-
Protein Expression and Purification Jun 2024Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently...
Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways.
PubMed: 38944221
DOI: 10.1016/j.pep.2024.106537 -
Gene Jun 2024The domestication of animals marks a pivotal moment in human history, profoundly influencing our demographic and cultural progress. This process has led to significant... (Review)
Review
The domestication of animals marks a pivotal moment in human history, profoundly influencing our demographic and cultural progress. This process has led to significant genetic, behavioral, and physical changes in livestock species compared to their wild ancestors. Understanding the evolutionary history and genetic diversity of livestock species is crucial, and mitochondrial DNA (mtDNA) has emerged as a robust marker for investigating molecular diversity in animals. Its highly conserved gene content across animal species, minimal duplications, absence of introns, and short intergenic regions make mtDNA analysis ideal for such studies. Mitochondrial DNA analysis has uncovered distinct cattle domestication events dating back to 8000years BC in Southwestern Asia. The sequencing of water buffalo mtDNA in 2004 provided important insights into their domestication history. Caprine mtDNA analysis identified three haplogroups, indicating varied maternal origins. Sheep, domesticated 12,000 years ago, exhibit diverse mtDNA lineages, suggesting multiple domestication events. Ovine mtDNA studies revealed clades A, B, C, and a fourth lineage, group D. The origins of domestic pigs were traced to separate European and Asian events followed by interbreeding. In camels, mtDNA elucidated the phylogeographic structure and genetic differentiation between wild and domesticated species. Horses, domesticated around 3500 BC, show significant mtDNA variability, highlighting their diverse origins. Yaks exhibit unique adaptations for high-altitude environments, with mtDNA analysis providing insights into their adaptation. Chicken mtDNA studies supported a monophyletic origin from Southeast Asia's red jungle fowl, with evidence of multiple origins. This review explores livestock evolution and diversity through mtDNA studies, focusing on cattle, water buffalo, goat, sheep, pig, camel, horse, yak and chicken. It highlights mtDNA's significance in unraveling maternal lineages, genetic diversity, and domestication histories, concluding with insights into its potential application in improving livestock production and reproduction dynamics.
PubMed: 38944163
DOI: 10.1016/j.gene.2024.148728 -
Plant Science : An International... Jun 2024During environmental changes, epigenetic processes can enable adaptive responses faster than natural selection. In plants, very little is known about the role of DNA...
During environmental changes, epigenetic processes can enable adaptive responses faster than natural selection. In plants, very little is known about the role of DNA methylation during long-term adaptation. Scots pine is a widely distributed coniferous species which must adapt to different environmental conditions throughout its long lifespan. Thus, epigenetic modifications may contribute towards this direction. We provide bisulfite next-generation sequencing data from the putative promoters and exons of eight adaptation-related genes (A3IP2, CCA1, COL1, COL2, FTL2, MFT1, PHYO, and ZTL) in three Scots pine populations located in northern and southern parts of Finland. DNA methylation levels were studied in the two seed tissues: the maternal megagametophyte which contributes to embryo viability, and the biparental embryo which represents the next generation. In most genes, differentially methylated cytosines (DMCs) were in line with our previously demonstrated gene expression differences found in the same Scots pine populations. In addition, we found a strong correlation of total methylation levels between the embryo and megagametophyte tissues of a given individual tree, which indicates that DNA methylation can be inherited from the maternal parent. In conclusion, our results imply that DNA methylation differences may contribute to the adaptation of Scots pine populations in different climatic conditions.
PubMed: 38944158
DOI: 10.1016/j.plantsci.2024.112173 -
Journal of Equine Veterinary Science Jun 2024Understanding normal microbial populations within areas of the respiratory tract is essential, as variable regional conditions create different niches for microbial...
