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International Journal of Molecular... Dec 2023Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are...
Optimized Recovery of Immature Germ Cells after Prepubertal Testicular Tissue Digestion and Multi-Step Differential Plating: A Step towards Fertility Restoration with Cancer-Cell-Contaminated Tissue.
Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal porcine ITT. Strategies for maximizing germ cell recovery, including a comparison of two enzymatic digestion protocols (P1 and P2) of ITT fragment sizes (4 mm and 8 mm and multi-step differential plating were explored. Cellular viability and yield, as well as numbers and proportions of DDX4+ germ cells, were assessed before incubating the cell suspensions overnight on uncoated plastics. Adherent cells were processed for immunocytochemistry (ICC) and floating cells were further incubated for three days on Poly-D-Lysine-coated plastics. Germ cell yield and cell types using ICC for SOX9, DDX4, ACTA2 and CYP19A1 were assessed at each step of the multi-step differential plating. Directly after digestion, cell suspensions contained >92% viable cells and 4.51% DDX4+ germ cells. Pooled results for fragment sizes revealed that the majority of DDX4+ cells adhere to uncoated plastics (P1; 82.36% vs. P2; 58.24%). Further incubation on Poly-D-Lysine-coated plastics increased germ cell recovery (4.80 ± 11.32 vs. 1.90 ± 2.07 DDX4+ germ cells/mm, respectively for P1 and P2). The total proportion of DDX4+ germ cells after the complete multi-step differential plating was 3.12%. These results highlight a reduced proportion and number of germ cells lost when compared to data reported with other methods, suggesting that multi-step differential plating should be considered for optimization of immature germ cell recovery. While Poly-D-Lysine-coating increased the proportions of recovered germ cells by 16.18% (P1) and 28.98% (P2), future studies should now focus on less cell stress-inducing enzymatic digestion protocols to maximize the chances of fertility restoration with low amounts of cryo-banked human ITT.
Topics: Humans; Animals; Swine; Neoplasms; Germ Cells; Fertility; Lysine; Plastics; Poly A; Digestion; Polylysine
PubMed: 38203691
DOI: 10.3390/ijms25010521 -
International Journal of Molecular... Dec 2023Delivery systems for biologically active substances such as proanthocyanidins (PCANs), produced in the form of electrospun nonwoven through the electrospinning method,...
Delivery systems for biologically active substances such as proanthocyanidins (PCANs), produced in the form of electrospun nonwoven through the electrospinning method, were designed using a polymeric blend of poly(L-lactide-co-glycolide) (PLGA)and poly[(R,S)-3-hydroxybutyrate] ((R,S)-PHB). The studies involved the structural and thermal characteristics of the developed electrospun three-dimensional fibre matrices unloaded and loaded with PCANs. In the next step, the hydrolytic degradation tests of these systems were performed. The release profile of PCANs from the electrospun nonwoven was determined with the aid of UV-VIS spectroscopy. Approximately 30% of the PCANs were released from the tested electrospun nonwoven during the initial 15-20 days of incubation. The chemical structure of water-soluble oligomers that were formed after the hydrolytic degradation of the developed delivery system was identified through electrospray ionization mass spectrometry. Oligomers of lactic acid and OLAGA oligocopolyester, as well as oligo-3-hydroxybutyrate terminated with hydroxyl and carboxyl end groups, were recognized as degradation products released into the water during the incubation time. It was also demonstrated that variations in the degradation rate of individual mat components influenced the degradation pattern and the number of formed oligomers. The obtained results suggest that the incorporation of proanthocyanidins into the system slowed down the hydrolytic degradation process of the poly(L-lactide--glycolide)/poly[(R,S)-3-hydroxybutyrate] three-dimensional fibre matrix. In addition, in vitro cytotoxicity and antimicrobial studies advocate the use of PCANs for biomedical applications with promising antimicrobial activity.
Topics: Humans; Polyesters; Periodontal Pocket; 3-Hydroxybutyric Acid; Proanthocyanidins; Drug Delivery Systems; Anti-Infective Agents; Hydroxybutyrates; Poly A; Water
PubMed: 38203673
DOI: 10.3390/ijms25010503 -
International Journal of Molecular... Dec 2023Poly(butylene sebacate-co-terephthalate) (PBSeT) copolyesters are prepared by melt polymerization via two-step transesterification and polycondensation using...
