440 research outputs found
Peptide-based solutions to reduce undesired cell culture media chemistry ā New options for stabilized media formulations
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Evolution of substrate-specific gene expression and RNA editing in brown rot wood-decaying fungi.
Fungi that decay wood have characteristic associations with certain tree species, but the mechanistic bases for these associations are poorly understood. We studied substrate-specific gene expression and RNA editing in six species of wood-decaying fungi from the 'Antrodia clade' (Polyporales, Agaricomycetes) on three different wood substrates (pine, spruce, and aspen) in submerged cultures. We identified dozens to hundreds of substrate-biased genes (i.e., genes that are significantly upregulated in one substrate relative to the other two substrates) in each species, and these biased genes are correlated with their host ranges. Evolution of substrate-biased genes is associated with gene family expansion, gain and loss of genes, and variation in cis- and trans- regulatory elements, rather than changes in protein coding sequences. We also demonstrated widespread RNA editing events in the Antrodia clade, which differ from those observed in the Ascomycota in their distribution, substitution types, and the genomic environment. Moreover, we found that substrates could affect editing positions and frequency, including editing events occurring in mRNA transcribed from wood-decay-related genes. This work shows the extent to which gene expression and RNA editing differ among species and substrates, and provides clues into mechanisms by which wood-decaying fungi may adapt to different hosts
Modulation of Mutant Huntingtin N-Terminal Cleavage and Its Effect on Aggregation and Cell Death
Huntingtonās disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntingtonās disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem brain, animal models, and cell culture models. It has been found that N-terminal fragments of the mutant huntingtin protein are more toxic than the full-length protein. Therefore, proteolytic processing of mutant huntingtin may play a key event in the pathogenesis of HD. Here, we present evidence that the region in huntingtin covering amino acids 116 to 125 is critical for N-terminal proteolytic processing. Within this region, we have identified mutations that either strongly reduce or enhance N-terminal cleavage. We took advantage of this effect and demonstrate that the mutation Ī121ā122 within the putative cleavage region enhances N-terminal cleavage of huntingtin and the aggregation of N-terminal fragments. Furthermore, this particular deletion increased the activation of apoptotic processes and decreased neuronal cell viability. Our data indicate that the N-terminal proteolytic processing of mutant huntingtin can be modulated with an effect on aggregation and cell death rate
Common signatures of differential microRNA expression in Parkinsonās and Alzheimerās disease brains
Dysregulation of microRNA gene expression has been implicated in many neurodegenerative diseases, including Parkinsonās disease. However, the individual dysregulated microRNAs remain largely unknown. Previous meta-analyses have highlighted several microRNAs being differentially expressed in post-mortem Parkinsonās disease and Alzheimer's disease brains versus controls, but they were based on small sample sizes. In this study, we quantified the expression of the most compelling Parkinsonās and Alzheimerās disease microRNAs from these meta-analyses (ācandidate miRNAsā) in one of the largest Parkinsonās/Alzheimerās disease caseācontrol post-mortem brain collections available (nĀ = 451), thereby quadruplicating previously investigated sample sizes. Parkinsonās disease candidate microRNA hsa-miR-132-3p was differentially expressed in our Parkinsonās (PĀ = 4.89Eā06) and Alzheimerās disease samples (PĀ = 3.20Eā24) compared with controls. Alzheimerās disease candidate microRNAs hsa-miR-132-5p (PĀ = 4.52Eā06) and hsa-miR-129-5p (PĀ = 0.0379) were differentially expressed in our Parkinsonās disease samples. Combining these novel data with previously published data substantially improved the statistical support (Ī± = 3.85Eā03) of the corresponding meta-analyses, clearly implicating these microRNAs in both Parkinsonās and Alzheimerās disease. Furthermore, hsa-miR-132-3p/-5p (but not