sRNA-Mediated Control of Transcription Termination in E. coli.

Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Hfq links translation repression to stress-induced mutagenesis in E. coli.

Mismatch repair (MMR) is a conserved mechanism exploited by cells to correct DNA replication errors both in growing cells and under nongrowing conditions. Hfq (host factor for RNA bacteriophage Qß replication), a bacterial Lsm family RNA-binding protein, chaperones RNA-RNA interactions between regulatory small RNAs (sRNAs) and target messenger RNAs (mRNAs), leading to alterations of mRNA translation and/or stability. Hfq has been reported to post-transcriptionally repress the DNA MMR gene mutS in stationary phase, possibly limiting MMR to allow increased mutagenesis. Here we report that Hfq deploys dual mechanisms to control mutS expression. First, Hfq binds directly to an (AAN)3 motif within the mutS 5' untranslated region (UTR), repressing translation in the absence of sRNA partners both in vivo and in vitro. Second, Hfq acts in a canonical pathway, promoting base-pairing of ArcZ sRNA with the mutS leader to inhibit translation. Most importantly, using pathway-specific mutS chromosomal alleles that specifically abrogate either regulatory pathway or both, we demonstrate that tight control of MutS levels in stationary phase contributes to stress-induced mutagenesis. By interacting with the mutS leader, Hfq serves as a critical switch that modulates bacteria from high-fidelity DNA replication to stress-induced mutagenesis.

Distinct developmental reprogramming footprint of macrophages during acute kidney injury across species.

Acute kidney injury (AKI) is a common finding in hospitalized patients, particularly those who are critically ill. The development of AKI is associated with several adverse outcomes including mortality, morbidity, progression to chronic kidney disease, and an increase in healthcare expenditure. Despite the well-established negative impact of AKI and rigorous efforts to better define, identify, and implement targeted therapies, the overall approach to the treatment of AKI continues to principally encompass supportive measures. This enduring challenge is primarily due to the heterogeneous nature of insults that activate many independent and overlapping molecular pathways. Consequently, it is evident that the identification of common mechanisms that mediate the pathogenesis of AKI, independent of etiology and engaged pathophysiological pathways, is of paramount importance and could lead to the identification of novel therapeutic targets. To better distinguish the commonly modulated mechanisms of AKI, we explored the transcriptional characteristics of human kidney biopsies from patients with acute tubular necrosis (ATN), and acute interstitial nephritis (AIN) using a NanoString inflammation panel. Subsequently, we used publicly available single-cell transcriptional resources to better interpret the generated transcriptional findings. Our findings identify robust acute kidney injury (AKI-induced) developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species. These results would expand the current understanding of the pathophysiology of AKI and potentially offer novel targets for additional studies to enhance the translational transition of AKI research.NEW & NOTEWORTHY Our findings identify robust acute kidney injury (AKI)-induced developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species.

MDMX inhibits casein kinase 1α activity and stimulates Wnt signaling.

Casein kinase 1 alpha (CK1α) is a serine/threonine kinase with numerous functions, including regulating the Wnt/ß-catenin and p53 pathways. CK1α has a well-established role in inhibiting the p53 tumor suppressor by binding to MDMX and stimulating MDMX-p53 interaction. MDMX purified from cells contains near-stoichiometric amounts of CK1α, suggesting that MDMX may in turn regulate CK1α function. We present evidence that MDMX is a potent competitive inhibitor of CK1α kinase activity (Ki  = 8 nM). Depletion of MDMX increases CK1α activity and ß-catenin S45 phosphorylation, whereas ectopic MDMX expression inhibits CK1α activity and ß-catenin phosphorylation. The MDMX acidic domain and zinc finger are necessary and sufficient for binding and inhibition of CK1α. P53 binding to MDMX disrupts an intramolecular auto-regulatory interaction and enhances its ability to inhibit CK1α. P53-null mice expressing the MDMXW200S/W201G mutant, defective in CK1α binding, exhibit reduced Wnt/ß-catenin target gene expression and delayed tumor development. Therefore, MDMX is a physiological inhibitor of CK1α and has a role in modulating cellular response to Wnt signaling. The MDMX-CK1α interaction may account for certain p53-independent functions of MDMX.

Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis.

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

S-Nitrosylation of the virulence regulator AphB promotes Vibrio cholerae pathogenesis.

Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. To colonize mammalian hosts, this pathogen must defend against host-derived toxic compounds, such as nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). RNS can covalently add an NO group to a reactive cysteine thiol on target proteins, a process called protein S-nitrosylation, which may affect bacterial stress responses. To better understand how V. cholerae regulates nitrosative stress responses, we profiled V. cholerae protein S-nitrosylation during RNS exposure. We identified an S-nitrosylation of cysteine 235 of AphB, a LysR-family transcription regulator that activates the expression of tcpP, which activates downstream virulence genes. Previous studies show that AphB C235 is sensitive to O2 and reactive oxygen species (ROS). Under microaerobic conditions, AphB formed dimer and directly repressed transcription of hmpA, encoding a flavohemoglobin that is important for NO resistance of V. cholerae. We found that tight regulation of hmpA by AphB under low nitrosative stress was important for V. cholerae optimal growth. In the presence of NO, S-nitrosylation of AphB abolished AphB activity, therefore relieved hmpA expression. Indeed, non-modifiable aphBC235S mutants were sensitive to RNS in vitro and drastically reduced colonization of the RNS-rich mouse small intestine. Finally, AphB S-nitrosylation also decreased virulence gene expression via debilitation of tcpP activation, and this regulation was also important for V. cholerae RNS resistance in vitro and in the gut. These results suggest that the modulation of the activity of virulence gene activator AphB via NO-dependent protein S-nitrosylation is critical for V. cholerae RNS resistance and colonization.

A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli.

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3' to 5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

Sensitivity and Uncertainty Analysis for Two-stream Capture-Recapture Methods in Disease Surveillance.

Capture-recapture methods are widely applied in estimating the number ( ) of prevalent or cumulatively incident cases in disease surveillance. Here, we focus the bulk of our attention on the common case in which there are 2 data streams. We propose a sensitivity and uncertainty analysis framework grounded in multinomial distribution-based maximum likelihood, hinging on a key dependence parameter that is typically nonidentifiable but is epidemiologically interpretable. Focusing on the epidemiologically meaningful parameter unlocks appealing data visualizations for sensitivity analysis and provides an intuitively accessible framework for uncertainty analysis designed to leverage the practicing epidemiologist's understanding of the implementation of the surveillance streams as the basis for assumptions driving estimation of . By illustrating the proposed sensitivity analysis using publicly available HIV surveillance data, we emphasize both the need to admit the lack of information in the observed data and the appeal of incorporating expert opinion about the key dependence parameter. The proposed uncertainty analysis is a simulation-based approach designed to more realistically acknowledge variability in the estimated associated with uncertainty in an expert's opinion about the nonidentifiable parameter, together with the statistical uncertainty. We demonstrate how such an approach can also facilitate an appealing general interval estimation procedure to accompany capture-recapture methods. Simulation studies illustrate the reliable performance of the proposed approach for quantifying uncertainties in estimating in various contexts. Finally, we demonstrate how the recommended paradigm has the potential to be directly extended for application to data from >2 surveillance streams.

Effects of pair housing on behavior, cortisol, and clinical outcomes during quarantine-like procedures for rhesus macaques (Macaca mulatta).

BACKGROUND: Compatible pair housing of macaques in research settings increases species-typical behaviors and facilitates beneficial social buffering. It is not yet established whether these benefits are maintained after intrafacility transfer and domestic quarantine, which are two stressors that can lead to behavioral and clinical abnormalities. METHODS: We evaluated 40 adolescent male rhesus macaques who were single- or pair-housed immediately following an intrafacility transfer. We measured behavior, fecal cortisol, body weight, and diarrhea occurrence. Body weight and diarrhea occurrence were also retrospectively analyzed in an additional 120 adolescent rhesus who underwent a similar transfer. RESULTS AND CONCLUSIONS: Pair-housed macaques exhibited less of some undesirable behaviors (e.g., self-clasping) and experienced less diarrhea than single-housed subjects; however, no significant differences in cortisol levels or alopecia measures were found. The demonstrated beneficial effects of pair housing for rhesus macaques following intrafacility transfer and adjustment suggest pairing upon arrival at a new facility will bolster animal welfare.

