The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated ...cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.
Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the ...pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX (BacteRiophage EXclusion) systems. These RM-like activities employ host protection by DNA modification, but immediate replication arrest occurs without evident of nuclease action on unmodified phage DNA. Here we show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a variant BREX system. A laboratory strain disabled for both the restriction and methylation activity of StySA nevertheless has wild type sequence in pglX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (μ) in their C-terminal domains. We further investigate this system in situ, replacing the mutated pglZμ and brxCμ genes with the WT counterpart. PglZ-WT supports methylation in the presence of either BrxCμ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. These results suggests that while BrxC, PglZ and PglX are principal components of the BREX modification activity, BrxL is required for restriction only. Furthermore, we show that a partial disruption of brxL disrupts transcription globally.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we ...develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
Abstract
DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of ...a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.
Background and aims
Hepatitis B virus (HBV) infection affects 300 million individuals worldwide, representing a major factor for the development of hepatic complications. Although existing antivirals ...are effective in suppressing replication, eradication of HBV is not achieved. Therefore, a multi-faceted approach involving antivirals and immunomodulatory agents is required. Non-human primates are widely used in pre-clinical studies due to their close evolutionary relationship to humans. Nonetheless, it is fundamental to identify the differences in immune response between humans and these models. Thus, we performed a transcriptomic characterization and interspecies comparison of the early immune responses to HBV in human and cynomolgus macaques.
Methods
We characterized early transcriptomic changes in human and cynomolgus B cells, T cells, myeloid and plasmacytoid dendritic cells (pDC) exposed to HBV
ex vivo
for 2 hours. Differentially-expressed genes were further compared to the profiles of HBV-infected patients using publicly-available single-cell data.
Results
HBV induced a wide variety of transcriptional changes in all cell types, with common genes between species representing only a small proportion. In particular, interferon gamma signaling was repressed in human pDCs. At the gene level, interferon gamma inducible protein 16 (
IFI16
) was upregulated in macaque pDCs, while downregulated in humans. Moreover,
IFI16
expression in pDCs from chronic HBV-infected patients anti-paralleled serum HBsAg levels.
Conclusion
Our characterization of early transcriptomic changes induced by HBV in humans and cynomolgus macaques represents a useful resource for the identification of shared and divergent host responses, as well as potential immune targets against HBV.
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we ...develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). Furthermore, we integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
To what extent do partisan media influence political attitudes and behavior? Although recent methodological advancements have improved scholars’ ability to identify the persuasiveness of partisan ...media, past studies typically rely on self-reported measures of media preferences, which may deviate from real-world news consumption. Integrating individual-level web-browsing data with a survey experiment, we contrast survey-based indicators of stated preferences with behavioral measures of revealed preferences based on the relative volume and slant of news individuals consume. Overall, we find that these measurement strategies generate differing conclusions regarding heterogeneity in partisan media’s persuasive impact. Whereas our stated preference measure raises the possibility of persuasion by crosscutting sources, our revealed preference measures suggest that, among consumers with more polarized media diets, partisan media exposure results in limited attitude change, with any observed effects driven primarily by politically concordant sources. Together, these findings underscore the importance of careful measurement for research on media persuasion.
To examine professional stakeholders' perspectives of barriers to behavioral health care (BHC) follow-up and telepsychology after pediatric traumatic brain injury (TBI).
Twenty-nine professionals ...participated in a focus group (FG) or key informant interview (KII) between January and March 2020. Professionals answered questions about facilitators and barriers to BHC follow-up and telepsychology. Given widespread telepsychology implementation since COVID-19, a follow-up survey assessing telehealth perceptions since the pandemic was sent out in December 2020. Nineteen professionals completed the survey.
Professionals identified individual (e.g., family factors, insurance coverage/finances, transportation/distance, availability, planning follow-up care) and system-level (e.g., lack of access to BHC providers) barriers to BHC post-injury. Possible solutions, like collaborative follow-up care, were also identified. Generally, clinical professionals have favorable impressions of telepsychology and utilized services as a delivery modality for clinical care. Though telepsychology could reduce barriers to care, professionals also expressed concerns (e.g., technology issues, security/safety) and challenges (e.g., funding, accessibility, training/licensure for clinicians) with implementing telepsychology.
Barriers identified highlight the need for context-specific solutions to increase BHC access, with telepsychology generally recognized as a beneficial modality for BHC. Future work should continue to focus on understanding barriers to BHC and potential solutions after pediatric TBI.
PDF
