Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, ...adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing.
The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and ...inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level.
► The E. coli RelB2E2 complex has an open V-shaped structure ► Isolated RelE is conformationally flexible ► The structure is not compatible with two copies binding adjacently on DNA ► The structure suggests a model for conditional cooperativity
Bacteria use "self-poisoning" to downregulate cellular processes and to adapt to changing environments. The toxins they use are regulated through tight binding to antitoxins, and Bøggild et al. present the structure of a key intermediate in this subtle regulation mechanism.
The increase in enzymatic rates with temperature up to an optimum temperature (T opt) is widely attributed to classical Arrhenius behavior, with the decrease in enzymatic rates above T opt ascribed ...to protein denaturation and/or aggregation. This account persists despite many investigators noting that denaturation is insufficient to explain the decline in enzymatic rates above T opt. Here we show that it is the change in heat capacity associated with enzyme catalysis (ΔC ‡ p ) and its effect on the temperature dependence of ΔG ‡ that determines the temperature dependence of enzyme activity. Through mutagenesis, we demonstrate that the T opt of an enzyme is correlated with ΔC ‡ p and that changes to ΔC ‡ p are sufficient to change T opt without affecting the catalytic rate. Furthermore, using X-ray crystallography and molecular dynamics simulations we reveal the molecular details underpinning these changes in ΔC ‡ p . The influence of ΔC ‡ p on enzymatic rates has implications for the temperature dependence of biological rates from enzymes to ecosystems.
...we selected promoters DaU6.3 and DaU6.5 to direct the gRNA expression for stable transformation of yam. Black bars indicate exons of the gene, PAM sequences in blue and underlined, and protospacer ...sequences in red. (d) Schematic presentation of pCas9_gRNA‐PDS used to generate genome‐edited events. (e) Transient gene expression in yam leaves agroinfiltrated with Agrobacterium harbouring pCas9_gRNA‐PDS. (e‐1) Leaf infiltrated with infection medium only, (e‐2&e‐3) leaf infiltrated with Agrobacterium showing bleached patches, (e‐4) microscopic examination of an infiltrated leaf section, (e‐5) green fluorescent micrograph of the infiltrated section, (e‐6) green fluorescent micrograph of an infiltrated section heat treated at 2 dpi and photographed at 4 dpi. The optimized agroinfiltration system with Agrobacterium strain EHA105 harbouring pCas9‐gRNA‐PDS (OD600 = 0.75) suspended in infiltration buffer (Murashige and Skoog medium salts and vitamins, 20 g/L sucrose, 1 mg/L 6‐benzylaminopurine, 0.2 µm CuSO4, pH 5.7) supplemented with 400 μm acetosyringone, infiltrated in the fully expanded young leaves and heat shock treatment at 37 °C for 30 min at 2 dpi showed the highest level of transient gene expression as bleached patches and a bright GFP fluorescence at 4 dpi (Figure 1e). The target region (300‐bp) of DrPDS from individual plants, with 4 leaves per plant separately sampled for DNA, was amplified by PCR using gene‐specific primers and the amplicons were subjected directly to Sanger sequencing.
Microbial dysbiosis, a shift from commensal to pathogenic microbiota, is often associated with mental health and the gut-brain axis, where dysbiosis in the gut may be linked to dysfunction in the ...brain. Many studies focus on dysbiosis induced by clinical events or traumatic incidents; however, many professions in austere or demanding environments may encounter continuously compounded stressors. This study seeks to explore the relationship between microbial populations and stress, both perceived and biochemical.
Eight individuals enrolled in the study to provide a longitudinal assessment of the impact of stress on gut health, with four individuals providing enough samples for analysis. Eleven core microbial genera were identified, although the relative abundance of these genera and other members of the microbial population shifted over time. Although our results indicate a potential relationship between perceived stress and microbial composition of the gut, no association with biochemical stress was observed. Increases in perceived stress seem to elucidate a change in potentially beneficial Bacteroides, with a loss in Firmicutes phyla. This shift occurred in multiple individuals, whereas using cortisol as a stress biomarker showed contradictory responses. These preliminary data provide a potential mechanism for gut monitoring, while identifying targets for downstream modulation.
Pharmaceuticals selected for exploration space missions must remain stable and effective throughout mission timeframes. Although there have been six spaceflight drug stability studies, there has not ...been a comprehensive analytical analysis of these data. We sought to use these studies to quantify the rate of spaceflight drug degradation and the time-dependent probability of drug failure resulting from the loss of active pharmaceutical ingredient (API). Additionally, existing spaceflight drug stability studies were reviewed to identify research gaps to be addressed prior to exploration missions. Data were extracted from the six spaceflight studies to quantify API loss for 36 drug products with long-duration exposure to spaceflight. Medications stored for up to 2.4 years in low Earth orbit (LEO) exhibit a small increase in the rate of API loss with a corresponding increase in risk of product failure. Overall, the potency for all spaceflight-exposed medications remains within 10% of terrestrial lot-matched control with a ~1.5 increase in degradation rate. All existing studies of spaceflight drug stability have focused primarily on repackaged solid oral medications, which is important because non-protective repackaging is a well-established factor contributing to loss of drug potency. The factor most detrimental to drug stability appears to be nonprotective drug repackaging, based on premature failure of drug products in the terrestrial control group. The result of this study supports a critical need to evaluate the effects of current repackaging processes on drug shelf life, and to develop and validate suitable protective repackaging strategies that help assure the stability of medications throughout the full duration of exploration space missions.
