Biology subjects

Mieczkowski, P. A.

Publications and source records attributed to Mieczkowski, P. A..

10 recordsLinked to original sources

bioRxiv2026

Sequential Penta-Omic Extraction Method Using Single Biospecimens of Post-mortem Human Brain

A multi-omic approach utilizing a single biospecimen is important to avoid intra-sample heterogeneity associated with testing multiple omic single-samples, and for more efficient use of small volumes of precious biopsies (<30 mg). This is especially true for the microanatomy of post-mortem human brain samples. Using post-mortem human brain biospecimens from the NIH NeuroBioBank, a penta-omic sequential extraction method is described, Simultaneous Metabolomic, Proteomic, Lipidomic - DNA, RNA Extraction (SiMPL-DREx). Each sequential omic extract was compared to those obtained by the gold standard single omic method. Preserving RIN is critical for brain and tissue banks, as it is a primary measure of tissue quality. For all five omic extracts, the tissue integrity numbers and omic profiles did not significantly differ from those obtained by the respective omic gold standard method. Unlike past multi-omic studies, this study quantified the relative solvent percentages and upstream losses for both the organic and aqueous phases, confirming an omics loss of under 5%.

biochemistry↗

bioRxiv2026

Comparative whole-genome analyses of articular chondrocytes and skin fibroblasts reveal distinct genome instability landscapes in mesenchymal cell types

DNA damage lesions can result in mutations and genome rearrangements that are associated with cellular aging and diseases. The landscape of somatic mutations in individual tissue and cell types are dictated by their unique physiological states, cellular functions, mutagenic exposures, and efficiency of DNA repair. Articular chondrocytes and skin fibroblasts are two cell types of mesodermal origin with distinct exposure to internal and external sources of DNA damage. While somatic genome instability features of skin fibroblasts have been well detailed, knowledge about mechanisms underlying genome changes in chondrocytes is scarce. Here, we took a whole-genome sequencing approach to evaluate the load, sources, and patterns of genome changes in 18 primary human chondrocyte clones from donors with and without osteoarthritis (OA). Findings in chondrocyte clones largely agreed with a recent study of 100 single-cell sequenced chondrocytes. We compared genome changes in chondrocytes with clonally-expanded human skin fibroblasts sequenced in our previous studies. We demonstrated that skin fibroblasts show a higher burden of somatic mutations, with an increased rate of mutation accumulation per cell division. Motif-centered analyses of mutation catalogues identified only endogenous sources of mutations in chondrocytes, as opposed to skin fibroblasts which also showed a heavy burden of UV-induced mutations. Spontaneous deamination of meCpG and mutagenesis by exposure to small epoxides and SN2 electrophiles showed higher mutagenic activities in chondrocytes compared to skin fibroblasts. Chondrocytes showed ubiquitous prevalence of indels in homonucleotide runs of [≥]5 bases, while skin fibroblasts showed high contributions of UV-associated deletions of [≥]5bp not in repeats. Structural variants in rearrangement hotspots colocalized with human common fragile sites in skin fibroblasts, but not in chondrocytes. Together, our study comprehensively recorded genome instability features in chondrocytes and highlighted the unique mutagenesis landscapes of two mesenchymal cell types.

genetics↗

bioRxiv2024

Specific Bacterial Taxa and Their Metabolite, DHPS, Linked to Alzheimers Disease, Parkinsons Disease, and Amyotrophic Lateral Sclerosis.

