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PLoS Pathogens Jun 2024The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been...
The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.
PubMed: 38935804
DOI: 10.1371/journal.ppat.1012333 -
PloS One 2024
Topics: Animals; Frontotemporal Dementia; Mice; Humans; tau Proteins; Mutation; Genotype; Cell Line; Central Nervous System
PubMed: 38935762
DOI: 10.1371/journal.pone.0305843 -
PLoS Biology Jun 2024Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these...
Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multiwell plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.
Topics: Caenorhabditis elegans; Animals; Chemotaxis; High-Throughput Screening Assays; Smell; Behavior, Animal; Software
PubMed: 38935621
DOI: 10.1371/journal.pbio.3002672 -
Cell Reports Jun 2024The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced...
The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression.
PubMed: 38935505
DOI: 10.1016/j.celrep.2024.114363 -
Hepatology Communications Jul 2024The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown.
BACKGROUND
The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown.
METHODS
Here, we established an animal model of gallbladder dysfunction and assessed the role of a diseased gallbladder in cholestasis-induced hepatic fibrosis (CIHF).
RESULTS
Mice with smooth muscle-specific deletion of Mypt1, the gene encoding the main regulatory subunit of myosin light chain phosphatase (myosin phosphatase target subunit 1 [MYPT1]), had apparent dysfunction of gallbladder motility. This dysfunction was evidenced by abnormal contractile responses, namely, inhibited cholecystokinin 8-mediated contraction and nitric oxide-resistant relaxation. As a consequence, the gallbladder displayed impaired bile filling and biliary tract dilation comparable to the alterations in CIHF. Interestingly, the mutant animals also displayed CIHF features, including necrotic loci by the age of 1 month and subsequently exhibited progressive fibrosis and hyperplastic/dilated bile ducts. This pathological progression was similar to the phenotypes of the animal model with bile duct ligation and patients with CIHF. The characteristic biomarker of CIHF, serum alkaline phosphatase activity, was also elevated in the mice. Moreover, we observed that the myosin phosphatase target subunit 1 protein level was able to be regulated by several reagents, including lipopolysaccharide, exemplifying the risk factors for gallbladder dysfunction and hence CIHF.
CONCLUSIONS
We propose that gallbladder dysfunction caused by myosin phosphatase target subunit 1 ablation is sufficient to induce CIHF in mice, resulting in impairment of the bile transport system.
Topics: Animals; Myosin-Light-Chain Phosphatase; Mice; Disease Models, Animal; Liver Cirrhosis; Cholestasis; Gallbladder Diseases; Gallbladder; Male; Mice, Knockout
PubMed: 38934703
DOI: 10.1097/HC9.0000000000000473 -
MSphere Jun 2024Bacterial ribonuclease E (RNase E) is vital for posttranscriptional regulation by degrading and processing RNA. The RraA protein inhibits RNase E activity through...
Bacterial ribonuclease E (RNase E) is vital for posttranscriptional regulation by degrading and processing RNA. The RraA protein inhibits RNase E activity through protein-protein interactions, exerting a global regulatory effect on gene expression. However, the specific role of RraA remains unclear. In this study, we investigated expression in ZJ-T and identified three promoters responsible for its expression, resulting in transcripts with varying 5'-UTR lengths. During the stationary phase, was significantly posttranscriptionally inhibited. Deletion of had no impact on bacterial growth in rich medium Luria-Bertani broth with salt (LBS) but resulted in decreased biofilm formation and increased resistance to polymyxin B. Transcriptome analysis revealed 350 differentially expressed genes (DEGs) between the wild type and the mutant, while proteome analysis identified 267 differentially expressed proteins (DEPs). Integrative analysis identified 55 genes common to both DEGs and DEPs, suggesting that RraA primarily affects gene expression at the posttranscriptional level. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis demonstrated that RraA facilitates the conversion of fatty acids, propionic acid, and branched-chain amino acids to acetyl-CoA while enhancing amino acid and peptide uptake. Notably, RraA positively regulates the expression of virulence-associated genes, including those involved in biofilm formation and the type VI secretion system. This study expands the understanding of the regulatory network of RraA through transcriptome analysis, emphasizing the importance of proteomic analysis in investigating posttranscriptional regulation.IMPORTANCERraA is an inhibitor protein of ribonuclease E that interacts with and suppresses its endonucleolytic activity, thereby playing a widespread regulatory role in the degradation and maturation of diverse mRNAs and noncoding small RNAs. However, the physiological functions and associated regulon of RraA in have not been fully elucidated. Here, we report that RraA impacts virulence-associated physiological processes, namely, antibiotic resistance and biofilm formation, in . By conducting an integrative analysis of both the transcriptome and proteome, we revealed the involvement of RraA in carbon metabolism, amino acid catabolism, and transport, as well as in the type VI secretion system. Collectively, these findings elucidate the regulatory influence of RraA on multiple pathways associated with metabolism and pathogenesis in .
PubMed: 38934599
DOI: 10.1128/msphere.00020-24 -
MSphere Jun 2024Phage therapy is increasing in relevance as an alternative treatment to combat antibiotic resistant bacteria. Phage cocktails are the state-of-the-art method of...
