Effect of "finite pool of worry" and COVID-19 on UK climate change perceptions.

Research reveals that a "finite pool of worry" constrains concern about and action on climate change. Nevertheless, a longitudinal panel survey of 1,858 UK residents, surveyed in April 2019 and June 2020, reveals little evidence for diminishing climate change concern during the COVID-19 pandemic. Further, the sample identifies climate change as a bigger threat than COVID-19. The findings suggest climate change has become an intransigent concern within UK public consciousness.

Endovascular Treatment for Acute Ischemic Stroke With or Without General Anesthesia: A Matched Comparison.

BACKGROUND: Endovascular treatment in large artery occlusion stroke reduces disability. However, the impact of anesthesia type on clinical outcomes remains uncertain. METHODS: We compared consecutive patients in the Swiss Stroke Registry with anterior circulation stroke receiving endovascular treatment with or without general anesthesia (GA). The primary outcome was disability on the modified Rankin Scale after 3 months, analyzed with ordered logistic regression. Secondary outcomes included dependency or death (modified Rankin Scale score ≥3), National Institutes of Health Stroke Scale after 24 hours, symptomatic intracranial hemorrhage with ≥4 points worsening on National Institutes of Health Stroke Scale within 7 days, and mortality. Coarsened exact matching and propensity score matching were performed to adjust for indication bias. RESULTS: One thousand two hundred eighty-four patients (GA: n=851, non-GA: n=433) from 8 Stroke Centers were included. Patients treated with GA had higher modified Rankin Scale scores after 3 months than patients treated without GA, in the unmatched (odds ratio [OR], 1.75 [1.42-2.16]; P<0.001), the coarsened exact matching (n=332-524, using multiple imputations of missing values; OR, 1.60 [1.08-2.36]; P=0.020), and the propensity score matching analysis (n=568; OR, 1.61 [1.20-2.15]; P=0.001). In the coarsened exact matching analysis, there were no significant differences in National Institutes of Health Stroke Scale after 1 day (estimated coefficient 2.61 [0.59-4.64]), symptomatic intracranial hemorrhage (OR, 1.06 [0.30-3.75]), dependency or death (OR, 1.42 [0.91-2.23]), or mortality (OR, 1.65 [0.94-2.89]). In the propensity score matching analysis, National Institutes of Health Stroke Scale after 24 hours (estimated coefficient, 3.40 [1.76-5.04]), dependency or death (OR, 1.49 [1.07-2.07]), and mortality (OR, 1.65 [1.11-2.45]) were higher in the GA group, whereas symptomatic intracranial hemorrhage did not differ significantly (OR, 1.77 [0.73-4.29]). CONCLUSIONS: This large study showed worse functional outcome after endovascular treatment of anterior circulation stroke with GA than without GA in a real-world setting. This finding appears to be independent of known differences in patient characteristics between groups.

OxyS small RNA induces cell cycle arrest to allow DNA damage repair.

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS-induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read-through transcription into a cryptic prophage encoding kilR The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS-mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.

Association of the COVID-19 outbreak with acute stroke care in Switzerland.

BACKGROUND AND PURPOSE: In Switzerland, the COVID-19 incidence during the first pandemic wave was high. Our aim was to assess the association of the outbreak with acute stroke care in Switzerland in spring 2020. METHODS: This was a retrospective analysis based on the Swiss Stroke Registry, which includes consecutive patients with acute cerebrovascular events admitted to Swiss Stroke Units and Stroke Centers. A linear model was fitted to the weekly admission from 2018 and 2019 and was used to quantify deviations from the expected weekly admissions from 13 March to 26 April 2020 (the "lockdown period"). Characteristics and 3-month outcome of patients admitted during the lockdown period were compared with patients admitted during the same calendar period of 2018 and 2019. RESULTS: In all, 28,310 patients admitted between 1 January 2018 and 26 April 2020 were included. Of these, 4491 (15.9%) were admitted in the periods March 13-April 26 of the years 2018-2020. During the lockdown in 2020, the weekly admissions dropped by up to 22% compared to rates expected from 2018 and 2019. During three consecutive weeks, weekly admissions fell below the 5% quantile (likelihood 0.38%). The proportion of intracerebral hemorrhage amongst all registered admissions increased from 7.1% to 9.3% (p = 0.006), and numerically less severe strokes were observed (median National Institutes of Health Stroke Scale from 3 to 2, p = 0.07). CONCLUSIONS: Admissions and clinical severity of acute cerebrovascular events decreased substantially during the lockdown in Switzerland. Delivery and quality of acute stroke care were maintained.

