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A new consortium of 27 partners coordinated by our department will tackle the challenge of discovery and characterisation of blood-brain barrier targets and transport mechanisms for brain delivery of therapeutics to treat neurodegenerative and metabolic diseases.
Estimated prevalence of hypertension and undiagnosed hypertension in a large inpatient population: A cross-sectional observational study.
INTRODUCTION: Hypertension is a major cause of morbidity and mortality. In community populations the prevalence of hypertension, both in diagnosed and undiagnosed states, has been widely reported. However, estimates for the prevalence of hospitalised patients with average blood pressures that meet criteria for the diagnosis of hypertension are lacking. We aimed to estimate the prevalence of patients in a UK hospital setting, whose average blood pressures meet current international guidelines for hypertension diagnosis. METHODS: We performed a retrospective cross-sectional observational study of patients admitted to adult wards in four acute hospitals in Oxford, UK, between March 2014 and April 2018. RESULTS: We identified 41,455 eligible admitted patients with a total of 1.7 million blood pressure measurements recorded during their hospital admissions. According to European ESC/ESH diagnostic criteria for hypertension, 21.4% (respectively 47% according to American ACC/AHA diagnostic criteria) of patients had a mean blood pressure exceeding the diagnostic threshold for either Stage 1, 2 or 3 hypertension. Similarly, 5% had a mean blood pressure exceeding the ESC/ESH (respectively 13% had a mean blood pressure exceeding the ACC/AHA) diagnostic criteria for hypertension, but no pre-existing diagnostic code for hypertension or a prescribed antihypertensive medication during their hospital stay. CONCLUSION: Large numbers of hospital inpatients have mean in-hospital blood pressures exceeding diagnostic thresholds for hypertension, with no evidence of diagnosis or treatment in the electronic record. Whether opportunistic screening for in -hospital high blood pressure is a useful way of detecting people with undiagnosed hypertension needs evaluation.
Purpose: The classic Kozak consensus is a critical genetic element included in gene therapy transgenes to encourage the translation of the therapeutic coding sequence. Despite optimizations of other transgene elements, the Kozak consensus has not yet been considered for potential tissue-specific sequence refinement. We screened the -9 to -1 region relative to the AUG start codon of retina-specific genes to identify whether a Kozak consensus that is different from the classic sequence may be more appropriate for inclusion in gene therapy transgenes that treat inherited retinal disease. Methods: Sequences for 135 genes known to cause nonsyndromic inherited retinal disease were extracted from the NCBI database, and the -9 to -1 nucleotides were compared. This panel was then refined to 75 genes with specific retinal functions, for which the -9 to -1 nucleotides were placed in front of a GFP transcript sequence and RNAfold predictions performed. These were compared with a GFP sequence with the classic Kozak consensus (GCCGCCACC), and sequences from retinal genes with minimum free energy (MFE) predictions greater than the reference sequence were selected to generate an optimized Kozak consensus sequence. The original Kozak consensus and the refined retina Kozak consensus were placed upstream of the Renilla luciferase coding sequence, which were used to transfect retinoblastoma cell lines Y-79 and WERI-RB-1 and HEK 293T/17 cells. Results: The nucleotide frequencies of the original panel of genes were determined to be comparable to the classic Kozak consensus. RNAfold analysis of a GFP transcript with the classic Kozak sequence in the 5' untranslated region (UTR) generated an MFE prediction of -503.3 kcal/mol. RNAfold analysis was then performed with a GFP transcript containing each -9 to -1 Kozak sequence of 75 retinal genes. Thirty-eight of the 75 genes provided a greater MFE value than -503.3 kcal/mol and exhibited an absence of stable secondary structures before the AUG codon. The -9 to -1 nucleotide frequencies of these genes identified a Kozak consensus of ACCGAGACC, differing from the classic Kozak consensus at positions -9, -5, and -4. Applying this sequence to the GFP transcript increased the MFE prediction to -500.1 kcal/mol. The newly identified retina Kozak sequence was also applied to Renilla luciferase plus the REP1 and RPGR transcripts used in current clinical trials. In all examples, the predicted transcript MFE score increased when compared with the current transcript sequences containing classic Kozak consensus sequences. In vitro transfections identified a 7%-9% increase in Renilla activity when incorporating the optimized Kozak sequence. Conclusions: The Kozak consensus is a critical element of eukaryotic genes; therefore, it is a required feature of gene therapy transgenes. To date, the classic sequence of GCCRCC (-6 to -1) has typically been incorporated in gene therapy transgenes, but the analysis described here suggests that, for vectors targeting the retina, using a Kozak consensus derived from retinal genes can provide increased expression of the target product.
