TOF mass spectrometry demonstrated the two b subunits had differences in peptide mass fingerprinting. These results revealed that the prepared R-phycocyanins were composed of one a and two b subunits. (α17:53 ß21:32 ß22:61) and (α17:53 ß21:31 ß22:62), which have a structural foundation to show their pIs too close for them to be definitely resolved by native IEF, are postulated to be the most possible trimeric forms of the R-phycocyanins prepared from the phycobilisomes of P. urceolata.

Isolation, purification and properties of an R-Phycocyanin from the phycobilisomes of a marine red macroalga Polysiphonia urceolata.

Phycobilisomes were prepared from a marine red macroalga Polysiphonia urceolata (P. urceolata) by sucrose step-gradient ultracentrifugation. From the prepared phycobilisomes, an R-phycocyanin was isolated by gel filtration on Sephadex G-150 and then purified by ion exchange chromatography on DEAE-Sepharose Fast Flow and native polyacrylamide gel electrophoresis (PAGE) performed in neutral buffer systems. The purified R-phycocyanins showed not only a homogeneous trimer of 136 kDa in gel filtration and a single band in native PAGE, but also exhibited one band at about pH 5.7 in native isoelectric focusing (IEF). By a gradient SDS-PAGE the purified R-phycocyanin was determined to contain one α subunit of 17.5 kDa (α (17.5)) and two ß subunits of 21.3 kDa and 22.6 kDa (ß (21.3) and ß (22.6)). The analysis from denaturing isoelectric focusing and two-dimension PAGE demonstrated that α (17.5), ß (21.3) and ß (22.6) had their pIs of 6.4, 5.3 and 5.4, respectively. Furthermore, mass spectroscopy analysis of ß (21.3) and ß (22.6) by MALDI-TOF mass spectrometry demonstrated the two ß subunits had differences in peptide mass fingerprinting. These results revealed that the prepared R-phycocyanins were composed of one α and two ß subunits. and , which have a structural foundation to show their pIs too close for them to be definitely resolved by native IEF, are postulated to be the most possible trimeric forms of the R-phycocyanins prepared from the phycobilisomes of P. urceolata.

The 42.1 and 53.7 kDa bands in SDS-PAGE of R-phycoerythrin from Polysiphonia urceolata.

In SDS-PAGE gels of three purified R-phycoerythrins (R-PEs) isolated from three species of red algae, two bands whose molecular weights were about 40 kDa and 50 kDa can stably be found when the sample loading amount was enough. It is important for structure study of R-PE to clarify what these bands represent and how they are formed. According to results of the second SDS-PAGE, as well as molecular weights, fluorescences under UV and abundance, the 42.1 kDa and 53.7 kDa bands in SDS-PAGE gels of R-PE from Polysiphonia urceolata were believed to be complexes of αß and ßγ1, respectively. Formation of these bands may be related to light and phycourobilins (PUB) in subunits; and appearance of these two bands provided some proofs on position of chromophores and directions of energy transfer in R-PE. R-PE containing γ1 subunit was obviously more stable than R-PE containing γ2 subunit when they were exposed to protein denaturants, so γ subunits of R-PE may play important roles in structural stability of R-PE aggregates and the main forces that maintain the stability of R-PE may be interactions between γ subunit and ß subunits.

Influence of ionic strength, pH, and SDS concentration on subunit analysis of phycoerythrins by SDS-PAGE.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often used for subunit analysis of proteins, but it is not efficient to make the alpha- and beta-subunits of phycoerythrins separated by normal SDS-PAGE. In this research, subunit components and subunit molecular weights of four purified phycoerythrins were analyzed by SDS-PAGE. Four factors including Tris concentration, pH, ammonium persulfate (APS), and SDS concentration were studied for their effects on SDS-PAGE of phycoerythrins. It showed that these factors can influence the separation of alpha- and beta-subunits, electrophoresis effect of gamma-subunits, apparent molecular weights of subunits, and mobility of marker proteins. The alpha- and beta-subunits separated better in the case of lower SDS concentration, lower Tris concentration, higher pH, and/or lower APS addition in separating gels. The molecular weights of alpha- and beta-subunits increased when Tris concentration increased in a certain range. It can be concluded that factors critical to subunit analysis by SDS-PAGE are SDS concentration and ionic strength, both of which are related to critical micelle concentration of SDS and ratio of SDS monomer to micelle in SDS-PAGE system. The ratio is postulated to influence SDS-PAGE by influencing the amount of SDS bound to polypeptides and shapes of polypeptide-SDS complexes.