Does Obesity Affect Preoperative Shoulder Balance in Adolescent Idiopathic Scoliosis?

BACKGROUND: Obese patients with adolescent idiopathic scoliosis (AIS) have been shown to present with larger curve magnitudes preoperatively. However, the effect of obesity on shoulder balance in AIS remains unknown. The purpose of our study was to determine if overweight and obese patients with AIS have worse radiographic shoulder balance on initial presentation when compared with normal weight patients. METHODS: AIS patients <18 years old, with Lenke 1 or 2 curves, who underwent a posterior spinal fusion between March 2013 and December 2018 were retrospectively evaluated. BMI-for-age percentiles as defined by the Center for Disease Control and Prevention were used: obese (≥95th percentile), overweight (85th to <95th percentile), and normal weight (5th to <85th percentile). Shoulder height was measured via the radiographic shoulder height (RSH) method, with an RSH ≤ 1 cm considered balanced. The primary outcome was preoperative shoulder balance. Secondary outcomes included postoperative shoulder balance, major curve correction, and UIV selection. RESULTS: One hundred eighty-four patients (116 [63%] normal weight and 68 [37%] overweight/obese) were included. The mean age at surgery was 13.1 ± 2 years, and mean follow-up was 17.4 ± 13 months. Preoperative shoulder imbalance was significantly greater in the overweight/obese group compared to the normal weight group (1.9 ± 1 cm vs. 1.5 ± 1 cm, p = 0.04). The odds ratio of presenting with unbalanced shoulders was 2.0 (95% CI: 1.02-3.83, p = 0.04) for the overweight/obese group. No significant differences were found for postoperative shoulder balance, UIV selection, or major curve correction. CONCLUSIONS: Overweight and obese patients with AIS are twice as likely to present with unbalanced shoulders preoperatively; however, this difference is not clinically relevant with a mean difference of 0.4 cm between cohorts. Finally, the preoperative BMI percentile did not show a significant effect on the chosen UIV or curve magnitude correction. LEVEL OF EVIDENCE: Level III: this is a retrospective case-control study.

Surgical Outcomes of Obese Patients With Adolescent Idiopathic Scoliosis From Endemic Areas of Obesity in the United States.

BACKGROUND: Obesity rates continue to rise among children and adolescents across the globe. A multicenter research consortium composed of institutions in the Southern US, located in states endemic for childhood obesity, was formed to evaluate the effect of obesity on pediatric musculoskeletal disorders. This study evaluates the effect of body mass index (BMI) percentile and socioeconomic status (SES) on surgical site infections (SSIs) and perioperative complications in patients with adolescent idiopathic scoliosis (AIS) treated with posterior spinal fusion (PSF). METHODS: Eleven centers in the Southern US retrospectively reviewed postoperative AIS patients after PSF between 2011 and 2017. Each center contributed data to a centralized database from patients in the following BMI-for-age groups: normal weight (NW, 5th to <85th percentile), overweight (OW, 85th to <95th percentile), and obese (OB, ≥95th percentile). The primary outcome variable was the occurrence of an SSI. SES was measured by the Area Deprivation Index (ADI), with higher scores indicating a lower SES. RESULTS: Seven hundred fifty-one patients were included in this study (256 NW, 235 OW, and 260 OB). OB and OW patients presented with significantly higher ADIs indicating a lower SES (P<0.001). In addition, SSI rates were significantly different between BMI groups (0.8% NW, 4.3% OW, and 5.4% OB, P=0.012). Further analysis showed that superficial and not deep SSIs were significantly different between BMI groups. These differences in SSI rates persisted even while controlling for ADI. Wound dehiscence and readmission rates were significantly different between groups (P=0.004 and 0.03, respectively), with OB patients demonstrating the highest rates. EBL and cell saver return were significantly higher in overweight patients (P=0.007 and 0.002, respectively). CONCLUSION: OB and OW AIS patients have significantly greater superficial SSI rates than NW patients, even after controlling for SES. LEVEL OF EVIDENCE: Level III.

Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo.

The molecular roles of many RNA-binding proteins in bacterial post-transcriptional gene regulation are not well understood. Approaches combining in vivo UV crosslinking with RNA deep sequencing (CLIP-seq) have begun to revolutionize the transcriptome-wide mapping of eukaryotic RNA-binding protein target sites. We have applied CLIP-seq to chart the target landscape of two major bacterial post-transcriptional regulators, Hfq and CsrA, in the model pathogen Salmonella Typhimurium. By detecting binding sites at single-nucleotide resolution, we identify RNA preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3'-located Rho-independent terminators as a universal motif involved in Hfq-RNA interactions. Additionally, Hfq preferentially binds 5' to sRNA-target sites in mRNAs, and 3' to seed sequences in sRNAs, reflecting a simple logic in how Hfq facilitates sRNA-mRNA interactions. Importantly, global knowledge of Hfq sites significantly improves sRNA-target predictions. CsrA binds AUGGA sequences in apical loops and targets many Salmonella virulence mRNAs. Overall, our generic CLIP-seq approach will bring new insights into post-transcriptional gene regulation by RNA-binding proteins in diverse bacterial species.

Hydrogen isotopes reveal evidence of migration of Miniopterus schreibersii in Europe.

BACKGROUND: The Schreiber's bat, Miniopterus schreibersii, is adapted to long-distance flight, yet long distance movements have only been recorded sporadically using capture-mark-recapture. In this study, we used the hydrogen isotopic composition of 208 wing and 335 fur specimens from across the species' European range to test the hypothesis that the species migrates over long distances. RESULTS: After obtaining the hydrogen isotopic composition (δ2H) of each sample, we performed geographic assignment tests by comparing the δ2H of samples with the δ2H of sampling sites. We found that 95 bats out of 325 showed evidence of long-distance movement, based on the analysis of either fur or wing samples. The eastern European part of the species range (Greece, Bulgaria and Serbia) had the highest numbers of bats that had moved. The assignment tests also helped identify possible migratory routes, such as movement between the Alps and the Balkans. CONCLUSIONS: This is the first continental-scale study to provide evidence of migratory behaviour of M. schreibersii throughout its European range. The work highlights the need for further investigation of this behaviour to provide appropriate conservation strategies.

A glutamine riboswitch is a key element for the regulation of glutamine synthetase in cyanobacteria.

As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5' UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.

Freiburg RNA tools: a central online resource for RNA-focused research and teaching.

The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de.

Emergency Department Door-to-Puncture Time Since 2014.

Background and Purpose- The impact of a mobile stroke unit (MSU) on access to intraarterial thrombectomy (IAT) is a prespecified BEST-MSU substudy (Benefits of Stroke Treatment Delivered Using a Mobile Stroke Unit Compared to Standard Management by Emergency Medical Services). On the MSU, IAT decision-making steps, such as computed tomography, neurological exam, and tPA (tissue-type plasminogen activator) treatment are completed before emergency department arrival. We hypothesized that such pre-ED assessment of potential IAT patients on an MSU improves the time from ED arrival to skin puncture time (door-to-puncture-time, DTPT). Methods- BEST-MSU is a prospective comparative effectiveness study of MSU versus standard management by emergency medical services (EMS). We compared ED DTPT among the following groups of MSU and EMS patients: all IAT patients, IAT patients post-tPA, and IAT patients post-tPA meeting thrombolytic adjudication criteria over the first 4 years of the study. Results- From August 2014 to July 2018, a total of 161 patients underwent IAT. Ninety-four patients presented to the ED via the MSU and 67 by EMS. One hundred forty patients received tPA before IAT, 85 in the MSU arm, and 55 in the EMS arm. One hundred twenty-six patients received tPA within thrombolytic adjudication criteria: 76 MSU and 50 EMS. DTPT in minutes was shorter for MSU patients (all IAT MSU versus EMS 89 versus 99, P=0.01; IAT post-tPA MSU versus EMS 93 versus 100, P=0.03; and IAT post-tPA within adjudicated criteria MSU versus EMS 93 versus 99.5, P=0.03). From 2014 to 2018, DTPT decreased at a faster rate for EMS compared with MSU-managed patients, improving by about an hour. Conclusions- Pre-ED IAT evaluation on an MSU results in faster DTPT compared with arrival by EMS. Since 2014, dramatic improvement in ED IAT metrics has attenuated this difference. However, DTPT in all groups indicates substantial room for improvement.