Spatio-temporal efficiency of fiscal environmental expenditure in reducing CO2 emissions in China's cities.

Although market-based CO2 emission control measures (e.g., carbon tax and carbon trading market) have been deeply discussed, government-based measures have received limited attention. This has led to increased uncertainty regarding the formulation of targeted emission reduction policies. Using a unique dataset, the non-radial directional distance function, a proposed spatial meta-frontier analysis method, and the log t convergence model, this study comprehensively investigates the spatio-temporal trends in fiscal environmental expenditure efficiency (FE) and corresponding causes for in a case study for 106 Chinese cities over 2007-2019. The results show that city-level FE presented a slow upward trend at a relatively low level, with a clearly skewed distribution. The technology gap effect between city groups and the overall best production technology, and the efficiency gap effect within city groups were the main drivers widening the overall FE gap. Convergence analysis indicated that three convergence clubs of FE were found, which were distributed across the country. This study highlights that, when constructing fiscal environmental expenditure policies, the government should focus on balancing the regional gap of FE while comprehensively improving FE.

HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα.

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.

Implementation of an EMS protocol to improve prehospital sepsis recognition.

PURPOSE: Optimal sepsis outcomes are achieved when sepsis is recognized early. Recognizing sepsis in the prehospital, EMS setting can be challenging and unreliable. The purpose of this study is to evaluate whether implementation of an EMS sepsis screening and prehospital alert protocol called PRESS (PREhospital SepsiS) is associated with improved sepsis recognition by EMS providers. DESIGN: We conducted a 12-month, before-after implementation study of the PRESS protocol in a large, public EMS system. The study intervention was a PRESS training program delivered to EMS providers. EMS patient inclusion criteria included: age ≥ 18 years, EMS systolic blood pressure < 110 mmHg, EMS heart rate > 90 bpm, and EMS respiratory rate > 20 bpm. Study exclusion criteria included the presence of any of following EMS conditions: trauma, cardiac arrest, pregnancy, toxic ingestion, or psychiatric emergency. Retrospective chart review was performed on all eligible EMS encounters during the study period. The primary outcome variable was the proportion of patients with sepsis who were identified by EMS providers. RESULTS: Approximately 300 EMS providers were trained to use PRESS. A total of 498 patient encounters met criteria for study inclusion; 222 were excluded, primarily due to trauma. A total of 276 patient encounters were analyzed. Sepsis recognition by EMS providers increased from 12% pre-PRESS protocol to 59% post-PRESS protocol (p < 0.001). In a post-hoc analysis of the post-PRESS cohort, septic patients who were identified by EMS received antibiotics 24 min faster than septic patients who were not identified by EMS [28 min (IQR 18-48) vs 52 (IQR 27-98), respectively, p = 0.021]. CONCLUSION: Implementation of an EMS sepsis screening and prehospital alert protocol was associated with an increase in sepsis recognition rates by EMS providers and a decrease in time to first antibiotic administration in the emergency department. Further studies are needed to evaluate the impact of this protocol in other populations.

Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding.

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT-DBD interaction.

Economic and intensity effects of coal consumption in China.

The increase in coal consumption and its impact on the environment has become a bottleneck that hinders sustainable development. This paper discusses the effect of economic growth and coal intensity on China's coal consumption during 2005-2017 using the Laspeyres index decomposition method. The decoupling of coal consumption from economic growth was examined in conjunction with the Tapio elasticity index, and the decoupling contributions of economic growth and coal intensity are further determined. The results indicated that economic growth drives an increase in coal consumption; however, the contribution rate declines gradually with decrease in economic growth rate in each province. Further research showed that the secondary industry is the main contributor to the increment, and the rapid development of tertiary industry increases indirect coal consumption. Coal intensity has a positive impact on curbing coal consumption, but it is not sufficient to offset the increment generated by the economic effect. Moreover, in each province, the curbing effect gradually decreased as the decline in coal intensity weakened in the secondary industry. Furthermore, coal consumption is weakly decoupled from economic growth over the long term, and the secondary industry will determine the future trend of decoupling.

