This study exploits time, the relatively unexplored fourth dimension of gene regulatory networks (GRNs), to learn the temporal transcriptional logic underlying dynamic nitrogen (N) signaling in ...plants. Our “just-in-time” analysis of time-series transcriptome data uncovered a temporal cascade of cis elements underlying dynamic N signaling. To infer transcription factor (TF)-target edges in a GRN, we applied a time-based machine learning method to 2,174 dynamic N-responsive genes. We experimentally determined a network precision cutoff, using TF-regulated genome-wide targets of three TF hubs (CRF4, SNZ, and CDF1), used to “prune” the network to 155 TFs and 608 targets. This network precision was reconfirmed using genome-wide TF-target regulation data for four additional TFs (TGA1, HHO5/6, and PHL1) not used in network pruning. These higher-confidence edges in the GRN were further filtered by independent TF-target binding data, used to calculate a TF “N-specificity” index. This refined GRN identifies the temporal relationship of known/validated regulators of N signaling (NLP7/8, TGA1/4, NAC4, HRS1, and LBD37/38/39) and 146 additional regulators. Six TFs—CRF4, SNZ, CDF1, HHO5/6, and PHL1—validated herein regulate a significant number of genes in the dynamic N response, targeting 54% of N-uptake/assimilation pathway genes. Phenotypically, inducible overexpression of CRF4 in planta regulates genes resulting in altered biomass, root development, and 15NO₃⁻ uptake, specifically under low-N conditions. This dynamic N-signaling GRN now provides the temporal “transcriptional logic” for 155 candidate TFs to improve nitrogen use efficiency with potential agricultural applications. Broadly, these time-based approaches can uncover the temporal transcriptional logic for any biological response system in biology, agriculture, or medicine.
The relative importance of regulation at the mRNA versus protein level is subject to ongoing debate. To address this question in a dynamic system, we mapped proteomic and transcriptomic changes in ...mammalian cells responding to stress induced by dithiothreitol over 30 h. Specifically, we estimated the kinetic parameters for the synthesis and degradation of RNA and proteins, and deconvoluted the response patterns into common and unique to each regulatory level using a new statistical tool. Overall, the two regulatory levels were equally important, but differed in their impact on molecule concentrations. Both mRNA and protein changes peaked between two and eight hours, but mRNA expression fold changes were much smaller than those of the proteins. mRNA concentrations shifted in a transient, pulse‐like pattern and returned to values close to pre‐treatment levels by the end of the experiment. In contrast, protein concentrations switched only once and established a new steady state, consistent with the dominant role of protein regulation during misfolding stress. Finally, we generated hypotheses on specific regulatory modes for some genes.
Synopsis
The contribution of mRNA and protein level regulation in the mammalian endoplasmic reticulum stress response is deconvoluted by analyzing time‐series protein and matching mRNA concentrations with a new statistical tool.
Protein and mRNA concentrations are quantified at different time points, generating a high‐confidence dataset of 1,237 genes/mRNAs.
A new statistical tool quantifies the contribution of regulatory processes and shows that mRNA and protein level regulation play similarly important roles.
mRNA and protein level regulation have different dynamics: mRNA concentrations spike in their change and return to pre‐perturbation levels, while protein concentrations switch in their behavior and reach a new steady‐state.
Hypotheses on modes of regulation for several groups of genes are presented.
The contribution of mRNA and protein level regulation in the mammalian endoplasmic reticulum stress response is deconvoluted by analyzing time‐series protein and matching mRNA concentrations with a new statistical tool.
Chromatin modification has gained increased attention for its role in the regulation of plant responses to environmental changes, but the specific mechanisms and molecular players remain elusive. ...Here, we show that the Arabidopsis (
) histone methyltransferase SET DOMAIN GROUP8 (SDG8) mediates genome-wide changes in H3K36 methylation at specific genomic loci functionally relevant to nitrate treatments. Moreover, we show that the specific H3K36 methyltransferase encoded by
is required for canonical RNA processing, and that RNA isoform switching is more prominent in the
deletion mutant than in the wild type. To demonstrate that SDG8-mediated regulation of RNA isoform expression is functionally relevant, we examined a putative regulatory gene,
,
, and
(
), whose nitrogen-responsive isoform-specific RNA expression is mediated by SDG8. We show by functional expression in shoot cells that the different RNA isoforms of
encode distinct regulatory proteins with different effects on genome-wide transcription. We conclude that SDG8 is involved in plant responses to environmental nitrogen supply, affecting multiple gene regulatory processes including genome-wide histone modification, transcriptional regulation, and RNA processing, and thereby mediating developmental and metabolic processes related to nitrogen use.
Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to ...parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.
The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun ...sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.
There are limited studies describing the epidemiology and outcomes in children and young adults receiving continuous kidney replacement therapy (CKRT). We aimed to describe associations between ...patient characteristics, CKRT prescription, and survival.
Retrospective multicenter cohort study.
980 patients aged from birth to 25 years who received CKRT between 2015 and 2021 at 1 of 32 centers in 7 countries participating in WE-ROCK (Worldwide Exploration of Renal Replacement Outcomes Collaborative in Kidney Diseases).
CKRT for acute kidney injury or volume overload.
Death before intensive care unit (ICU) discharge.
Descriptive statistics.
Median age was 8.8 years (IQR, 1.6-15.0), and median weight was 26.8 (IQR, 11.6-55.0) kg. CKRT was initiated a median of 2 (IQR, 1-6) days after ICU admission and lasted a median of 6 (IQR, 3-14) days. The most common CKRT modality was continuous venovenous hemodiafiltration. Citrate anticoagulation was used in 62%, and the internal jugular vein was the most common catheter placement location (66%). 629 participants (64.1%) survived at least until ICU discharge. CKRT dose, filter type, and anticoagulation were similar in those who did and did not survive to ICU discharge. There were apparent practice variations by institutional ICU size.
Retrospective design; limited representation from centers outside the United States.
In this study of children and young adults receiving CKRT, approximately two thirds survived at least until ICU discharge. Although variations in dialysis mode and dose, catheter size and location, and anticoagulation were observed, survival was not detected to be associated with these parameters.
In this large contemporary epidemiological study of children and young adults receiving continuous kidney replacement therapy in the intensive care unit, we observed that two thirds of patients survived at least until ICU discharge. However, patients with comorbidities appeared to have worse outcomes. Compared with previously published reports on continuous kidney replacement therapy practice, we observed greater use of continuous venovenous hemodiafiltration with regional citrate anticoagulation.
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Continuous renal replacement therapy (CRRT) is used for the symptomatic management of acute kidney injury (AKI) and fluid overload (FO). Contemporary reports on pediatric CRRT are small and single ...center in design. Large international studies evaluating CRRT practice and outcomes are lacking. Herein, we describe the design of a multinational collaborative.
The Worldwide Exploration of Renal Replacement Outcomes Collaborative in Kidney Disease (WE-ROCK) is an international collaborative of pediatric specialists whose mission is to improve short- and long-term outcomes of children treated with CRRT. The aims of this multicenter retrospective study are to describe the epidemiology, liberation patterns, association of fluid balance and timing of CRRT initiation, and CRRT prescription with outcomes.
We included children (n = 996, 0–25 years) admitted to an intensive care unit (ICU) and treated with CRRT for AKI or FO at 32 centers (in 7 countries) from 2018 to 2021. Demographics and clinical characteristics before CRRT initiation, during the first 7 days of both CRRT, and liberation were collected. Outcomes include the following: (i) major adverse kidney events at 90 days (mortality, dialysis dependence, and persistent kidney dysfunction), and (ii) functional outcomes (functional stats scale).
The retrospective WE-ROCK study represents the largest international registry of children receiving CRRT for AKI or FO. It will serve as a broad and invaluable resource for the field of pediatric critical care nephrology that will improve our understanding of practice heterogeneity and the association of CRRT with clinical and patient-centered outcomes. This will generate preliminary data for future interventional trials in this area.
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Continuous kidney replacement therapy (CKRT) is increasingly used in youths with critical illness, but little is known about longer-term outcomes, such as persistent kidney dysfunction, continued ...need for dialysis, or death.
