Competition between maturation and degradation drives human snRNA 3' end quality control.

Polymerases and exonucleases act on 3' ends of nascent RNAs to promote their maturation or degradation but how the balance between these activities is controlled to dictate the fates of cellular RNAs remains poorly understood. Here, we identify a central role for the human DEDD deadenylase TOE1 in distinguishing the fates of small nuclear (sn)RNAs of the spliceosome from unstable genome-encoded snRNA variants. We found that TOE1 promotes maturation of all regular RNA polymerase II transcribed snRNAs of the major and minor spliceosomes by removing posttranscriptional oligo(A) tails, trimming 3' ends, and preventing nuclear exosome targeting. In contrast, TOE1 promotes little to no maturation of tested U1 variant snRNAs, which are instead targeted by the nuclear exosome. These observations suggest that TOE1 is positioned at the center of a 3' end quality control pathway that selectively promotes maturation and stability of regular snRNAs while leaving snRNA variants unprocessed and exposed to degradation in what could be a widespread mechanism of RNA quality control given the large number of noncoding RNAs processed by DEDD deadenylases.

Sm complex assembly and 5' cap trimethylation promote selective processing of snRNAs by the 3' exonuclease TOE1.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity toward canonical snRNAs through their Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

Rapid detection of Ebolavirus using isothermal recombinase-aided amplification.

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.

Sellar and parasellar lesions in the transition age: a retrospective Italian multi-centre study.

BACKGROUND: Sellar/parasellar lesions have been studied in the adult and paediatric age range, but during the transition age their epidemiology, clinical manifestations, management and treatment outcomes have been poorly investigated. MATERIALS AND METHODS: An Italian multicentre cohort study, in which hospital records of patients with diagnosis of sellar/parasellar lesions during the transition age and young adulthood (15-25 years), were reviewed in terms of prevalence, clinical and hormonal features at diagnosis, and outcomes where available. Both pituitary neuroendocrine tumours (pituitary tumours, Group A) and non-endocrine lesions (Group B) were included. RESULTS: Among Group A (n = 170, 46.5% macroadenomas), the most frequent were prolactin and GH-secreting tumours, with a female predominance. Among Group B (n = 28), germinomas and Rathke cells cysts were the most common. In Group A, the most frequent hormonal deficiency was gonadal dysfunction. Galactorrhoea and amenorrhoea were relatively common in female patients with prolactinomas. Pre-surgical diabetes insipidus was only seen in Group B, in which also hormone deficiencies were more frequent and numerous. Larger lesions were more likely to be seen in Group B. Patients in Group B were more frequently male, younger, and leaner than those of Group A, whereas at last follow-up they showed more obesity and dyslipidaemia. In our cohort, the percentage of patients with at least one pituitary deficiency increased slightly after surgery. CONCLUSIONS: The management of sellar/parasellar lesions is challenging in the transition age, requiring an integrated and multidisciplinary approach. Hormone and metabolic disorders can occur many years after treatment, therefore long-term follow-up is mandatory.

Nisin variants from Streptococcus and Staphylococcus successfully express in NZ9800.

AIMS: Increases in antimicrobial resistance have meant that the antimicrobial potential of lantibiotics is now being investigated irrespective of the nature of the producing organism. The aim of this study was to investigate whether natural nisin variants produced by non-Generally Recognized as Safe (GRAS) strains, such as nisin H, nisin J and nisin P, could be expressed in a well-characterized GRAS host. METHODS AND RESULTS: This study involved cloning the nisin A promoter and leader sequence fused to nisin H, nisin J or nisin P structural gene sequences originally produced by Streptococcus hyointestinalis DPC 6484, Staphylococcus capitis APC 2923 and Streptococcus agalactiae DPC 7040, respectively. This resulted in their expression in Lactococcus lactis NZ9800, a genetically modified strain that does not produce nisin A. CONCLUSIONS: Induction of the nisin controlled gene expression system demonstrates that these three nisin variants could be acted on by nisin A machinery provided by the host strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Describes the first successful heterologous production of three natural nisin variants by a GRAS strain, and demonstrates how such systems could be harnessed not only for lantibiotic production but also in the expansion of their structural diversity and development for use as future biotherapeutics.

Salivary gland tissues and derived primary and metastatic neoplasms: unusual pitfalls in the work-up of sellar lesions. A systematic review.