hsa-miR-129-5p) showed association with Ī±-synuclein neuropathological Braak staging (PĀ = 3.51Eā03/PĀ = 0.0117), suggesting that hsa-miR-132-3p/-5p play a role in Ī±-synuclein aggregation beyond the early disease phase. Our study represents the largest independent assessment of recently highlighted candidate microRNAs in Parkinsonās and Alzheimerās disease brains, to date. Our results implicate hsa-miR-132-3p/-5p and hsa-miR-129-5p to be differentially expressed in both Parkinsonās and Alzheimerās disease, pinpointing shared pathogenic mechanisms across these neurodegenerative diseases. Intriguingly, based on publicly available high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation data, hsa-miR-132 may interact withĀ SNCAĀ messenger RNA in the human brain, possibly pinpointing novel therapeutic approaches in fighting Parkinsonās disease
Differential microRNA expression analyses across two brain regions in Alzheimerās disease
Dysregulation of microRNAs (miRNAs) is involved in the pathogenesis of neurodegenerative diseases, including Alzheimerās disease (AD). Hitherto, sample sizes from differential miRNA expression studies in AD are exceedingly small aggravating any biological inference. To overcome this limitation, we investigated six candidate miRNAs in a large collection of brain samples. Brain tissue was derived from superior temporal gyrus (STG) and entorhinal cortex (EC) from 99 AD patients and 91 controls. MiRNA expression was examined by qPCR (STG) or small RNA sequencing (EC). Brain region-dependent differential miRNA expression was investigated in a transgenic AD mouse model using qPCR and FISH. Total RNA sequencing was used to assess differential expression of miRNA target genes. MiR-129-5p, miR-132-5p, and miR-138-5p were significantly downregulated in AD vs. controls both in STG and EC, while miR-125b-5p and miR-501-3p showed no evidence for differential expression in this dataset. In addition, miR-195-5p was significantly upregulated in EC but not STG in AD patients. The brain region-specific pattern of miR-195-5p expression was corroborated in vivo in transgenic AD mice. Total RNA sequencing identified several novel and functionally interesting target genes of these miRNAs involved in synaptic transmission (GABRB1), the immune-system response (HCFC2) or AD-associated differential methylation (SLC16A3). Using two different methods (qPCR and small RNA-seq) in two separate brain regions in 190 individuals we more than doubled the available sample size for most miRNAs tested. Differential gene expression analyses confirm the likely involvement of miR-129-5p, miR-132-5p, miR-138-5p, and miR-195-5p in AD pathogenesis and highlight several novel potentially relevant target mRNAs
RPL24: a potential therapeutic target whose depletion or acetylation inhibits polysome assembly and cancer cell growth.
Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by \u3e70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis
Genome-wide meta-analysis of short-tandem repeats for Parkinsonās disease risk using genotype imputation
Idiopathic Parkinsonās disease is determined by a combination of genetic and environmental factors. Recently, the first genome-wide association study on short-tandem repeats in Parkinsonās disease reported on eight suggestive short-tandem repeat-based risk loci (Ī± = 5.3 Ć 10ā6), of which four were novel, i.e. they had not been implicated in Parkinsonās disease risk by genome-wide association analyses of single-nucleotide polymorphisms before. Here, we tested these eight candidate short-tandem repeats in a large, independent Parkinsonās disease caseācontrol dataset (n = 4757). Furthermore, we combined the results from both studies by meta-analysis resulting in the largest Parkinsonās disease genome-wide association study of short-tandem repeats to date (n = 43 844). Lastly, we investigated whether leading short-tandem repeat risk variants exert functional effects on gene expression regulation based on methylation quantitative trait locus data in human āpost-mortemā brain (n = 142). None of the eight previously reported short-tandem repeats were significantly associated with Parkinsonās disease in our independent dataset after multiple testing correction (Ī± = 6.25 Ć 10ā3). However, we observed modest support for short-tandem repeats near CCAR2 and NCOR1 in the updated meta-analyses of all available data. While the genome-wide meta-analysis