Purpose: A local colony of inbred mice (129S6/SvEvTac origin), in isolation for over a decade, were found to have absent light-adapted electroretinogram (ERG) responses. We investigated the inheritance and genetic basis of this phenotype of cone photoreceptor function loss. Methods: An affected 129S6/SvEvTac colony animal was outcrossed to a C57BL/6J mouse and intercrossed to investigate inheritance in the F2 generation. We performed ERG testing and targeted resequencing on genes of interest (Gnat2, Cnga3, Cngb3, Pde6c, Hcn1, Syne2). The eyes of a subset of animals underwent histologic immunostaining. Results: All 129S6/SvEvTac colony animals tested lacked cone pathway function by ERG testing (n = 12), although rod pathway-based ERG responses remained unaffected. Outcross-intercross breeding showed a recessive inheritance pattern. A novel missense mutation was identified in the Cngb3 gene, which causes an amino acid substitution at a conserved residue (NM_013927)c.692G>A; p.(R231H). The recessive phenotype only affected homozygotes (χ2 = 39, P = 3.2e-10). Cones had normal morphology at postnatal day (PND) 70, but cone cell counts declined from PND 30 to PND 335 (P = 0.038), indicating progressive cone photoreceptor death. Conclusions: We identified the spontaneous occurrence of a 10th model of cone photoreceptor function loss (cpfl10) in an isolated line of inbred mice. Our results indicate that this is caused by a novel missense mutation in the Cngb3 gene, with a fully recessive inheritance pattern. This mouse may provide a more appropriate background against which to assess CNGB3 achromatopsia gene therapy for missense mutations.
AAV Induced Expression of Human Rod and Cone Opsin in Bipolar Cells of a Mouse Model of Retinal Degeneration
Vision loss caused by inherited retinal degeneration affects millions of people worldwide, and clinical trials involving gene supplementation strategies are ongoing for select forms of the disease. When early therapeutic intervention is not possible and patients suffer complete loss of their photoreceptor cells, there is an opportunity for vision restoration techniques, including optogenetic therapy. This therapy provides expression of light-sensitive molecules to surviving cell types of the retina, enabling light perception through residual neuronal pathways. To this end, the bipolar cells make an obvious optogenetic target to enable upstream processing of visual signal in the retina. However, while AAV transduction of the bipolar cells has been described, the expression of human opsins in these cell types within a model of retinal degeneration (rd1) has been less successful. In this study, we have expanded the optogenetic toolkit and shown successful expression of human rhodopsin driven by an ON-bipolar cell promoter (Grm6) in the rd1 mouse model using modified AAV capsids (AAV2.4YF, AAV8.BP2, and AAV2.7m8) delivered via intraocular injection. We also show the first presentation of ectopic expression of human cone opsin in the bipolar cells of rd1 mice. These data provide evidence of an expansion of the optogenetic toolkit with the potential to restore useful visual function, setting the stage for future trials in human patients.
The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario.
Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.
Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).
AbstractWhile it is well established that dopamine transmission is integral in mediating the influence of reward expectations on reward-seeking actions, the precise causal role of dopamine transmission in moment-to-moment cue-driven behavioural control remains contentious. This is a particular issue in situations where it is necessary to refrain from responding to achieve a beneficial outcome. To examine this, we manipulated dopamine transmission pharmacologically as rats performed a Go/No-Go task that required them to either make or withhold action to gain either a small or large reward. Stimulation of D1Rs, both globally and locally in the nucleus accumbens core (NAcC) region consistently disrupted No-Go performance, potentiating inappropriate responses that clustered strongly just after cue presentation. D1R blockade did not, however, improve rats’ ability to withhold responses, but instead primarily disrupted performance on Go trials. While global D1R blockade caused a general reduction of invigoration of reward seeking actions, intra-NAcC administration of the D1R antagonist by contrast increased the likelihood that Go trial performance was in an “unfocused” state. Such a state was characterised, both on and off drug, by a reduction in the precision and speed of responding even though the appropriate action sequence was often executed. These findings suggests that the balance of activity at NAcC D1Rs plays a key role in enabling the rapid activation of a focused, reward-seeking state to enable animals to efficiently and accurately achieve their goal.
Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons
Abstractα-Synuclein is critical in the pathogenesis of Parkinson’s disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced α-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson’s disease or multiple system atrophy. Increased α-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in α-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal α-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson’s disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of α-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.
Educational Initiatives and Implementation of Electroencephalography into the Acute Care Environment: a protocol of a Systematic Review
Abstract Background: Use of electroencephalography (EEG) is currently recommended by the American Clinical Neurophysiology Society for a wide range of indications, including diagnosis of nonconvulsive status epilepticus and evaluation of unexplained disorders of consciousness. Data interpretation usually occurs by expert personnel (e.g. epileptologists, neurophysiologists), with information relayed to the primary care team. However, data cannot always be read in time-sensitive fashion, leading to potential delays in EEG interpretation and patient management. Multiple training programs have recently been described to enable non-experts to rapidly interpret EEG at the bedside. A comprehensive review of these training programs, including the tools used, outcomes obtained, and potential pitfalls, is currently lacking. Therefore, the optimum training program and implementation strategy remain unknown. Methods: We will conduct a systematic review of descriptive studies, case series, cohort studies and randomized controlled trials assessing training programs for EEG interpretation by non-experts. Our primary objective is to comprehensively review educational programs in this domain and report their structure, patterns of implementation, limitations, and trainee feedback. Our secondary objective will be to compare the performance of non-experts for EEG interpretation with a gold standard (e.g. interpretation by a certified electroencephalographers). Studies will be limited to those performed in acute care settings in both adult and paediatric populations (intensive care unit, emergency department, or post-anesthesia care units). Comprehensive search strategies will be developed for MEDLINE, EMBASE, WoS, CINAHL, and CENTRAL to identify studies for review. The gray literature will be scanned for further eligible studies. Two reviewers will independently screen the search results to identify studies for inclusion. A standardized data extraction form will be used to collect important data from each study. If possible, we will attempt to meta-analyse the quantitative data. If heterogeneity between studies is too high, we will present meaningful quantitative comparisons of secondary outcomes as per the synthesis without meta-analysis (SWiM) reporting guidelines. Discussion: We will aim to summarize the current literature in this domain to understand the structure, patterns, and pitfalls of EEG training programs for non-experts. This review is undertaken with a view to inform future education designs, potentially enabling rapid detection of EEG abnormalities and timely intervention by the treating physician. PROSPERO registration: Submitted and undergoing review (URL currently unavailable).
Almost any injury to the brain may initiate disturbances in local and systemic coagulation. This statement is particularly true for subarachnoid hemorrhage, traumatic brain injuries, and cranial and spinal dural arteriovenous fistulae. The pathogenesis of coagulative disorders is highlighted from both a genetic and a structural biology perspective, along with a discussion of predictive biomarkers and clinical risk factors to guide the selection of eligible patients for primary prevention. This comprehensive review allows a better understanding of the coagulative disorders affecting patients harboring CNS diseases and accordingly offers new insights into their pharmacological and surgical treatment.
In critically ill patients, the activation of blood coagulation occurs in parallel with the release of inflammatory mediators, which characterizes systemic inflammatory response syndrome and sepsis and sepsis-related conditions such as severe sepsis and septic shock. As a consequence, the overall hemostatic balance is shifted toward the activation of the coagulation, due to either the activation of the clotting system or the downregulation of the anticoagulant pathways. Systemic inflammation during sepsis leads to the generation of proinflammatory cytokines that, among other things, orchestrate coagulation and fibrinolytic activation. Abundant intravascular fibrin formation leads to microvascular thrombosis, which causes widespread ischemic organ damage up to organ necrosis and clinically impresses as widespread skin necrosis and multiple organ dysfunction syndrome. On the basis of the pathophysiologic concepts and the striking anticoagulant and anti-inflammatory properties of coagulation inhibitors in models for severe sepsis, administration of these inhibitors has been considered an attractive therapeutic approach for human sepsis.