Phage therapy is increasing in relevance as an alternative treatment to combat antibiotic resistant bacteria. Phage cocktails are the state-of-the-art method of administering phages in clinical settings, preferred over monophage treatment because of their ability to eliminate multiple bacterial strains and reduce resistance formation. In our study, we compare monophage applications and phage cocktails to our chosen method of phage sequential treatments. To do so, we isolated four novel bacteriophages capable of infecting T3, a close relative of , and characterized them using sequencing and transmission electron microscopy. While investigating monophage treatments, we observed that different phage concentrations had a strong impact on the timing and amount of resistance formation. When using phage cocktails, we observed that were capable of forming resistance in the same timespan it took them to become resistant to single phages. We isolated mutants resistant to each single phage as well as mutants exposed to phage cocktails, resulting in bacteria resistant to all four phages at once. Sequencing these mutants showed that different treatments yielded unique single nucleotide polymorphism mutation patterns. In order to combat resistance formation, we added phages one by one in intervals of 24 h, thus managing to delay resistance development and keeping bacterial growth significantly lower compared to phage cocktails.IMPORTANCEWHO declared antimicrobial resistance a top threat to global health; while antibiotics have stood at the forefront in the fight against bacterial infection, the increasing number of multidrug-resistant bacteria highlights a need to branch out in order to address the threat of antimicrobial resistance. Bacteriophages, viruses solely infecting bacteria, could present a solution due to their abundance, versatility, and adaptability. For this study, we isolated new phages infecting a fast-mutating strain capable of forming resistance within 30 h. By using a sequential treatment approach of adding one phage after another, we were able to curb bacterial growth significantly more compared to state-of-the-art phage cocktails.
PubMed: 38934592
DOI: 10.1128/msphere.00707-23 -
Neural Regeneration Research Jun 2024Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by extended CAG trinucleotide repeats in the androgen receptor (AR) gene, which encodes a...
Reduced mesencephalic astrocyte-derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology.
Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by extended CAG trinucleotide repeats in the androgen receptor (AR) gene, which encodes a ligand-dependent transcription factor. The mutant AR protein, characterized by polyglutamine expansion, is prone to misfolding and forms aggregates in both the nucleus and cytoplasm in the brain in SBMA patients. These aggregates alter protein-protein interactions and compromise transcriptional activity. In this study, we reported that in both cultured N2a cells and mouse brain, mutant AR with polyglutamine expansion causes reduced expression of mesencephalic astrocyte-derived neurotrophic factor (MANF). Overexpression of MANF ameliorated the neurotoxicity of mutant AR through the inhibition of mutant AR aggregation. Conversely, knocking down endogenous MANF in the mouse brain exacerbated neuronal damage and mutant AR aggregation. Our findings suggest that inhibition of MANF expression by mutant AR is a potential mechanism underlying neurodegeneration in SBMA.
PubMed: 38934406
DOI: 10.4103/NRR.NRR-D-23-01666 -
Frontiers in Cellular and Infection... 2024This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of...
BACKGROUND
This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in , a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive.
METHODS
We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLAT.
RESULTS
We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within LPS. Notably, the predicted regulator gene , serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile.
CONCLUSION
The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.
Topics: Porphyromonas gingivalis; Lipopolysaccharides; Signal Transduction; Virulence Factors; Gene Expression Regulation, Bacterial; Energy Metabolism; Dinucleoside Phosphates; Fatty Acids; Humans; Bacterial Proteins
PubMed: 38933693
DOI: 10.3389/fcimb.2024.1418651 -
Frontiers in Plant Science 2024Autotetraploid rice holds high resistance to abiotic stress and substantial promise for yield increase, but it could not be commercially used because of low fertility....
INTRODUCTION
Autotetraploid rice holds high resistance to abiotic stress and substantial promise for yield increase, but it could not be commercially used because of low fertility. Thus, our team developed neo-tetraploid rice with high fertility and hybrid vigor when crossed with autotetraploid rice. Despite these advances, the molecular mechanisms underlying this heterosis remain poorly understood.
METHODS
An elite autotetraploid rice line (HD11) was used to cross with neo-tetraploid rice, and 34 hybrids were obtained to evaluate agronomic traits related to yield. WE-CLSM, RNA-seq, and CRISPR/Cas9 were employed to observe endosperm structure and identify candidate genes from two represent hybrids.
RESULTS AND DISCUSSION
These hybrids showed high seed setting and an approximately 55% increase in 1000-grain weight, some of which achieved grain yields comparable to those of the diploid rice variety. The endosperm observations indicated that the starch grains in the hybrids were more compact than those in paternal lines. A total of 119 seed heterosis related genes (SHRGs) with different expressions were identified, which might contribute to high 1000-grain weight heterosis in neo-tetraploid hybrids. Among them, 12 genes had been found to regulate grain weight formation, including , and . Haplotype analyses of these 12 genes revealed the various effects on grain weight among different haplotypes. The hybrids could polymerize more dominant haplotypes of above grain weight regulators than any homozygous cultivar. Moreover, two SHRGs ( and ) mutants displayed a significant reduction in 1000-grain weight and an increase in grain chalkiness, indicating that and positively regulate grain weight. Our research has identified a valuable autotetraploid germplasm for generating strong yield heterosis in combination with neo-tetraploid lines and gaining molecular insights into the regulatory processes of heterosis in tetraploid rice.
PubMed: 38933462
DOI: 10.3389/fpls.2024.1421207