Poly(butylene sebacate-co-terephthalate) (PBSeT) copolyesters are prepared by melt polymerization via two-step transesterification and polycondensation using pentaerythritol (PE) as a branching agent. The effects of the incorporated PE on its chemical, thermal, mechanical, and degradation properties, along with the rheological properties of its melt, are investigated. The highest molecular weight and intrinsic viscosity along with the lowest melt flow index were achieved at a PE content of 0.2 mol%, with minimal reduction in the tensile strength and the highest tear strength. The addition of PE did not significantly influence the thermal behavior and stability of the PBSeT copolyesters; however, the elongation at break decreased with increasing PE content. The sample with 0.2 mol% PE exhibited a higher storage modulus and loss modulus as well as a lower loss angle tangent than the other samples, indicating improved melt elasticity. The incorporation of more than 0.2 mol% PE enhanced the enzymatic degradation of copolyesters. In summary, including within 0.2 mol%, PE effectively improved both the processability-related characteristics and degradation properties of PBSeT copolyesters, suggesting their potential suitability for use in agricultural and packaging materials.
Topics: Poly A; Esterification; Fatty Acids; Propylene Glycols; Phthalic Acids; Alkenes
PubMed: 38203226
DOI: 10.3390/ijms25010055 -
Cells Dec 2023Small ubiquitin-related modifiers (SUMOs) function as post-translational protein modifications and regulate nearly every aspect of cellular function. While a single... (Review)
Review
Small ubiquitin-related modifiers (SUMOs) function as post-translational protein modifications and regulate nearly every aspect of cellular function. While a single ubiquitin protein is expressed across eukaryotic organisms, multiple SUMO paralogues with distinct biomolecular properties have been identified in plants and vertebrates. Five SUMO paralogues have been characterized in humans, with SUMO1, SUMO2 and SUMO3 being the best studied. SUMO2 and SUMO3 share 97% protein sequence homology (and are thus referred to as SUMO2/3) but only 47% homology with SUMO1. To date, thousands of putative sumoylation substrates have been identified thanks to advanced proteomic techniques, but the identification of SUMO1- and SUMO2/3-specific modifications and their unique functions in physiology and pathology are not well understood. The SUMO2/3 paralogues play an important role in proteostasis, converging with ubiquitylation to mediate protein degradation. This function is achieved primarily through SUMO-targeted ubiquitin ligases (STUbLs), which preferentially bind and ubiquitylate poly-SUMO2/3 modified proteins. Effects of the SUMO1 paralogue on protein solubility and aggregation independent of STUbLs and proteasomal degradation have also been reported. Consistent with these functions, sumoylation is implicated in multiple human diseases associated with disturbed proteostasis, and a broad range of pathogenic proteins have been identified as SUMO1 and SUMO2/3 substrates. A better understanding of paralogue-specific functions of SUMO1 and SUMO2/3 in cellular protein quality control may therefore provide novel insights into disease pathogenesis and therapeutic innovation. This review summarizes current understandings of the roles of sumoylation in protein quality control and associated diseases, with a focus on the specific effects of SUMO1 and SUMO2/3 paralogues.
Topics: Humans; Animals; Proteomics; Ubiquitin; Protein Processing, Post-Translational; Eukaryota; Poly A; Small Ubiquitin-Related Modifier Proteins; SUMO-1 Protein
PubMed: 38201212
DOI: 10.3390/cells13010008 -
G3 (Bethesda, Md.) Mar 2024The decay of messenger RNA with a premature termination codon by nonsense-mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway...
The decay of messenger RNA with a premature termination codon by nonsense-mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in Saccharomyces cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2, and UPF3), as well as NMD4 and EBS1, we identify factors known to function in posttermination recycling and characterize their contribution to NMD. These observations in S. cerevisiae expand on data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD by showing that perturbations in factors implicated in 40S recycling also correlate with a loss of NMD.
Topics: Animals; Saccharomyces cerevisiae; RNA Helicases; Nonsense Mediated mRNA Decay; Ribosomes; RNA, Messenger; Mammals
PubMed: 38198768
DOI: 10.1093/g3journal/jkad295 -
Nucleic Acids Research Apr 2024LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two...
LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'→3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.
Topics: Humans; HeLa Cells; Long Interspersed Nucleotide Elements; Retroelements; RNA; RNA, Messenger
PubMed: 38197223
DOI: 10.1093/nar/gkad1251 -
Translational Cancer Research Dec 2023Breast cancer is one of the main causes of death among women. RNA binding proteins (RBPs) play a crucial role in the progression of breast cancer, with increasingly...