Hypothermia describes a state in which the body's mechanism for temperature regulation is overwhelmed in the face of a cold stressor. Hypothermia is classified as accidental or intentional, primary or secondary, and according to its severity.
As seen in the previous section, nanotechnology holds the promise to redesign the realm of medicine as we know it nowadays, because it basically provides new dramatic tools to prevent diseases, promote health, and alleviate human sufiering, which are surely among our strongest mandate. Having this huge impact, a potential for both great good or even great harm, the nanofuture certainly represents the latest stage toward which ethics is called to focus. Physicist Freeman Dyson once stated, “Progress of science is destined to bring enormous confusion andmisery tomankind unless it is accompanied by progress in ethics” . Ethics, in fact, has always provided a rational approach to moral dilemmas, and bioethics has expanded rapidly in recent decades to specifically address them in life sciences. Dilemmas in innovation technology are common, and no big surprise if they are also gradually arising as part of the initial difiusion of nanotechnology-driven solutions to healthcare needs . Aiming to better analyze and understand those dilemmas, while moving our scientific research at the maximum speed for the sake of its benefits, in this section of the book we will cover the themes of risk assessment, risk management, risk communication, and research and development regulations, and finally we will discuss the concerns associated with human enhancement.
Genes and environment in multiple sclerosis: Impact of temporal changes in the sex ratio on recurrence risks.
OBJECTIVE: To evaluate the impact of temporal increase of female to male (F:M) sex ratio for persons with multiple sclerosis (MS) on the familial risk (empiric recurrence risks or RRs) for biological relatives of affected individuals. METHODS: Detailed family histories were systematically obtained from people with MS attending the University of British Columbia Hospital MS Clinic. The study cohort was born in 1970 or more recently. Data were collected from 1 September 2015 to 31 January 2019. The study was designed to allow only one proband per family. Age-corrected RRs for biological relatives of probands were calculated based on a modification of the maximum-likelihood approach. RESULTS: Data analyses were possible for 746 unique probands (531 females; 215 males) and 19,585 of their biological relatives. RRs were temporally impacted. CONCLUSION: Both genetic sharing and environmental factors are important in determining RRs. It appears that there is an increase in MS risk due to environmental factors in later life (i.e. not shared family environment). Environmental exposures in genetically predisposed individuals might be driving the MS risk. The increase in F:M ratio of RRs for sisters/brothers of female probands over time is likely due to environmental differences.
Adjuvant External Beam Radiotherapy Following Enucleation of Eyes With Extraocular Extension From Uveal Melanoma.
PURPOSE: To report local disease control and all-cause mortality in patients with extraocular extension (EOE) of uveal melanoma undergoing enucleation followed by observation or external beam radiotherapy (EBRT). METHODS: Charts of patients enucleated between January 1, 1997 and December 31, 2019, with histopathological evidence of EOE of uveal melanoma were reviewed. RESULTS: The cohort comprised 51 patients with a mean age of 67 ± 15 years, 22 (43%) of whom underwent adjuvant postenucleation EBRT. Risk factors for metastasis included presence of epithelioid cells (29/45; 88%), closed loops (20/43; 47%), monosomy 3 (16/25; 64%), and gain of 8q (20/22; 91%). Patients undergoing EBRT had more extensive EOE (median: 5.1 mm vs. 2.6 mm, p = 0.008) and surgical excision was less likely to be histologically complete (2/20; 10% vs. 14/25; 56%, p = 0.002). Local side effects following EBRT were seen in 64% (14/22). At latest follow up, 59% of patients (30/51) were alive, with a median follow up of 1.8 years (interquartile range: 2.9; range: 0.1-6.5]. By Kaplan-Meier survival analysis, the 5- and 10-year overall survival rates were 56% and 12%, respectively. There was no difference in all-cause mortality between those receiving adjuvant EBRT and those who were observed (log rank, p = 0.273). No cases of orbital recurrence were documented. CONCLUSIONS: Orbital EBRT causes significant morbidity. Cases with relatively small EOE undergoing enucleation can be safely observed, without adjuvant EBRT. Multicenter studies are required to better assess the role of EBRT when EOE is more extensive.