Targeting Argonaute to chromatin.

In many eukaryotes, siRNAs bound to Argonaute proteins guide chromatin-modifying enzymes to complementary loci, resulting in transcriptional gene silencing. Multiple lines of evidence indicate that siRNAs base-pair with longer RNAs produced at target loci, but the possibility that siRNAs base-pair directly with DNA remains an attractive hypothesis. In a recent study, Shimada et al. (pp. 2571-2580) conducted experiments that address these alternative hypotheses, yielding additional evidence that fission yeast siRNA-Argonaute silencing complexes are recruited to target loci exclusively via interactions with nascent transcripts.

Natural variation in DNA methylation homeostasis and the emergence of epialleles.

In plants and mammals, DNA methylation plays a critical role in transcriptional silencing by delineating heterochromatin from transcriptionally active euchromatin. A homeostatic balance between heterochromatin and euchromatin is essential to genomic stability. This is evident in many diseases and mutants for heterochromatin maintenance, which are characterized by global losses of DNA methylation coupled with localized ectopic gains of DNA methylation that alter transcription. Furthermore, we have shown that genome-wide methylation patterns in Arabidopsis thaliana are highly stable over generations, with the exception of rare epialleles. However, the extent to which natural variation in the robustness of targeting DNA methylation to heterochromatin exists, and the phenotypic consequences of such variation, remain to be fully explored. Here we describe the finding that heterochromatin and genic DNA methylation are highly variable among 725 A. thaliana accessions. We found that genic DNA methylation is inversely correlated with that in heterochromatin, suggesting that certain methylation pathway(s) may be redirected to genes upon the loss of heterochromatin. This redistribution likely involves a feedback loop involving the DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), H3K9me2, and histone turnover, as highly expressed, long genes with a high density of CMT3-preferred CWG sites are more likely to be methylated. Importantly, although the presence of CG methylation in genes alone may not affect transcription, genes containing CG methylation are more likely to become methylated at non-CG sites and silenced. These findings are consistent with the hypothesis that natural variation in DNA methylation homeostasis may underlie the evolution of epialleles that alter phenotypes.

The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation.

In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV's largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

RNA polymerases IV and V influence the 3' boundaries of Polymerase II transcription units in Arabidopsis.

Nuclear multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved in plants as specialized forms of Pol II. Their functions are best understood in the context of RNA-directed DNA methylation (RdDM), a process in which Pol IV-dependent 24 nt siRNAs direct the de novo cytosine methylation of regions transcribed by Pol V. Pol V has additional functions, independent of Pol IV and 24 nt siRNA biogenesis, in maintaining the repression of transposons and genomic repeats whose silencing depends on maintenance cytosine methylation. Here we report that Pol IV and Pol V play unexpected roles in defining the 3' boundaries of Pol II transcription units. Nuclear run-on assays reveal that in the absence of Pol IV or Pol V, Pol II occupancy downstream of poly A sites increases for approximately 12% of protein-coding genes. This effect is most pronounced for convergently transcribed gene pairs. Although Pols IV and V are detected near transcript ends of the affected Pol II - transcribed genes, their role in limiting Pol II read-through is independent of siRNA biogenesis or cytosine methylation for the majority of these genes. Interestingly, we observed that splicing was less efficient in pol IV or pol V mutant plants, compared to wild-type plants, suggesting that Pol IV or Pol V might affect pre-mRNA processing. We speculate that Pols IV and V (and/or their associated factors) play roles in Pol II transcription termination and pre-mRNA splicing by influencing polymerase elongation rates and/or release at collision sites for convergent genes.

Moving towards the detection of frailty with biomarkers: A population health study.

Aging adults experience increased health vulnerability and compromised abilities to cope with stressors, which are the clinical manifestations of frailty. Frailty is complex, and efforts to identify biomarkers to detect frailty and pre-frailty in the clinical setting are rarely reproduced across cohorts. We developed a predictive model incorporating biological and clinical frailty measures to identify robust biomarkers across data sets. Data were from two large cohorts of older adults: "Invecchiare in Chianti (Aging in Chianti, InCHIANTI Study") (n = 1453) from two small towns in Tuscany, Italy, and replicated in the Atherosclerosis Risk in Communities Study (ARIC) (n = 6508) from four U.S. communities. A complex systems approach to biomarker selection with a tree-boosting machine learning (ML) technique for supervised learning analysis was used to examine biomarker population differences across both datasets. Our approach compared predictors with robust, pre-frail, and frail participants and examined the ability to detect frailty status by race. Unique biomarker features identified in the InCHIANTI study allowed us to predict frailty with a model accuracy of 0.72 (95% confidence interval (CI) 0.66-0.80). Replication models in ARIC maintained a model accuracy of 0.64 (95% CI 0.66-0.72). Frail and pre-frail Black participant models maintained a lower model accuracy. The predictive panel of biomarkers identified in this study may improve the ability to detect frailty as a complex aging syndrome in the clinical setting. We propose several concrete next steps to keep research moving toward detecting frailty with biomarker-based detection methods.

Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii.

Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease outbreaks.