PURPOSE: Salivary gland (SG) tissue and derived neoplasms may occur in the sellar region. As the current literature is mostly limited to case reports, the puzzling case of an inflammatory SG removed by transsphenoidal surgery (TS) and mimicking a prolactinoma prompted us to perform the first systematic review of these unusual conditions. METHODS: A systematic literature search was conducted according to the PRISMA guidelines. Forty-four individual cases-non-neoplastic enlarged salivary glands (NNESG, n = 15), primary benign (n = 7) and malignant (n = 8) ectopic salivary tumours (ST) and sellar metastasis from eutopic primary ST (n = 14)-were suitable for the analysis of clinical, radiological and pathological characteristics. Therapeutic outcome was reviewed as a secondary endpoint. RESULTS: All cases were diagnosed after surgery. NNESG commonly affected young and/or female patients, typically leading to headaches and hyperprolactinemia and originating close to the neurohypophysis. Submucosal SG should be excluded before concluding to an intrasellar NNESG after TS. No gender or age predominance was found for primary ectopic ST, which present as large tumors, with histological phenotypes similar to common ST. Hypopituitarism and diabetes insipidus were more frequent in ST than in NNESG. NNESG and benign ectopic ST rarely recur. Malignant ectopic ST should be distinguished from secondary localizations of eutopic ST reaching the sella by contiguity or metastatic spread; both share a frequent unfavorable outcome. CONCLUSION: Sellar neoplasms derived from SG are rare but misleading conditions and pituitary dysfunction is likely to be more common than currently reported. Appropriate pathological evaluation and multidisciplinary approach are required.

The prevalence of silent acromegaly in prolactinomas is very low.

PURPOSE: The aim of this study was to evaluate the somatotroph axis in a large series of patients with prolactinoma to verify the prevalence of silent acromegaly in this population. METHODS: A hundred and forty-four patients were enrolled in a multicenter study: 90 were already on cabergoline (CAB) and enrolled in a cross-sectional arm (group A) with random PRL, GH and IGF-I determination on treatment (≥ 3 months), whereas 54 untreated patients were enrolled at diagnosis in a prospective arm (group B) with PRL, GH and IGF-I measurement before and after 6 and 12 months of treatment. In the presence of high IGF-I, CAB was withdrawn for 3 months and GH, IGF-I, PRL and GH during an oral Glucose Tolerance Test (OGTT) were obtained. RESULTS: High IGF-I levels (ULN 1.01-1.56) were observed in 9 patients (6.25%, 5F). After CAB withdrawal, IGF-I levels normalized in 5/9 patients, GH was < 0.4 ng/ml after OGTT in 7/9 cases or at random GH determination in one case. After CAB re-introduction, IGF-I levels re-increased in a single case. Overall, a single young female patient harboring a macroadenoma in group A was diagnosed with silent acromegaly and underwent successful transsphenoidal removal of a GH/PRL-secreting adenoma. CONCLUSION: The prevalence of silent acromegaly in prolactinomas (0.7%) is lower than previously reported and OGTT is helpful to recognize silent acromegaly. We suggest that the somatotroph axis should be evaluated at diagnosis in all cases and not systematically during follow-up.

Dephosphorylation activates the interferon-stimulated Schlafen family member 11 in the DNA damage response.

Human Schlafen 11 (SLFN11) is an interferon-stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus's distinct codon preference. Additionally, lack of SLFN11 expression has been linked to the resistance of cancer cells to DNA-damaging agents (DDAs). We recently resolved the underlying mechanism, finding that it involves SLFN11-mediated cleavage of select tRNAs predominantly employed in the translation of the ATR and ATM Ser/Thr kinases, thereby establishing SLFN11 as a novel tRNA endonuclease. Even though SLFN11 is thus involved in two of the most prominent diseases of our time, cancer and HIV infection, its regulation remained thus far unresolved. Using MS and bioinformatics-based approaches combined with site-directed mutagenesis, we show here that SLFN11 is phosphorylated at three different sites, which requires dephosphorylation for SLFN11 to become fully functionally active. Furthermore, we identified protein phosphatase 1 catalytic subunit γ (PPP1CC) as the upstream enzyme whose activity is required for SLFN11 to cleave tRNAs and thereby act as a selective translational inhibitor. In summary, our work has identified both the mechanism of SLFN11 activation and PPP1CC as the enzyme responsible for its activation. Our findings open up future studies of the PPP1CC subunit(s) involved in SLFN11 activation and the putative kinase(s) that inactivates SLFN11.

Rapid estrogen receptor-α signaling mediated by ERK activation regulates vascular tone in male and ovary-intact female mice.