Neurodegenerative diseases (NDDs) are multifactorial disorders frequently associated with gut dysbiosis, oxidative stress, and inflammation; however, the pathophysiological mechanisms remain poorly understood. We investigated bacterial and metabolic dyshomeostasis in the gut microbiome associated with early disease stages across three NDDs, amyotrophic lateral sclerosis (ALS), Alzheimers Disease (AD), Parkinsons Disease (PD), and healthy controls (HC) and discovered a previously unrecognized link between a microbial-derived metabolite with an unknown role in human physiology, 2,3-dihydroxypropane-1-sulfonate (DHPS), and NDDs. DHPS was downregulated in AD, ALS, and PD, while Eubacterium and Desulfovibrio, capable of metabolizing this metabolite,1-4 were increased in all disease cohorts. Additionally, select taxa within the Clostridia class had strong negative correlations to DHPS suggesting a potential role in DHPS metabolism. Hydrogen sulfide is a catabolic product of DHPS,1,5 and hydrogen sulfide promotes inflammation,6-8 oxidative stress,9 mitochondrial damage,10 and gut dysbiosis,2,11 known hallmarks of NDD. These findings suggest that cryptic sulfur metabolism via DHPS is a missing link in our current understanding of NDD onset and progression. To the best of our knowledge, we are the first to provide evidence of a conserved gut-brain axis linkage of specific bacterial taxa and their metabolism of DHPS shared by three neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Investigating cocaine- and abstinence-induced effects on astrocyte gene expression in the nucleus accumbens

In recent years, astrocytes have been increasingly implicated in cellular mechanisms of substance use disorders (SUD). Astrocytes are structurally altered following exposure to drugs of abuse; specifically, astrocytes within the nucleus accumbens (NAc) exhibit significantly decreased surface area, volume, and synaptic colocalization after operant self-administration of cocaine and extinction or protracted abstinence (45 days). However, the mechanisms that elicit these morphological modifications are unknown. The current study aims to elucidate the molecular modifications that lead to observed astrocyte structural changes in rats across cocaine abstinence using astrocyte-specific RiboTag and RNA-seq, as an unbiased, comprehensive approach to identify genes whose transcription or translation change within NAc astrocytes following cocaine self-administration and extended abstinence. Using this method, our data reveal cellular processes including cholesterol biosynthesis that are altered specifically by cocaine self-administration and abstinence, suggesting that astrocyte involvement in these processes is changed in cocaine-abstinent rats. Overall, the results of this study provide insight into astrocyte functional adaptations that occur due to cocaine exposure or during cocaine withdrawal, which may pinpoint further mechanisms that contribute to cocaine-seeking behavior.

neuroscience↗

bioRxiv2024

MutLα suppresses error-prone DNA mismatch repair and preferentially protects noncoding DNA from mutations

The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutL is an important eukaryotic MMR factor. We have examined the contributions of MutL to maintaining genome stability. We show here that loss of MutL in yeast increases the genome-wide mutation rate by [~]130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutL leads to error-prone MMR that produces T>C base substitutions in 5-ATA-3 sequences. In agreement with this finding, our examination of human whole genome DNA sequencing data has revealed that loss of MutL in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T>C mutations in 5-NTN-3 sequences. Our further analysis has shown that MutL-independent MMR plays a role in suppressing base substitutions in N3 homopolymeric runs. In addition, we describe that MutL preferentially defends noncoding DNA from mutations. Our study defines the contributions of MutL-dependent and independent mechanisms to genome-wide MMR.

genomics↗

bioRxiv2024

Multiple DNA repair pathways prevent acetaldehyde-induced mutagenesis in yeast

Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used Saccharomyces cerevisiae to systematically delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We found that deletion of the nucleotide excision repair (NER) genes, RAD1 or RAD14, led to an increase in mutagenesis upon acetaldehyde exposure. Acetaldehyde-induced mutations were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair in{Delta} rad1 strains. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C[->]A changes coupled with an enrichment of gCn[->]A changes in the acetaldehyde-treated{Delta} rad1 isolates. The gCn[->]A mutation signature has been shown to be diagnostic of acetaldehyde exposure in yeast and in human cancers. We also demonstrated that the deletion of the two DNA-protein crosslink (DPC) repair proteases, WSS1 and DDI1, also led to increased acetaldehyde-induced mutagenesis. Defects in base excision repair (BER) led to a mild increase in mutagenesis, while defects in mismatch repair (MMR), homologous recombination repair (HR) and post replicative repair pathways did not impact mutagenesis upon acetaldehyde exposure. Our results in yeast were further corroborated upon analysis of whole exome sequenced liver cancers, wherein, tumors with defects in ERCC1 and ERCC4 (NER), FANCD2 (ICL repair) or SPRTN (DPC repair) carried a higher gCn[->]A mutation load than tumors with no deleterious mutations in these genes. Our findings demonstrate that multiple DNA repair pathways protect against acetaldehyde-induced mutagenesis.