Integrating Care Coordination and Mental Health Research Into Dialysis Practice: Stakeholder Perspectives, Methods, and Outcomes.

Rationale & Objective: Despite many studies suggesting beneficial innovations for patients, few make it into clinical practice. This study aims to enhance patient care by facilitating effective dissemination of patient-centered outcomes research to health care workers in outpatient dialysis facilities, aided by the Patient-Centered Outcomes Research Institute's (PCORI) dissemination and implementation framework. Study Design: Dissemination and implementation project. Setting & Population: Outpatient hemodialysis facilities in the United States. Methods: We brought together panels of key stakeholders, which included researchers, patient subject matter experts, and dialysis personnel. Their role was to provide guidance on the content and methods for disseminating research findings. With a focus on 2 critical patient safety areas-care coordination or care transitions and mental or behavioral health-we developed virtual education modules. These modules were then made available to outpatient dialysis facilities by the national 5-Diamond Patient Safety Program. Results: In 2022, the training was used by more than 2,500 dialysis facilities and approximately 40,000 dialysis staff in the care coordination module, and by more than 300 dialysis facilities and 5,000 staff for the mental health module. Cumulatively, the modules affected more than 179,000 patients. Evidence of efficacy was the significant increase in trainee knowledge of research findings and implementation considerations (P ≤ 0.05). Limitations: Potential selection bias because dialysis facilities that did not participate in the program may differ significantly from those that did, which may affect generalizability. In addition, variable timing in release of the different modules may have influenced uptake by facilities. Conclusions: By using key stakeholder guidance and accessible virtual education modules, the implementation framework shows promise in effectively disseminating research findings within outpatient dialysis settings. This method potentially carries implications for broader health care settings as well. Plain-Language Summary: Our study addresses a common health care challenge-many promising ideas for improving patient care never actually reach the patients. We aimed to bridge the dissemination gap by sharing research with health care workers in outpatient dialysis, promoting evidence-based practice. We collaborated with experts, patients, and dialysis personnel to develop easy-to-understand educational materials focused on 2 critical topics: care coordination and mental health. In 2022, our training benefited more than 2,500 facilities and 40,000 staff for care coordination, and 300 facilities with 5,000 staff for mental health, positively affecting more than 179,000 patients. We found that the training significantly increased knowledge among staff. Our approach shows promise for sharing research effectively in dialysis centers and potentially in other health care settings.

In vitro efficacy of antibiotics commonly used to treat human plague against intracellular Yersinia pestis.

Yersinia pestis initiates infection as a facultative intracellular parasite in host macrophages; however, little is known about the efficacy of antibiotics commonly used to treat human plague against intracellular Y. pestis. Intracellular minimal bactericidal concentrations (MBCs) were determined using a high-throughput broth microdilution assay in which human THP-1 macrophage-like cells were infected with Y. pestis strain KIM6-2053.1+ and exposed to 2-fold serial dilutions of antibiotics for 24 h in 96-well plates. The numbers of CFU, upon which minimal bactericidal concentrations were based, were determined by counting "microcolonies" in wells of 96-well plates following lysis of tissue culture cells to release surviving Y. pestis, replica dilution, and plating in soft tryptic soy broth agar. For THP-1 cells, streptomycin and ciprofloxacin had comparable efficacies for intra- and extracellular Y. pestis, but the MBCs for chloramphenicol, gentamicin, doxycycline, and amoxicillin were two-, three-, four-, and five 2-fold serial dilutions greater, respectively, for intracellular than for extracellular Y. pestis. During the initial stage of plague, intracellular Y. pestis may be less susceptible to antibiotic killing by particular antibiotics recommended for treatment of plague, such as gentamicin or doxycycline, whereas others, such as streptomycin and ciprofloxacin, may have similar efficacies against extracellular or intracellular Y. pestis. This may be of particular importance in the selection of antibiotics for prophylactic treatment in the case of a bioterrorism event.

Evolutionary and Experimental Loss of Gene Body Methylation and Its Consequence to Gene Expression.

In flowering plants, gene body methylation (gbM) is associated with a subset of constitutively expressed genes. It has been proposed that gbM modulates gene expression. Here, we show that there are no consistent and direct differences to expression following the loss of gbM. By comparing expression of gbM genes in Arabidopsis thaliana accessions to orthologous genes in two Eutrema salsugineum genotypes, we identified both positive and negative expression differences associated with gbM loss. However, expression is largely unaffected by gbM loss in E. salsugineum Expression differences between species were within the variation of expression observed within A. thaliana accessions that displayed variation in gbM. Furthermore, experimentally induced loss of gbM did not consistently lead to differences in expression compared to wild type. To date, there is no convincing data to support a direct causal link between the presence/absence of gbM and the modulation of expression in flowering plants.

Epimutations are associated with CHROMOMETHYLASE 3-induced de novo DNA methylation.

