11,988 research outputs found

    Small newborns in post-conflict Northern Uganda: Burden and interventions for improved outcomes

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    Introduction: A small newborn can be the result of either a low birthweight (LBW), or a preterm birth (PB), or both. LBW can be due to either a preterm appropriate-for gestational-age (preterm-AGA), or a term small-for-gestational age (term-SGA) or intrauterine growth restriction (IUGR). An IUGR is a limited in-utero foetal growth rates or foetal weight < 10th percentile. Small newborns have an increased risk of dying, particularly in low-resource settings. We set out to assess the burden, the modifiable risk factors and health outcomes of small newborns in the post-conflict Northern Ugandan district of Lira. In addition, we studied the use of video-debriefing when training health staff in Helping Babies Breathe. Subjects and methods: In 2018-19, we conducted a community-based cohort study on 1556 mother-infant dyads, nested within a cluster randomized trial. In our cohort study, we estimated the incidence and risk factors for LBW and PB and the association of LBW with severe outcomes. We explored the prevalence of and factors associated with neonatal hypoglycaemia, as well as any association between neonatal death and hypoglycaemia. In addition, we conducted a cluster randomized trial to compare Helping Babies Breathe (HBB) training in combination with video debriefing to the traditional HBB training alone on the attainment and retention of health worker neonatal resuscitation competency. Results: The incidence of LBW and PB in our cohort was lower than the global estimates, 7.3% and 5.0%, respectively. Intermittent preventive treatment for malaria was associated with a reduced risk of LBW. HIV infection was associated with an increased risk of both LBW and PB, while maternal formal education (schooling) of ≥7 years was associated with a reduced risk of LBW and PB. The proportions of neonatal deaths were many-folds higher among LBW infants compared to their non-LBW counterparts. The proportion of neonatal deaths among LBW was 103/1000 live births compared to 5/1000 among the non-LBW. The prevalence of neonatal hypoglycaemia in our cohort was 2.5%. LBW and PB each independently were associated with an increased risk of neonatal hypoglycaemia. Neonatal hypoglycaemia was associated with an increased risk of hospitalisation and severe outcomes. We demonstrated that neonatal resuscitation training with video debriefing, improved competence attainment and retention among health workers, compared to traditional HBB training alone. Conclusion: In northern Uganda, small infants still have a many-fold higher risk of dying compared to normal infants. In addition, small infants are also at more risk of neonatal hypoglycaemia compared to normal infants. Efforts are needed to secure essential newborn care, should we reach the target of Sustainable Development Goal number 3.2 of reducing infant mortality to less than 12/1000 live births by 2030

    In vitro investigation of the effect of disulfiram on hypoxia induced NFκB, epithelial to mesenchymal transition and cancer stem cells in glioblastoma cell lines

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Glioblastoma multiforme (GBM) is one of the most aggressive and lethal cancers with a poor prognosis. Advances in the treatment of GBM are limited due to several resistance mechanisms and limited drug delivery into the central nervous system (CNS) compartment by the blood-brain barrier (BBB) and by actions of the normal brain to counteract tumour-targeting medications. Hypoxia is common in malignant brain tumours such as GBM and plays a significant role in tumour pathobiology. It is widely accepted that hypoxia is a major driver of GBM malignancy. Although it has been confirmed that hypoxia induces GBM stem-like-cells (GSCs), which are highly invasive and resistant to all chemotherapeutic agents, the detailed molecular pathways linking hypoxia, GSC traits and chemoresistance remain obscure. Evidence shows that hypoxia induces cancer stem cell phenotypes via epithelial-to-mesenchymal transition (EMT), promoting therapeutic resistance in most cancers, including GBM. This study demonstrated that spheroid cultured GBM cells consist of a large population of hypoxic cells with CSC and EMT characteristics. GSCs are chemo-resistant and displayed increased levels of HIFs and NFκB activity. Similarly, the hypoxia cultured GBM cells manifested GSC traits, chemoresistance and invasiveness. These results suggest that hypoxia is responsible for GBM stemness, chemoresistance and invasiveness. GBM cells transfected with nuclear factor kappa B-p65 (NFκB-p65) subunit exhibited CSC and EMT markers indicating the essential role of NFκB in maintaining GSC phenotypes. The study also highlighted the significance of NFκB in driving chemoresistance, invasiveness, and the potential role of NFκB as the central regulator of hypoxia-induced stemness in GBM cells. GSC population has the ability of self-renewal, cancer initiation and development of secondary heterogeneous cancer. The very poor prognosis of GBM could largely be attributed to the existence of GSCs, which promote tumour propagation, maintenance, radio- and chemoresistance and local infiltration. In this study, we used Disulfiram (DS), a drug used for more than 65 years in alcoholism clinics, in combination with copper (Cu) to target the NFκB pathway, reverse chemoresistance and block invasion in GSCs. The obtained results showed that DS/Cu is highly cytotoxic to GBM cells and completely eradicated the resistant CSC population at low dose levels in vitro. DS/Cu inhibited the migration and invasion of hypoxia-induced CSC and EMT like GBM cells at low nanomolar concentrations. DS is an FDA approved drug with low toxicity to normal tissues and can pass through the BBB. Further research may lead to the quick translation of DS into cancer clinics and provide new therapeutic options to improve treatment outcomes in GBM patients