Effective Connectivity Measured Using Optogenetically Evoked Hemodynamic Signals Exhibits Topography Distinct from Resting State Functional Connectivity in the Mouse.

Brain connectomics has expanded from histological assessment of axonal projection connectivity (APC) to encompass resting state functional connectivity (RS-FC). RS-FC analyses are efficient for whole-brain mapping, but attempts to explain aspects of RS-FC (e.g., interhemispheric RS-FC) based on APC have been only partially successful. Neuroimaging with hemoglobin alone lacks specificity for determining how activity in a population of cells contributes to RS-FC. Wide-field mapping of optogenetically defined connectivity could provide insights into the brain's structure-function relationship. We combined optogenetics with optical intrinsic signal imaging to create an efficient, optogenetic effective connectivity (Opto-EC) mapping assay. We examined EC patterns of excitatory neurons in awake, Thy1-ChR2 transgenic mice. These Thy1-based EC (Thy1-EC) patterns were evaluated against RS-FC over the cortex. Compared to RS-FC, Thy1-EC exhibited increased spatial specificity, reduced interhemispheric connectivity in regions with strong RS-FC, and appreciable connection strength asymmetry. Comparing the topography of Thy1-EC and RS-FC patterns to maps of APC revealed that Thy1-EC more closely resembled APC than did RS-FC. The more general method of Opto-EC mapping with hemoglobin can be determined for 100 sites in single animals in under an hour, and is amenable to other neuroimaging modalities. Opto-EC mapping represents a powerful strategy for examining evolving connectivity-related circuit plasticity.

IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions.

The IntaRNA algorithm enables fast and accurate prediction of RNA-RNA hybrids by incorporating seed constraints and interaction site accessibility. Here, we introduce IntaRNAv2, which enables enhanced parameterization as well as fully customizable control over the prediction modes and output formats. Based on up to date benchmark data, the enhanced predictive quality is shown and further improvements due to more restrictive seed constraints are highlighted. The extended web interface provides visualizations of the new minimal energy profiles for RNA-RNA interactions. These allow a detailed investigation of interaction alternatives and can reveal potential interaction site multiplicity. IntaRNAv2 is freely available (source and binary), and distributed via the conda package manager. Furthermore, it has been included into the Galaxy workflow framework and its already established web interface enables ad hoc usage.

CTGF/CCN2 is an autocrine regulator of cardiac fibrosis.

Cardiac fibrosis is a common pathologic consequence of stress insult to the heart and is characterized by abnormal deposition of fibrotic extracellular matrix that compromises cardiac function. Cardiac fibroblasts are key mediators of fibrotic remodeling and are regulated by secreted stress-response proteins. The matricellular protein connective tissue growth factor (CTGF), or CCN2, is strongly produced by injured cardiomyocytes and although it is considered a pro-fibrotic factor in many organ systems, its role in cardiac fibrosis is controversial. Here we adopted a cell-specific genetic approach to conditionally delete CCN2 in either cardiomyocytes or activated fibroblasts. Fibrosis was induced by angiotensin II-based neurohumoral stimulation, an insult that strongly induces CCN2 expression from cardiomyocytes and to a lesser extent in fibroblasts. Remarkably, only CCN2 deletion from activated fibroblasts inhibited the fibrotic remodeling while deletion from cardiomyocytes (the main source of CCN2 in the heart) had no effects. In vitro experiments revealed that although efficiently secreted by both fibroblasts and cardiomyocytes, only fibroblast-derived CCN2 is proficient in its ability to fully activate fibroblasts. These results overall indicate that although secreted into the extracellular matrix, CCN2 acts in an autocrine fashion. Secretion of CCN2 by cardiomyocytes is not pro-fibrotic, while fibroblast-derived CCN2 can modulate fibrosis in the heart. In conclusion we found that cardiomyocyte-derived CCN2 is dispensable for cardiac fibrosis, while inhibiting CCN2 induction in activated fibroblasts is sufficient to abrogate the cardiac fibrotic response to angiotensin II. Hence, CCN2 is an autocrine factor in the heart.