Understanding normal microbial populations within areas of the respiratory tract is essential, as variable regional conditions create different niches for microbial flora, and proliferation of commensal microbes likely contributes to clinical respiratory disease. The objective was to describe microbial population variability between respiratory tract locations in healthy horses. Samples were collected from four healthy adult horses by nasopharyngeal lavage (NPL), transtracheal aspirate (TTA), and bronchoalveolar lavage (BAL) of six distinct regions within the lung. Full-length 16S ribosomal DNA sequencing and microbial profiling analysis was performed. There was a large amount of diversity, with over 1797 ASVs identified, reduced to 94 taxa after tip agglomeration and prevalence filtering. Number of taxa and diversity were highly variable across horses, sample types, and BAL locations. Firmicutes, proteobacteria, and actinobacteria were the predominant phyla. There was a significant difference in richness (Chao1, p=0.02) and phylogenetic diversity (FaithPD, p = 0.01) between NPL, TTA, and BAL. Sample type (p=0.03) and horse (p=0.005) contributed significantly to Bray-Curtis compositional diversity, while Weighted Unifrac metric was only affected by simplified sample type (NPL and TTA vs BAL, p=0.04). There was no significant effect of BAL locations within the lung with alpha or beta diversity statistical tests. Overall findings support diverse microbial populations that were variable between upper and lower respiratory tract locations, but with no apparent difference in microbial populations of the six biogeographic regions of the lung, suggesting that BAL fluid obtained blindly by standard clinical techniques may be sufficient for future studies in healthy horses.
PubMed: 38944129
DOI: 10.1016/j.jevs.2024.105141 -
The Journal of Biological Chemistry Jun 2024Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins...
Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the Nucleocytoviricota phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.
PubMed: 38944123
DOI: 10.1016/j.jbc.2024.107504 -
The Journal of Biological Chemistry Jun 2024Mitochondria are the nexus of cellular energy metabolism and major signaling hubs that integrate information from within and without the cell to implement cell function.... (Review)
Review
Mitochondria are the nexus of cellular energy metabolism and major signaling hubs that integrate information from within and without the cell to implement cell function. Mitochondria harbor a distinct polyploid genome, mitochondrial DNA (mtDNA), that encodes respiratory chain components required for energy production. MtDNA mutation and depletion have been linked to obesity and metabolic syndrome in humans. At the cellular and subcellular levels, mtDNA synthesis is coordinated by membrane contact sites implicated in lipid transfer from the endoplasmic reticulum, tying genome maintenance to lipid storage and homeostasis. Here, we examine the relationship between mtDNA and lipid trafficking, the influence of lipotoxicity on mtDNA integrity, and how lipid metabolism may be disrupted in primary mtDNA disease.
PubMed: 38944117
DOI: 10.1016/j.jbc.2024.107498 -
International Journal of Biological... Jun 2024The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack...
The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.
PubMed: 38944094
DOI: 10.1016/j.ijbiomac.2024.133463 -
Briefings in Functional Genomics Jun 2024Acute myeloid leukemia (AML) is a type of blood cancer with diverse genetic variations and DNA methylation alterations. By studying the interaction of gene mutations,...
Acute myeloid leukemia (AML) is a type of blood cancer with diverse genetic variations and DNA methylation alterations. By studying the interaction of gene mutations, expression, and DNA methylation, we aimed to gain valuable insights into the processes that lead to block differentiation in AML. We analyzed TCGA-LAML data (173 samples) with RNA sequencing and DNA methylation arrays, comparing FLT3 mutant (48) and wild-type (125) cases. We conducted differential gene expression analysis using cBioPortal, identified DNA methylation differences with ChAMP tool, and correlated them with gene expression changes. Gene set enrichment analysis (g:Profiler) revealed significant biological processes and pathways. ShinyGo and GeneCards were used to find potential transcription factors and their binding sites among significant genes. We found significant differentially expressed genes (DEGs) negatively correlated with their most significant methylation probes (Pearson correlation coefficient of -0.49, P-value <0.001) between FLT3 mutant and wild-type groups. Moreover, our exploration of 450 k CpG sites uncovered a global hypo-methylated status in 168 DEGs. Notably, these methylation changes were enriched in the promoter regions of Homebox superfamily gene, which are crucial in transcriptional-regulating pathways in blood cancer. Furthermore, in FLT3 mutant AML patient samples, we observed overexpress of WT1, a transcription factor known to bind homeobox gene family. This finding suggests a potential mechanism by which WT1 recruits TET2 to demethylate specific genomic regions. Integrating gene expression and DNA methylation analyses shed light on the impact of FLT3 mutations on cancer cell development and differentiation, supporting a two-hit model in AML. This research advances understanding of AML and fosters targeted therapeutic strategy development.