Estrogen has been shown to affect vascular reactivity. Here, we assessed the estrogen receptor-α (ERα) dependency of estrogenic effects on vasorelaxation via a rapid nongenomic pathway in both male and ovary-intact female mice. We compared the effect of a primary estrogen, 17ß-estradiol (E2) or 4,4',4″-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT; selective ERα agonist). We found that E2 and PPT induced greater aortic relaxation in female mice than in male mice, indicating ERα mediation, which was further validated by using ERα antagonism. Treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydrochloride; ERα antagonist) attenuated PPT-mediated vessel relaxation in both sexes. ERα-mediated vessel relaxation was further validated by the absence of significant PPT-mediated relaxation in aortas isolated from ERα knockout mice. Treatment with a specific ERK inhibitor, PD-98059, reduced E2-induced vessel relaxation in both sexes but to a lesser extent in female mice. Furthermore, PD-98059 prevented PPT-induced vessel relaxation in both sexes. Both E2 and PPT treatment activated ERK as early as 5-10 min, which was attenuated by PD-98059 in aortic tissue, cultured primary vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). Aortic rings denuded of endothelium showed no differences in vessel relaxation after E2 or PPT treatment, implicating a role of ECs in the observed sex differences. Here, our results are unique to show estrogen-stimulated rapid ERα signaling mediated by ERK activation in aortic tissue, as well as VSMCs and ECs in vitro, in regulating vascular function by using side-by-side comparisons in male and ovary-intact female mice in response to E2 or PPT. NEW & NOTEWORTHY Here, we assessed the estrogen receptor-α dependency of estrogenic effects in vasorelaxation of both male and ovary-intact female mice by performing side-by-side comparisons. Also, we describe the connection between estrogen-stimulated rapid estrogen receptor-α signaling and downstream ERK activation in regulating vascular function in male and ovary-intact female mice.

Toward the Development of Standards for Yellow Flashing Lights Used in Work Zones.

Flashing yellow warning lights are important for worker and driver safety in work zones. Current standards for these lights do not address whether and how they should be coordinated to provide course-way information to drivers navigating through work zones. A field study in which the intensities and flash patterns of warning lights along a simulated work zone were varied during daytime and nighttime, was conducted to assess drivers' responses to different configurations, leading to several conclusions. During the daytime, driver responses were relatively insensitive to warning light characteristics, although they preferred sequential and synchronized flash patterns over random, uncoordinated flashing. At nighttime, a temporal peak intensity of 25 cd with a sequential flash pattern was optimal for providing course-way information. A single initial warning light having a higher intensity may help drivers detect the work zone without creating unacceptable visual discomfort.

The altered gut microbiota in adults with cystic fibrosis.

BACKGROUND: Cystic Fibrosis (CF) is an autosomal recessive disease that affects the function of a number of organs, principally the lungs, but also the gastrointestinal tract. The manifestations of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the gastrointestinal tract, as well as frequent antibiotic exposure, undoubtedly disrupts the gut microbiota. To analyse the effects of CF and its management on the microbiome, we compared the gut microbiota of 43 individuals with CF during a period of stability, to that of 69 non-CF controls using 454-pyrosequencing of the 16S rRNA gene. The impact of clinical parameters, including antibiotic therapy, on the results was also assessed. RESULTS: The CF-associated microbiome had reduced microbial diversity, an increase in Firmicutes and a reduction in Bacteroidetes compared to the non-CF controls. While the greatest number of differences in taxonomic abundances of the intestinal microbiota was observed between individuals with CF and the healthy controls, gut microbiota differences were also reported between people with CF when grouped by clinical parameters including % predicted FEV1 (measure of lung dysfunction) and the number of intravenous (IV) antibiotic courses in the previous 12 months. Notably, CF individuals presenting with severe lung dysfunction (% predicted FEV1 ≤ 40%) had significantly (p < 0.05) reduced gut microbiota diversity relative to those presenting with mild or moderate dysfunction. A significant negative correlation (-0.383, Simpson's Diversity Index) was also observed between the number of IV antibiotic courses and gut microbiota diversity. CONCLUSIONS: This is one of the largest single-centre studies on gut microbiota in stable adults with CF and demonstrates the significantly altered gut microbiota, including reduced microbial diversity seen in CF patients compared to healthy controls. The data show the impact that CF and it's management have on gut microbiota, presenting the opportunity to develop CF specific probiotics to minimise microbiota alterations.

Genetic, enzymatic and metabolite profiling of the Lactobacillus casei group reveals strain biodiversity and potential applications for flavour diversification.

AIMS: The Lactobacillus casei group represents a widely explored group of lactic acid bacteria, characterized by a high level of biodiversity. In this study, the genetic and phenotypic diversity of a collection of more than 300 isolates of the Lact. casei group and their potential to produce volatile metabolites important for flavour development in dairy products, was examined. METHODS AND RESULTS: Following confirmation of species by 16S rRNA PCR, the diversity of the isolates was determined by pulsed-field gel electrophoresis. The activities of enzymes involved in the proteolytic cascade were assessed and significant differences among the strains were observed. Ten strains were chosen based on the results of their enzymes activities and they were analysed for their ability to produce volatiles in media with increased concentrations of a representative aromatic, branched chain and sulphur amino acid. Volatiles were assessed using gas chromatography coupled with mass spectrometry. Strain-dependent differences in the range and type of volatiles produced were evident. CONCLUSIONS: Strains of the Lact. casei group are characterized by genetic and metabolic diversity which supports variability in volatile production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a screening approach for the knowledge-based selection of strains potentially enabling flavour diversification in fermented dairy products.