did not reveal additional study-wide significant (Ī± = 6.3 Ć 10ā7) short-tandem repeat signals, we identified seven novel suggestive Parkinsonās disease short-tandem repeat risk loci (Ī± = 5.3 Ć 10ā6). Of these, especially a short-tandem repeat near MEIOSIN showed consistent evidence for association across datasets. CCAR2, NCOR1 and one novel suggestive locus identified here (LINC01012) emerged from colocalization analyses showing evidence for a shared causal short-tandem repeat variant affecting both Parkinsonās disease risk and cis DNA methylation in brain. Larger studies, ideally using short-tandem repeats called from whole-sequencing data, are needed to more fully investigate their role in Parkinsonās disease
The sodium iodide symporter (NIS) as theranostic gene: its emerging role in new imaging modalities and non-viral gene therapy
Cloning of the sodium iodide symporter (NIS) in 1996 has provided an opportunity to use NIS as a powerful theranostic transgene. Novel gene therapy strategies rely on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of therapeutic radionuclides. This review highlights the remarkable progress during the last two decades in the development of the NIS gene therapy concept using selective non-viral gene delivery vehicles including synthetic polyplexes and genetically engineered mesenchymal stem cells. In addition, NIS is a sensitive reporter gene and can be monitored by high resolution PET imaging using the radiotracers sodium [ 124 I]iodide ([ 124 I]NaI) or [ 18 F]tetrafluoroborate ([ 18 F]TFB). We performed a small preclinical PET imaging study comparing sodium [ 124 I]iodide and in-house synthesized [ 18 F]TFB in an orthotopic NIS-expressing glioblastoma model. The results demonstrated an improved image quality using [ 18 F]TFB. Building upon these results, we will be able to expand the NIS gene therapy approach using non-viral gene delivery vehicles to target orthotopic tumor models with low volume disease, such as glioblastoma
Aging impairs the osteocytic regulation of collagen integrity and bone quality
Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFĪ² signaling (TĪ²RI
Steatosis drives monocyte-derived macrophage accumulation in human metabolic dysfunction-associated fatty liver disease
BACKGROUND & AIMS: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common complication of obesity with a hallmark feature of hepatic steatosis. Recent data from animal models of MAFLD have demonstrated substantial changes in macrophage composition in the fatty liver. In humans, the relationship between liver macrophage heterogeneity and liver steatosis is less clear.
METHODS: Liver tissue from 21 participants was collected at time of bariatric surgery and analysed using flow cytometry, immunofluorescence, and H&E microscopy. Single-cell RNA sequencing was also conducted on a subset of samples (n = 3). Intrahepatic triglyceride content was assessed via MRI and tissue histology. Mouse models of hepatic steatosis were used to investigate observations made from human liver tissue.
RESULTS: We observed variable degrees of liver steatosis with minimal fibrosis in our participants. Single-cell RNA sequencing revealed four macrophage clusters that exist in the human fatty liver encompassing Kupffer cells and monocyte-derived macrophages (MdMs). The genes expressed in these macrophage subsets were similar to those observed in mouse models of MAFLD. Hepatic CD14
CONCLUSIONS: The human liver in MAFLD contains macrophage subsets that align well with those that appear in mouse models of fatty liver disease. Recruited myeloid cells correlate well with the degree of liver steatosis in humans. MdMs appear to participate in lipid uptake during early stages of MALFD.
IMPACT AND IMPLICATIONS: Metabolic dysfunction associated fatty liver disease (MAFLD) is extremely common; however, the early inflammatory responses that occur in human disease are not well understood. In this study, we investigated macrophage heterogeneity in human livers during early MAFLD and demonstrated that similar shifts in macrophage subsets occur in human disease that are similar to those seen in preclinical models. These findings are important as they establish a translational link between mouse and human models of disease, which is important for the development and testing of new therapeutic approaches for MAFLD
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