Sustainable intensification of agriculture in Africa is essential for accomplishing food and nutritional security and addressing the rising concerns of climate change. There is an urgent need to ...close the yield gap in staple crops and enhance food production to feed the growing population. In order to meet the increasing demand for food, more efficient approaches to produce food are needed. All the tools available in the toolbox, including modern biotechnology and traditional, need to be applied for crop improvement. The full potential of new breeding tools such as genome editing needs to be exploited in addition to conventional technologies. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing has rapidly become the most prevalent genetic engineering approach for developing improved crop varieties because of its simplicity, efficiency, specificity, and easy to use. Genome editing improves crop variety by modifying its endogenous genome free of any foreign gene. Hence, genome-edited crops with no foreign gene integration are not regulated as genetically modified organisms (GMOs) in several countries. Researchers are using CRISPR/Cas-based genome editing for improving African staple crops for biotic and abiotic stress resistance and improved nutritional quality. Many products, such as disease-resistant banana, maize resistant to lethal necrosis, and sorghum resistant to the parasitic plant Striga and enhanced quality, are under development for African farmers. There is a need for creating an enabling environment in Africa with science-based regulatory guidelines for the release and adoption of the products developed using CRISPR/Cas9-mediated genome editing. Some progress has been made in this regard. Nigeria and Kenya have recently published the national biosafety guidelines for the regulation of gene editing. This article summarizes recent advances in developments of tools, potential applications of genome editing for improving staple crops, and regulatory policies in Africa.
Yam (Dioscorea spp.) is an economically important crop grown in the tropical and subtropical regions, producing tuberous roots that serve as a staple food, an income source, and an excellent source ...of various pharmaceutical precursors. Yam production is constrained by disease and pest infestations and a range of abiotic stresses. Genetic improvement can significantly mitigate these challenges, improve productivity, expand the yam markets, and increase economic gains. However, several intrinsic attributes of the crop have curtailed progress in yam breeding. Advanced genetic engineering such as genome editing by sequence‐specific nucleases has emerged as complementary approaches to conventional breeding techniques. Mainly, the clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein (CRISPR/Cas) system for genome editing has provided robust platforms for gene function analysis and crop improvement in the post‐genomic era. Despite its significance, research towards improving the yam species remains under‐represented compared to other staple tuber crops such as cassava and sweet potato. Thus, it is critical to explore avenues for increasing the genetic gains from this under‐exploited crop. The present review focuses on the progress and prospects for applying the CRISPR/Cas technology for yam improvement. The study elaborates on the currently available CRISPR/Cas tool for yam genome engineering and explores the potential applications of this toolkit in mitigating the various challenges encountered in yam production and consumption. Furthermore, we have delved into the challenges associated with this technology and the improvements made to minimize these challenges. The insights presented herein provide a guide for yam improvement to increase genetic gains from this under‐researched and under‐utilized resource.
Yam is an economically important crop grown in the tropical and subtropical world, producing tuberous root as a staple food, and an income source for millions of farmers. Yam production is constrained by disease and pest infestations and a range of abiotic factors. Advanced genetic engineering tools such as CRISPR/Cas‐based genome editing have the potential for developing improved varieties of yam with diseases and pests resistance.
The use of mechanical ventilation in the ED Easter, Benjamin D., MD; Fischer, Christopher, MD; Fisher, Jonathan, MD
The American journal of emergency medicine,
09/2012, Letnik:
30, Številka:
7
Journal Article
Recenzirano
Abstract Objectives Although EDs are responsible for the initial care of critically ill patients and the amount of critical care provided in the ED is increasing, there are few data examining ...mechanical ventilation (MV) in the ED. In addition, characteristics of ED-based ventilation may affect planning for ventilator shortages during pandemic influenza or bioterrorist events. The study examined the epidemiology of MV in US EDs, including demographic, clinical, and hospital characteristics; indications for MV; ED length of stay (LOS); and in-hospital mortality. Methods This study was a retrospective review of the 1993 to 2007 National Hospital Ambulatory Medical Care Survey ED data sets. Ventilated patients were compared with ED patients admitted to the intensive care unit (ICU) and to all other ED visits. Results There were 3.6 million ED MV visits (95% confidence interval CI, 3.2-4.0 million) over the study period. Sex, age, race, and payment source were similar for mechanically ventilated and ICU patients ( P > .05 for all). Approximately 12.5% of ventilated patients underwent cardiopulmonary resuscitation compared with 1.7% of ICU admissions and 0.2% of all other ED visits ( P < .0001). Accordingly, in-hospital mortality was significantly higher for ventilated patients (24%; 95% CI, 13.1%-34.9%) than both comparison groups (9.3% and 2.5%, respectively). Median LOS for ventilated patients was 197 minutes (interquartile range, 112-313 minutes) compared with 224 minutes for ICU admissions and 140 minutes for all other ED visits. Conclusions Patients undergoing ED MV have particularly high in-hospital mortality rates, but their ED LOS is sufficient for implementation of evidence-based ventilator interventions.
Abstract
The 3′-ends of eukaryotic pre-mRNAs are processed in the nucleus by a large multiprotein complex, the cleavage and polyadenylation factor (CPF). CPF cleaves RNA, adds a poly(A) tail and ...signals transcription termination. CPF harbors four enzymatic activities essential for these processes, but how these are coordinated remains poorly understood. Several subunits of CPF, including two protein phosphatases, are also found in the related 'associated with Pta1′ (APT) complex, but the relationship between CPF and APT is unclear. Here, we show that the APT complex is physically distinct from CPF. The 21 kDa Syc1 protein is associated only with APT, and not with CPF, and is therefore the defining subunit of APT. Using ChIP-seq, PAR-CLIP and RNA-seq, we show that Syc1/APT has distinct, but possibly overlapping, functions from those of CPF. Syc1/APT plays a more important role in sn/snoRNA production whereas CPF processes the 3′-ends of protein-coding pre-mRNAs. These results define distinct protein machineries for synthesis of mature eukaryotic protein-coding and non-coding RNAs.
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