PubMed: 38944027
DOI: 10.1093/bfgp/elae028 -
Pathology, Research and Practice Jun 2024Usual Interstitial Pneumonia (UIP) a fibrosing pneumonia is associated with idiopathic pulmonary fibrosis, chronic autoimmune disease (AID), or hypersensitivity...
BACKGROUND
Usual Interstitial Pneumonia (UIP) a fibrosing pneumonia is associated with idiopathic pulmonary fibrosis, chronic autoimmune disease (AID), or hypersensitivity pneumonia. Oxygen radicals, due to tobacco smoke, can damage DNA and might upregulate PARP1. Cytosolic DNA from dying pneumocytes activate cytosolic GMP-AMP-synthase-stimulator of interferon genes (cGAS-STING) pathway and TREX1. Prolonged inflammation induces senescence, which might be inhibited by phagocytosis, eliminating nuclear debris. We aimed to evaluate activation of cGAS-STING-TREX1 pathway in UIP, and if phagocytosis and anti-phagocytosis might counteract inflammation.
METHODS
44 cases of UIP with IPF or AID were studied for the expression of cGAS, pSTING, TREX1 and PARP1. LAMP1 and Rab7 expression served as phagocytosis markers. CD47 protecting phagocytosis and p16 to identify senescent cells were also studied.
RESULTS
Epithelial cells in remodeled areas and macrophages expressed cGAS-pSTING, TREX1; epithelia but not macrophages stained for PARP1. Myofibroblasts, endothelia, and bronchial/bronchiolar epithelial cells were all negative except early myofibroblastic foci expressing cGAS. Type II pneumocytes expressed cGAS and PARP1, but less pSTING. TREX1 although expressed was not activated. Macrophages and many regenerating epithelial cells expressed LAMP1 and Rab7. CD47, the 'don't-eat-me-signal', was expressed by macrophages and epithelial cells including senescence cells within the remodeled areas.
CONCLUSIONS
The cGAS-STING pathway is activated in macrophages and epithelial cells within remodeled areas. LikelyTREX1 because not activated cannot sufficiently degrade DNA fragments. PARP1 activation points to smoking-induced oxygen radical release, prolonging inflammation and leading to fibrosis. By expressing CD47 epithelial cells within remodeled areas protect themselves from being eliminated by phagocytosis.
PubMed: 38944022
DOI: 10.1016/j.prp.2024.155432 -
Veterinary Immunology and... Jun 2024Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils...
Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils are crucial in combating infections by migrating into milk and deploying various defense strategies, including the release of neutrophil extracellular traps (NETs) composed of DNA, histones, and bactericidal proteins. This study investigated whether NETs are released by goat neutrophils stimulated in vitro by Staphylococcus aureus and Staphylococcus warneri, two common pathogens of goat mastitis. PMNs were isolated from blood from healthy adult goats. We evaluated goat NET formation by stimulating cells with: phorbol 12-myristate 13-acetate (PMA) as a positive control, cytochalasin for inhibition of actin polymerization, S. aureus, and S. warneri. NET formation was observed in response to chemical stimulation and bacterial presence, effectively trapping pathogens. Variations in NET formation between S. aureus and S. warneri suggest pathogen-specific responses. These findings suggest that the formation of NETs may be an important complementary mechanism in the defense against mastitis in goats. In conclusion, this study unveils a novel defense mechanism in goats, indicating the role of NETs against S. aureus and S. warneri in mastitis.
PubMed: 38943998
DOI: 10.1016/j.vetimm.2024.110793