Minor class splicing shapes the zebrafish transcriptome during development.

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

Research Note: A self-luminous light table for persons with Alzheimer's disease.

Light can be used to consolidate sleep in individuals with Alzheimer's disease and related dementias, but the light delivery method is one of the greatest challenges for successful treatment. Based on our field observations, it was hypothesised that a self-luminous light table would be a practical way to deliver light because persons with Alzheimer's disease typically spend a significant amount of time sitting at tables. Compared to a baseline week, sleep percent and efficiency significantly increased and agitation and depression scores significantly decreased during the four intervention weeks. The self-luminous light table was an effective and practical method to deliver circadian-effective light to persons with Alzheimer's disease.

Comparisons of three practical field devices used to measure personal light exposures and activity levels.

This paper documents the spectral and spatial performance characteristics of two new versions of the Daysimeter, devices developed and calibrated by the Lighting Research Center to measure and record personal circadian light exposure and activity levels, and compares them to those of the Actiwatch Spectrum (Philips Healthcare). Photometric errors from the Daysimeters and the Actiwatch Spectrum were also determined for various types of light sources. The Daysimeters had better photometric performance than the Actiwatch Spectrum. To assess differences associated with measuring light and activity levels at different locations on the body, older adults wore four Daysimeters and an Actiwatch Spectrum for five consecutive days. Wearing the Daysimeter or Actiwatch Spectrum on the wrist compromises accurate light measurements relative to locating a calibrated photosensor at the plane of the cornea.

The 3' exonuclease TOE1 selectively processes snRNAs through recognition of Sm complex assembly and 5' cap trimethylation.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity towards canonical snRNAs by recognizing Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing.

Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.

Modulation, microbiota and inflammation in the adult CF gut: A prospective study.

BACKGROUND: Cystic Fibrosis (CF) has prominent gastrointestinal and pancreatic manifestations. The aim of this study was to determine the effect of Cystic fibrosis transmembrane conductance regulator (CFTR) modulation on, gastrointestinal inflammation, pancreatic function and gut microbiota composition in people with cystic fibrosis (CF) and the G551D-CFTR mutation. METHODS: Fourteen adult patients with the G551D-CFTR mutation were assessed clinically at baseline and for up to 1 year after treatment with ivacaftor. The change in gut inflammatory markers (calprotectin and lactoferrin), exocrine pancreatic status and gut microbiota composition and structure were assessed in stool samples. RESULTS: There was no significant change in faecal calprotectin nor lactoferrin in patients with treatment while all patients remained severely pancreatic insufficient. There was no significant change in gut microbiota diversity and richness following treatment. CONCLUSION: There was no significant change in gut inflammation after partial restoration of CFTR function with ivacaftor, suggesting that excess gut inflammation in CF is multi-factorial in aetiology. In this adult cohort, exocrine pancreatic function was irreversibly lost. Longer term follow-up may reveal more dynamic changes in the gut microbiota and possible restoration of CFTR function.

Impact of Cadmium Mediated by Tobacco Use in Musculoskeletal Diseases.

Tobacco use has a negative impact on health due to its relationship with the development of high-mortality diseases, such as pulmonary cancer. However, the effect of cadmium (Cd), present in tobacco smoke, on the development of joint diseases has been scarcely studied. The objective of this review is to discuss the evidence regarding the mechanisms by which Cd exposure, through tobacco smoke, may lead to the development of osteoarthritis (OA), osteoporosis (OP), and rheumatoid arthritis (RA). There's evidence suggesting a string association between moderate to severe OA development and tobacco use, and that a higher blood concentration of Cd can trigger oxidative stress (OS) and inflammation, favoring cartilage loss. At the bone level, the Cd that is inhaled through tobacco smoke affects bone mineral density, resulting in OP mediated by a decrease in the antioxidant enzymes, which favors the bone resorption process. In RA, tobacco use promotes the citrullination process through Cd exposure and increases OS and inflammation. Understanding how tobacco use can increase the damage at the articular level mediated by a toxic metal, i.e., Cd, is important. Finally, we propose prevention, control, and treatment strategies for frequently disabling diseases, such as OA, OP, and RA to reduce its prevalence in the population.