To characterize the incidence and risk factors, including liberation patterns, associated with major adverse kidney events 90 days after CKRT initiation (MAKE-90) in children, adolescents, and young adults.
This international, multicenter cohort study was conducted among patients aged 0 to 25 years from The Worldwide Exploration of Renal Replacement Outcomes Collaborative in Kidney Disease (WE-ROCK) registry treated with CKRT for acute kidney injury or fluid overload from 2015 to 2021. Exclusion criteria were dialysis dependence, concurrent extracorporeal membrane oxygenation use, or receipt of CKRT for a different indication. Data were analyzed from May 2 to December 14, 2023.
Patient clinical characteristics and CKRT parameters were assessed. CKRT liberation was classified as successful, reinstituted, or not attempted. Successful liberation was defined as the first attempt at CKRT liberation resulting in 72 hours or more without return to dialysis within 28 days of CKRT initiation.
MAKE-90, including death or persistent kidney dysfunction (dialysis dependence or ≥25% decline in estimated glomerular filtration rate from baseline), were assessed.
Among 969 patients treated with CKRT (529 males 54.6%; median IQR age, 8.8 1.7-15.0 years), 630 patients (65.0%) developed MAKE-90. On multivariable analysis, cardiac comorbidity (adjusted odds ratio aOR, 1.60; 95% CI, 1.08-2.37), longer duration of intensive care unit admission before CKRT initiation (aOR for 6 days vs 1 day, 1.07; 95% CI, 1.02-1.13), and liberation pattern were associated with MAKE-90. In this analysis, patients who successfully liberated from CKRT within 28 days had lower odds of MAKE-90 compared with patients in whom liberation was attempted and failed (aOR, 0.32; 95% CI, 0.22-0.48) and patients without a liberation attempt (aOR, 0.02; 95% CI, 0.01-0.04).
In this study, MAKE-90 occurred in almost two-thirds of the population and patient-level risk factors associated with MAKE-90 included cardiac comorbidity, time to CKRT initiation, and liberation patterns. These findings highlight the high incidence of adverse outcomes in this population and suggest that future prospective studies are needed to better understand liberation patterns and practices.
Membrane lipid homeostasis is required for bacteria to survive in a spectrum of host environments. This homeostasis is achieved by regulation of fatty acid chain length and of the ratio of ...unsaturated to saturated fatty acids. In the pathogen Streptococcus pneumoniae, fatty acid biosynthesis is carried out by a cluster of fatty acid biosynthesis (fab) genes (FASII locus) whose expression is controlled by the FabT repressor. Encoded immediately downstream of the FASII locus is BriC, a competence-induced, cell-cell communication peptide that promotes biofilm development as well as nasopharyngeal colonization in a murine model of pneumococcal carriage. Here, we demonstrate that briC is cotranscribed with genes of the fab gene cluster and that a reduction of briC levels, caused by decoupling its transcription from fab gene cluster, negatively affects biofilm development. BriC elevates fabT transcription, which is predicted to alter the balance of unsaturated and saturated fatty acids produced by the pathway. We find that briC inactivation results in a decreased production of unsaturated fatty acids. This affects the membrane properties by decreasing the abundance of di-unsaturated phosphatidylglycerol molecular species. We propose that the link between BriC, FabT, and phospholipid composition contributes to the ability of S. pneumoniae to alter membrane homeostasis in response to the production of a quorum-sensing peptide. IMPORTANCE Adaptation of bacteria to their host environment is a key component of colonization and pathogenesis. As an essential component of bacterial membranes, fatty acid composition contributes to host adaptation. Similarly, cell-cell communication, which enables population level responses, also contributes to host adaptation. While much is known about the pathways that control the biosynthesis of fatty acids, many questions remain regarding regulation of these pathways and consequently the factors that affect the balance between unsaturated and saturated fatty acids. We find that BriC, a cell-cell communication peptide implicated in biofilm regulation and colonization, both is influenced by a fatty acid biosynthesis pathway and affects this same pathway. This study identifies a link between cell-cell communication, fatty acid composition, and biofilms and, in doing so, suggests that these pathways are integrated into the networks that control pneumococcal colonization and host adaptation.
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