Animal model of coronary microembolization under transthoracic echocardiographic guidance in rats.

The aim of the study was to develop a model of coronary microembolization (CME) in rats at a lower cost. We developed a novel rat model without thoracotomy and ventilation under the guidance of echocardiography. Rats were sacrificed at 3 h, 24 h and 1 month postoperatively in both the Echo-CME and Open-chest CME groups for the comparison of the modeling accuracy, mortality, cardiopulmonary circulation, pleural adhesion and ventilation-induced lung injury (VILI). Results showed that the coronary microthrombus formed at 3 h and reached its peak at 24 h postoperatively, which included platelet aggregation and fibrin web. The Echo-group increases success rates, decreased mortality, postoperative complications including pleural adhesion, cardiopulmonary dysfunction and VILI postoperatively than the Open-chest group at 1month postoperatively. The ejection fraction of the CME group decreased to 50% and obvious cardiac fibrosis formed at 3 months postoperatively. Our unique surgical method provided a platform to study molecular mechanisms and potential new pathways for CME treatment.

Potential Role of Fiscal Decentralization on Interprovincial Differences in CO2 Emissions in China.

Spatial differences in CO2 emissions must be taken into account in CO2 mitigation. In this work, a spatial within-between logarithmic mean Divisia index decomposition model was developed by using cluster analysis to evaluate the potential role of fiscal decentralization in driving interprovincial differences in CO2 emissions in China. The results revealed that the direct impact of fiscal decentralization emerged as a major emission driver after 2009. The differences of provincial CO2 emissions from the national average can be mainly attributed to emission differences between the distinct provincial clusters. The direct and indirect impacts of fiscal decentralization contributed to the shaping of differences in CO2 emission between provinces and their provincial cluster average, and between provincial cluster average and the national average. Reducing the differences in CO2 emission between distinct provincial clusters should be considered a breakthrough for the Chinese government. The provinces with CO2 emissions below the national average and above the average emissions of its provincial cluster still have the potential for further mitigation. Optimizing the expenditure authority of the central and provincial governments and improving the energy efficiency of the provincial fiscal expenditure are the two effective ways to further promote CO2 mitigation.

Novel ERCC2 variant in trichothiodystrophy infant: the first case report in China.

BACKGROUND: Trichothiodystrophy (TTD) is a rare, autosomal recessive, multisystem disorder most commonly caused by variants in ERCC2. CASE PRESENTATION: Here, we describe the first Chinese patient with a novel variant in ERCC2. A male infant, who was born to a healthy non-consanguineous couple, exhibited brittle hair, hair loss ichthyosis, eczema, retinal pigmentation and hypospadias. He carried a novel heterozygous ERCC2 variant. The maternal variant (c.2191-18_2213del) is a previous described genomic deletion that affects the splicing of intron 22. The paternal variant (c.1666-1G > A), that occurs in the splice site of intron 17 and likely alters ERCC2 gene function through aberrant splicing, has not been reported previously. CONCLUSIONS: Our case reported a novel pathogenic variant in ERCC2, which expanded the known genetic variants associated with TTD.

MDMX acidic domain inhibits p53 DNA binding in vivo and regulates tumorigenesis.

The MDM2 homolog MDMX oncoprotein is indispensable for inhibition of p53 during normal embryonic development and malignant transformation, yet how MDMX harnesses p53 functions is unclear. In addition to a canonical N-terminal p53-binding domain, recent work suggests the central acidic domain of MDMX regulates p53 interaction through intramolecular mimicry and engages in second-site interaction with the p53 core domain in vitro. To test the physiological relevance of these interactions, we generated an MDMX knockin mouse having substitutions in a conserved WW motif necessary for these functions (W201S/W202G). Notably, MDMXSG cells have normal p53 level but increased p53 DNA binding and target gene expression, and rapidly senesce. In vivo, MDMXSG inhibits early-phase disease in Eµ-Myc transgenic mice but accelerates the onset of lethal lymphoma and shortens overall survival. Therefore, MDMX is an important regulator of p53 DNA binding, which complements the role of MDM2 in regulating p53 level. Furthermore, the results suggest that the WW motif has dual functions that regulate p53 and inhibit Myc-driven lymphomas independent of p53.