In many plant species, a subset of transcribed genes are characterized by strictly CG-context DNA methylation, referred to as gene body methylation (gbM). The mechanisms that establish gbM are unclear, yet flowering plant species naturally without gbM lack the DNA methyltransferase, CMT3, which maintains CHG (H = A, C, or T) and not CG methylation at constitutive heterochromatin. Here, we identify the mechanistic basis for gbM establishment by expressing CMT3 in a species naturally lacking CMT3. CMT3 expression reconstituted gbM through a progression of de novo CHG methylation on expressed genes, followed by the accumulation of CG methylation that could be inherited even following loss of the CMT3 transgene. Thus, gbM likely originates from the simultaneous targeting of loci by pathways that promote euchromatin and heterochromatin, which primes genes for the formation of stably inherited epimutations in the form of CG DNA methylation.

Specifications of Targeting Heterochromatin Modifications in Plants.

Plants encode a diverse repertoire of DNA methyltransferases that have specialized to target cytosines for methylation in specific sequence contexts. These include the de novo methyltransferase, DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which methylates cytosines in all sequence contexts through an RNA-guided process, the CHROMOMETHYLASES (CMTs), which methylate CHH and CHG cytosines (where H is A, T, or C), and METHYLTRANSFERASE 1 (MET1), which maintains methylation of symmetrical CG contexts. In this review, we discuss the sequence specificities and targeting of each of these pathways. In particular, we highlight recent studies that indicate CMTs preferentially target CWG or CWA/CAW motifs (where W is A or T), and discuss how self-reinforcing feedback loops between DNA methyltransferases and histone modifications characteristic of heterochromatin specify targeting. Finally, the initiating events that lead to gene body methylation are discussed as a model illustrating how interdependent targeting of different silencing pathways can potentiate the establishment of off-target epialleles.

The RNAs of RNA-directed DNA methylation.

RNA-directed chromatin modification that includes cytosine methylation silences transposable elements in both plants and mammals, contributing to genome defense and stability. In Arabidopsis thaliana, most RNA-directed DNA methylation (RdDM) is guided by small RNAs derived from double-stranded precursors synthesized at cytosine-methylated loci by nuclear multisubunit RNA Polymerase IV (Pol IV), in close partnership with the RNA-dependent RNA polymerase, RDR2. These small RNAs help keep transposons transcriptionally inactive. However, if transposons escape silencing, and are transcribed by multisubunit RNA polymerase II (Pol II), their mRNAs can be recognized and degraded, generating small RNAs that can also guide initial DNA methylation, thereby enabling subsequent Pol IV-RDR2 recruitment. In both pathways, the small RNAs find their target sites by interacting with longer noncoding RNAs synthesized by multisubunit RNA Polymerase V (Pol V). Despite a decade of progress, numerous questions remain concerning the initiation, synthesis, processing, size and features of the RNAs that drive RdDM. Here, we review recent insights, questions and controversies concerning RNAs produced by Pols IV and V, and their functions in RdDM. We also provide new data concerning Pol V transcript 5' and 3' ends. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation.

Plant multisubunit RNA polymerase V (Pol V) transcription recruits Argonaute-small interfering RNA (siRNA) complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive C-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro yet essential in vivo. One CTD subdomain (DeCL) is required for Pol V function at virtually all loci. Other CTD subdomains have locus-specific effects. In a yeast two-hybrid screen, the 3'→ 5' exoribonuclease RRP6L1 was identified as an interactor with the DeCL and glutamine-serine (QS)-rich subdomains located downstream of an Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3' ends of Pol V transcripts sliced by Argonaute 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife.

Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.

Characterization of recombinant B. abortus strain RB51SOD toward understanding the uncorrelated innate and adaptive immune responses induced by RB51SOD compared to its parent vaccine strain RB51.

Brucella abortus is a Gram-negative, facultative intracellular pathogen for several mammals, including humans. Live attenuated B. abortus strain RB51 is currently the official vaccine used against bovine brucellosis in the United States and several other countries. Overexpression of protective B. abortus antigen Cu/Zn superoxide dismutase (SOD) in a recombinant strain of RB51 (strain RB51SOD) significantly increases its vaccine efficacy against virulent B. abortus challenge in a mouse model. An attempt has been made to better understand the mechanism of the enhanced protective immunity of RB51SOD compared to its parent strain RB51. We previously reported that RB51SOD stimulated enhanced Th1 immune response. In this study, we further found that T effector cells derived from RB51SOD-immunized mice exhibited significantly higher cytotoxic T lymphocyte activity than T effector cells derived from RB51-immunized mice against virulent B. abortus-infected target cells. Meanwhile, the macrophage responses to these two strains were also studied. Compared to RB51, RB51SOD cells had a lower survival rate in macrophages and induced lower levels of macrophage apoptosis and necrosis. The decreased survival of RB51SOD cells correlates with the higher sensitivity of RB51SOD, compared to RB51, to the bactericidal action of either Polymyxin B or sodium dodecyl sulfate (SDS). Furthermore, a physical damage to the outer membrane of RB51SOD was observed by electron microscopy. Possibly due to the physical damage, overexpressed Cu/Zn SOD in RB51SOD was found to be released into the bacterial cell culture medium. Therefore, the stronger adaptive immunity induced by RB51SOD did not correlate with the low level of innate immunity induced by RB51SOD compared to RB51. This unique and apparently contradictory profile is likely associated with the differences in outer membrane integrity and Cu/Zn SOD release.