BACKGROUND
Breast cancer is one of the main causes of death among women. RNA binding proteins (RBPs) play a crucial role in the progression of breast cancer, with increasingly detailed understanding of RBP functional molecular mechanisms in breast cancer, the functional research of RBPs may help elucidate the potential mechanisms of tumor occurrence, development, invasion, metastasis and prognosis. DDB1- and CUL4-associated factor 13 (DCAF13) is an RBPs has been identified as a substrate receptor for the CUL4-DDB1 E3 ligase complex. Its expression is related to the prognosis of certain cancer. We tried to explore both co-expressed network and biological functions of DCAF13 in breast cancer.
METHODS
The Cancer Genome Atlas (TCGA) database was used to analyze the different expression of DCAF13 messenger RNA (mRNA) between normal breast tissue and breast carcinoma tissue, and the clinical data about 960 samples were downloaded from the cBio Cancer Genomics Portal (cBioPortal). The expression level of DCAF13, co-expression network, and survival were analyzed. Those with a fold change ≥1 and FDR <0.05 were considered to have statistical significance. Unsupervised clustering of differentially expressed RBPs was performed based on log2-transformed FPKM values using the "pheatmap" package in R. Genes with a Spearman score >0.55 were regarded as moderately co-expressed genes. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to construct a co-expression network. Meanwhile, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to identify the biological process cluster and pathway cluster, respectively.
RESULTS
Compared with normal breast tissue, DCAF13 mRNA expression was significantly increased in breast cancer tissue (P<0.01). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to identify the functions of the co-expressed network. These genes were mainly enriched in mitosis, nuclear division, metabolic process, recombination, replication and repair of DNA, double-strand break repair, posttranscriptional regulation of gene expression, regulation of cell cycle, division and proliferation, regulation of protein stability and also participation in in regulation of poly(A) RNA binding, mRNA binding, tRNA binding, adenosine triphosphate (ATP) binding. KEGG pathway analysis revealed that the genes were mainly enriched in cell cycle, oocyte meiosis and oxidative phosphorylation. According to survival analysis, upregulation of DCAF13 mRNA was significant for overall survival (OS) (P=0.0163).
CONCLUSIONS
DCAF13 is up-regulated in breast cancer, the OS of patients with DCAF13 up-regulation was obviously reduced. DCAF13 was used as a diagnostic marker and therapeutic target for breast cancer. By building a co-expression network of DCAF13 and conducting bioinformatics analysis, it is possible to find the biomarker to evaluate patient prognosis. This finding provides a new target in mechanism and cell research of breast cancer.
PubMed: 38197079
DOI: 10.21037/tcr-23-1923 -
Doklady. Biochemistry and Biophysics Feb 2024The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the...
The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm.
Topics: Animals; Active Transport, Cell Nucleus; Histones; Drosophila melanogaster; RNA, Messenger; Nuclear Proteins; Cell Nucleus
PubMed: 38189888
DOI: 10.1134/S160767292370059X -
RSC Advances Jan 2024As one of the most promising types of label-free nanopores has great potential for DNA sequencing fast detection of different DNA bases. As one of the most promising...
As one of the most promising types of label-free nanopores has great potential for DNA sequencing fast detection of different DNA bases. As one of the most promising types of label-free nanopores, two-dimensional nanopore materials have been developed over the past two decades. However, how to detect different DNA bases efficiently and accurately is still a challenging problem. In the present work, the translocation of four homogeneous DNA strands (, poly(A), poly(C), poly(G), and poly(T)) through two-dimensional transition-metal carbide (MXene) membrane nanopores with different surface terminal groups is investigated all-atom molecular dynamics simulations. Interestingly, it is found that the four types of bases can be distinguished by different ion currents and dwell times when they are transported through the TiC(OH) nanopore. This is mainly attributed to the different orientation and position distributions of the bases, the hydrogen bonding inside the MXene nanopore, and the interaction of the ssDNA with the nanopore. The present study enhances the understanding of the interaction between DNA strands and MXene nanopores with different functional groups, which may provide useful guidelines for the design of MXene-based devices for DNA sequencing in the future.
PubMed: 38188982
DOI: 10.1039/d3ra05432b -
Microbial Cell Factories Jan 2024The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and...
BACKGROUND
The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins.
RESULTS
We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels.
CONCLUSION
The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories.
Topics: 5' Untranslated Regions; Base Sequence; Kluyveromyces; RNA, Messenger
PubMed: 38172836
DOI: 10.1186/s12934-023-02271-3