    Mathematical models to evaluate the impact of increasing serotype coverage in pneumococcal conjugate vaccines

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    Of over 100 serotypes of Streptococcus pneumoniae, only 7 were included in the first pneumo- coccal conjugate vaccine (PCV). While PCV reduced the disease incidence, in part because of a herd immunity effect, a replacement effect was observed whereby disease was increasingly caused by serotypes not included in the vaccine. Dynamic transmission models can account for these effects to describe post-vaccination scenarios, whereas economic evaluations can enable decision-makers to compare vaccines of increasing valency for implementation. This thesis has four aims. First, to explore the limitations and assumptions of published pneu- mococcal models and the implications for future vaccine formulation and policy. Second, to conduct a trend analysis assembling all the available evidence for serotype replacement in Europe, North America and Australia to characterise invasive pneumococcal disease (IPD) caused by vaccine-type (VT) and non-vaccine-types (NVT) serotypes. The motivation behind this is to assess the patterns of relative abundance in IPD cases pre- and post-vaccination, to examine country-level differences in relation to the vaccines employed over time since introduction, and to assess the growth of the replacement serotypes in comparison with the serotypes targeted by the vaccine. The third aim is to use a Bayesian framework to estimate serotype-specific invasiveness, i.e. the rate of invasive disease given carriage. This is useful for dynamic transmission modelling, as transmission is through carriage but a majority of serotype-specific pneumococcal data lies in active disease surveillance. This is also helpful to address whether serotype replacement reflects serotypes that are more invasive or whether serotypes in a specific location are equally more invasive than in other locations. Finally, the last aim of this thesis is to estimate the epidemiological and economic impact of increas- ing serotype coverage in PCVs using a dynamic transmission model. Together, the results highlight that though there are key parameter uncertainties that merit further exploration, divergence in serotype replacement and inconsistencies in invasiveness on a country-level may make a universal PCV suboptimal.Open Acces

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies

    Quantifying the Indirect Effect of Wolves on Aspen in Northern Yellowstone National Park: Evidence for a Trophic Cascade?

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    Yellowstone National Park is renowned for its incredible wildlife, and perhaps the most famous of these species is the gray wolf, which was reintroduced to the Park in the mid-1990s. After reintroduction, it was highly publicized by scientists, journalists, and environmentalists that the wolf both decreased elk density and changed elk behavior in a way that reduced elk effects on plants, a process known as a “trophic cascade.” Aspen, which is eaten by elk in winter, is one species at the forefront of Yellowstone trophic cascade research because it has been in decline across the Park for over a century. However, due to the challenges of measuring trophic cascades, there is continued uncertainty regarding the effects of wolves on aspen in northern Yellowstone. Thus, the purpose of my dissertation was to provide a comprehensive test of a trophic cascade in this system. Specifically, I used 20 years of data on aspen, elk, and wolves in Yellowstone to: 1) clarify annual trends in browsing and height of young aspen (a proxy for regeneration) after wolf reintroduction, 2) assess the influence of wolves scaring elk on aspen (“trait-mediated indirect effects”), and 3) evaluate the effect of wolves killing elk on aspen (“density-mediated indirect effects”). My research suggests that wolves indirectly contributed to increased aspen over story recruitment following their reintroduction by helping to reduce the elk population size, but elk response to the risk of wolf predation did not reduce elk foraging in a way that measurably increased aspen recruitment. Additionally, hunter harvest of elk north of the park was twice as important as wolf predation in causing increased aspen recruitment. However, despite wolves and hunters limiting elk abundance, it is still uncommon for young aspen to grow past peak browsing height (120-cm), indicating that many stands remain vulnerable to elk herbivory nearly 30 years after wolf reintroduction. These results highlight that the strength and mechanism of predator effects on plant communities are context-specific. Thus, using predator reintroduction as a tool for ecosystem restoration without considering the many factors that shape trophic cascades may result in different management and conservation outcomes than intended

    Emergency Advanced Clinical Practitioners: Quality and Acceptability

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    Evidence gathering in support of sustainable Scottish inshore fisheries: work package (4) final report: a pilot study to define the footprint and activities of Scottish inshore fisheries by identifying target fisheries, habitats and associated fish stocks

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    [Extract from Executive Summary] This work was conducted under Work package 4 of the European Fisheries Funded program “Evidence Gathering in Support of Sustainable Scottish Inshore Fisheries”. The overall aim of the program was to work in partnership with Marine Scotland Fisheries Policy and with the Scottish Inshore Fisheries Groups to help develop inshore fisheries management. Specifically the program aims were to establish the location of fishing activities within inshore areas; to identify catch composition and associated fishery impacts; to define the environmental footprint and availability of stocks; to develop economic value within local fisheries and; to establish an information resource base to assist the development of inshore fisheries management provisions.Publisher PD

    Genome-wide identification of the genetic basis of amyotrophic lateral sclerosis

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    Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases

    Structural basis of translational recycling and bacterial ribosome rescue

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    In the last step of gene expression, a messenger RNA (mRNA) sequence is translated into a polypeptide. This highly regulated and dynamic process is carried out by the ribosome, a ribonucleoprotein complex composed of two unequal subunits. The translation cycle is initiated when the small ribosomal subunit (SSU) binds to an mRNA and recognizes the start codon of the open reading frame (ORF). Then the large ribosomal subunit (LSU) joins and the ribosome starts moving along the mRNA. A protein is synthesized until the ribosome reaches a stop codon. A cell needs thousands (prokaryotes) or millions (eukaryotes) of ribosomes for protein production and spends enormous amounts of energy on the assembly of this macromolecular machinery. Therefore, it is crucial to recycle the machinery after each successful round of translation. The recycling step allows release of mRNA, transfer RNA (tRNA) and the synthesized polypeptide from ribosomal subunits and subsequent binding of the next mRNA for protein synthesis. The first part of this dissertation includes studies of the highly conserved and essential ribosome recycling factor ATP binding cassette (ABC) Subfamily E Member 1 (ABCE1). In eukaryotes and archaea, ABCE1 binds the ribosome and in concert with an A-site factor and splits the ribosome into large and small subunits. ABCE1 harbors two nucleotide binding sites (NBSs), which are formed at the interface of two nucleotide binding domains (NBDs). Prior to this work, the ABCE1-bound pre-splitting complex, as well as the ABCE1-bound post-splitting complex, had been visualized by cryo-electron microscopy (cryo-EM) at medium resolution. This structural analysis combined with functional studies led to a model for the mechanism of the splitting event. ATP-binding and the closure of the NBSs lead to repositioning of the iron-sulfur cluster domain, which results in collision with the A-site factor and ribosome splitting. Yet, how conformational changes during the splitting event are triggered and communicated to the NBSs of ABCE1, was not understood. To gain molecular insights into this process, a structure of a fully nucleotide-occluded (closed) state of ABCE1 bound to the archaeal 30S post-splitting complex was solved by cryo-EM. At a resolution of 2.8 Å a detailed molecular analysis of ABCE1 was performed and confirmed by a combination of mutational and functional studies. This allowed to propose a refined model of how the ATPase cycle is linked to ribosome splitting and which role the different domains of ABCE1 play. In eukaryotes, the recycling phase is directly linked to translation initiation via the SSU. After being released from the mRNA 3’ end, the SSU can engage with another or even the same mRNA at the 5’ end. The recycling factor ABCE1 was found to be associated with initiation complexes, but whether it plays a role in initiation was not clear. Using cryo-EM, structures of native ABCE1-containing initiation complexes were solved and intensive 3D classification allowed to distinguish different stages of initiation, during which ABCE1 may play a role. Surprisingly, ABCE1 adopted a previously unknown state for ABC-type ATPases that was termed “hybrid state”. Here, the NBSI is in a half open state with ADP bound and the NBSII is in a closed state with ATP bound. Further, eukaryotic initiation factor 3j (eIF3j) was found to stabilize this hybrid conformation via its N-terminus. Since eIF3j had already been described to assist ABCE1 in ribosome dissociation, in vitro splitting assays were performed demonstrating that eiF3j indeed actively enhances the splitting reaction. On top of this, the high-resolution structure allowed to describe the interaction network of eIF3j with the ribosome, initiation factors (IFs), and ABCE1. Independent of ABCE1, the structures presented here allowed to provide an improved molecular model of the human 43S pre-initiation complex (PIC) and to analyze its sophisticated interaction network. In particular, new molecular insights into the large eIF3 complex encircling the 43S PIC, and the eIF2 ternary complex delivering the initiator tRNA are provided. Equally important as canonical recycling is the recognition and recycling of ribosomes that result from translational failure. Aberrant translation elongation and ribosome stalling can be caused by a plethora of different stresses. In bacterial cells, multiple rescue systems are known such as trans-translation or alternative ribosome rescue factor-mediated termination, which act on ribosome nascent chain complexes with an empty A-site (non-stop complexes). It has been a long standing question how ribosomes that are stalled in the middle of an ORF (no-go complexes) are recognized and recycled. The second part of this dissertation reports a new bacterial rescue system that acts on no-go complexes. In eukaryotes, the concept of ribosome collisions as a trigger for ribosome rescue has been studied extensively. Here, it was found that a similar mechanism exists in bacteria and thus a structural analysis of collided disomes in E. coli and B. subtilis was conducted. In a genetic screen, the endonuclease SmrB was identified as one candidate for a collision sensor. Structural analysis of SmrB-bound disomes elucidated how this rescue factor is recruited to collided ribosomes. Its SMR domain binds to the disome interface between the stalled and the collided ribosome in close proximity to the mRNA and in a position ideal to perform endonucleolytic cleavage. Such cleavage then results in non-stop complexes that can be recycled by the pathways mentioned above. In conclusion, this work provides mechanistic insights into how a cell distinguishes stalled ribosomes from actively translating ribosomes and characterizes a novel ribosome rescue pathway
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