genetics↗

bioRxiv2022

Acetaldehyde makes a distinct mutation signature in single-stranded DNA

Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG[->]TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the genotoxic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA is highly mutagenic and makes strand-biased mutations on guanines (G[->]T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Pol{zeta}. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption.

genetics↗

bioRxiv2021

The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

genetics↗

bioRxiv2020

Characterization of systemic genomic instability in budding yeast

Conventional models of genome evolution are centered around the principle that mutations form independently of each other and build up slowly over time. We characterized the occurrence of bursts of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, providing support for an additional non-independent and faster mode of mutation accumulation. We initially characterized a yeast clone isolated for carrying an LOH event at a specific chromosome site, and surprisingly, found that it also carried multiple unselected rearrangements elsewhere in its genome. Whole genome analysis of over 100 additional clones selected for carrying primary LOH tracts revealed that they too contained unselected structural alterations more often than control clones obtained without any selection. We also measured the rates of coincident LOH at two different chromosomes and found that double LOH formed at rates 14-150 fold higher than expected if the two underlying single LOH events occurred independently of each other. These results were consistent across different strain backgrounds, and in mutants incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population can experience discrete episodes of systemic genomic instability, when the entire genome becomes vulnerable and multiple chromosomal alterations can form over a narrow time window. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in the cancer and genomic disorder contexts. The experimental model we describe provides a system to further dissect the fundamental biological processes responsible for punctuated bursts of structural genomic variation. SIGNIFICANCE STATEMENTMutations are generally thought to accumulate independently and gradually over many generations. Here, we combined complementary experimental approaches in budding yeast to track the appearance of chromosomal changes resulting in loss-of-heterozygosity (LOH). In contrast to the prevailing model, our results provide evidence for the existence of a path for non-independent accumulation of multiple chromosomal alteration events over few generations. These results are analogous to recent reports of bursts of genomic instability in human cells. The experimental model we describe provides a system to further dissect the fundamental biological processes underlying such punctuated bursts of mutation accumulation.

genetics↗

bioRxiv2017

Mitotic systemic genomic instability in yeast

Conventional models of genome evolution generally include the assumption that mutations accumulate gradually and independently over time. We characterized the occurrence of sudden spikes in the accumulation of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, suggesting the existence of a mitotic systemic genomic instability process (mitSGI). We characterized the emergence of a rough colony morphology phenotype resulting from an LOH event spanning a specific locus (ACE2/ace2-A7). Surprisingly, half of the clones analyzed also carried unselected secondary LOH tracts elsewhere in their genomes. The number of secondary LOH tracts detected was 20-fold higher than expected assuming independence between mutational events. Secondary LOH tracts were not detected in control clones without a primary selected LOH event. We then measured the rates of single and double LOH at different chromosome pairs and found that coincident LOH accumulated at rates 30-100 fold higher than expected if the two underlying single LOH events occurred independently. These results were consistent between two different strain backgrounds, and in mutant strains incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population experience systemic genomic instability episodes, resulting in multiple chromosomal rearrangements over one or few generations. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in the cancer and genomic disorder contexts, all of which challenge the idea of gradual accumulation of structural genomic variation. Our experimental approach provides a model to further dissect the fundamental mechanisms responsible for mitSGI.\n\nSIGNIFICANCE STATEMENTPoint mutations and alterations in chromosome structure are generally thought to accumulate gradually and independently over many generations. Here, we combined complementary genetic approaches in budding yeast to track the appearance of chromosomal changes resulting in loss-of-heterozygosity (LOH). Contrary to expectations, our results provided evidence for the occurrence of non-independent accumulation of multiple LOH events over one or a few cell generations. These results are analogous to recent reports of bursts of chromosomal instability in humans. Our experimental approach provides a framework to further dissect the fundamental mechanisms underlying systemic chromosomal instability processes, including in the human cancer and genomic disorder contexts.

genetics↗