Computational analysis of CLIP-seq data.

CLIP-seq experiments are currently the most important means for determining the binding sites of RNA binding proteins on a genome-wide level. The computational analysis can be divided into three steps. In the first pre-processing stage, raw reads have to be trimmed and mapped to the genome. This step has to be specifically adapted for each CLIP-seq protocol. The next step is peak calling, which is required to remove unspecific signals and to determine bona fide protein binding sites on target RNAs. Here, both protocol-specific approaches as well as generic peak callers are available. Despite some peak callers being more widely used, each peak caller has its specific assets and drawbacks, and it might be advantageous to compare the results of several methods. Although peak calling is often the final step in many CLIP-seq publications, an important follow-up task is the determination of binding models from CLIP-seq data. This is central because CLIP-seq experiments are highly dependent on the transcriptional state of the cell in which the experiment was performed. Thus, relying solely on binding sites determined by CLIP-seq from different cells or conditions can lead to a high false negative rate. This shortcoming can, however, be circumvented by applying models that predict additional putative binding sites.

A stress-induced small RNA modulates alpha-rhizobial cell cycle progression.

Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

TGF-ß1 affects cell-cell adhesion in the heart in an NCAM1-dependent mechanism.

The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFß1). While TGFß1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFß1 were generated to model the observed increase in activity in the failing heart. Activation of TGFß1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFß1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFß1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFß1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFß1 activity in the human cardiac ventricle. Overall, we found that TGFß1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFß1 and reduce heart failure severity.

Differentiation of ncRNAs from small mRNAs in Escherichia coli O157:H7 EDL933 (EHEC) by combined RNAseq and RIBOseq - ryhB encodes the regulatory RNA RyhB and a peptide, RyhP.

BACKGROUND: While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. RESULTS: Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. CONCLUSION: Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.

Photorhabdus-nematode symbiosis is dependent on hfq-mediated regulation of secondary metabolites.

Photorhabdus luminescens maintains a symbiotic relationship with the nematodes Heterorhabditis bacteriophora and together they infect and kill insect larvae. To maintain this symbiotic relationship, the bacteria must produce an array of secondary metabolites to assist in the development and replication of nematodes. The regulatory mechanisms surrounding production of these compounds are mostly unknown. The global post-transcriptional regulator, Hfq, is widespread in bacteria and performs many functions, one of which is the facilitation of sRNA binding to target mRNAs, with recent research thoroughly exploring its various pleiotropic effects. Here we generate and characterize an hfq deletion mutant and show that in the absence of hfq, the bacteria are no longer able to maintain a healthy symbiosis with nematodes due to the abolishment of the production of all known secondary metabolites. RNAseq led us to produce a second deletion of a known repressor, HexA, in the same strain, which restored both metabolite production and symbiosis.

Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on Basal Oas Gene Expression and Independent of Virus-Induced Interferon.

UNLABELLED: The oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2',5'-oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2',5'-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2(H126R)) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2(H126R) replicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient (Ifnar1(-/-)) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions of Oas gene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2(H126R) activated RNase L in Ifih1(-/-) BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2(H126R) failed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as in Ifnar1(-/-) BMM, both expressing low basal levels of Oas genes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basal Oas gene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels. IMPORTANCE: The oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basal Oas gene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.