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Nasal nitric oxide (nNO) measurements are used in the assessment of patients suspected of having primary ciliary dyskinesia (PCD), but recommendations for performing such measurements have not focused on children and do not include all current practices. To guide the development of a European Respiratory Society-supported technical standard for nNO measurement in children, an international online survey was conducted to better understand current measurement practices among providers involved in PCD diagnostics.78 professionals responded, representing 65 centres across 18 countries, mainly in Europe and North America. Nearly all centres measured nNO in children and more than half performed measurements before 5 years of age. The test was often postponed in children with signs of acute airway infection. In Europe, the electrochemical technique was more frequently used than chemiluminescence. A similar proportion of centres performed measurements during exhalation against a resistance (49 out of 65) or during tidal breathing (50 out of 65); 15 centres used only exhalation against a resistance and 15 used only tidal breathing. The cut-off values used to discriminate PCD were consistent across centres using chemiluminescence analysers; these centres reported results as an output (nL·min−1). Cut-off values were highly variable across centres using electrochemical devices, and nNO concentrations were typically reported as ppb.This survey is the first to determine real-world use of nNO measurements globally and revealed remarkable variability in methodology, equipment and interpretation. These findings will help standardise methods and training.

Third-generation HIV-1-derived lentiviral vectors are successfully used as therapeutic agents in various clinical applications. To further promote their use, we attempted to enhance vector infectivity by targeting the dimerization and packaging properties of the RNA transfer vector based on the premise that these two processes are tightly linked. We rationally designed mutant vectors to favor the dimeric conformation, potentially enhancing genome packaging. Initial assessments using standard assays generated outputs of variable reproducibility, sometimes with conflicting results. Therefore, we developed a novel competitive qRT-PCR assay in a co-transfection setting to measure the relative packaging efficiencies of wild-type and mutant transfer vectors. Here we report the effect of the dimerization-stabilizing mutations on infectious and physical titers of lentiviral vectors together with their packaging efficiency, measured using our novel assay. Enhancing dimerization did not automatically lead to better vector RNA packaging, suggesting that, for vector functionality, sufficient flexibility of the RNA to adopt different conformations is more important than the dimerization capacity. Our novel competitive qPCR assay enables a more stringent analysis of RNA packaging efficiency, allowing a much more precise understanding of the links between RNA structure, packaging, and infectious titers that will be invaluable for future vector development., Graphical Abstract, To optimize lentiviral vectors by enhancing genome packaging, Vamva et al. developed a competitive qRT-PCR assay to enable quantification of lentiviral RNA packaging with unprecedented accuracy. This assay has wide application in the gene therapy field for validation and quantitative titration of vectors for use in investigative and therapeutic studies.

ObjectiveThe efficacy of spoken language comprehension therapies for persons with aphasia remains equivocal. We investigated the efficacy of a self-led therapy app, ‘Listen-In’, and examined the relation between brain structure and therapy response.MethodsA cross-over randomised repeated measures trial with five testing time points (12-week intervals), conducted at the university or participants' homes, captured baseline (T1), therapy (T2-T4) and maintenance (T5) effects. Participants with chronic poststroke aphasia and spoken language comprehension impairments completed consecutive Listen-In and standard care blocks (both 12 weeks with order randomised). Repeated measures analyses of variance compared change in spoken language comprehension on two co-primary outcomes over therapy versus standard care. Three structural MRI scans (T2-T4) for each participant (subgroup, n=25) were analysed using cross-sectional and longitudinal voxel-based morphometry.ResultsThirty-five participants completed, on average, 85 hours (IQR=70–100) of Listen-In (therapy first, n=18). The first study-specific co-primary outcome (Auditory Comprehension Test (ACT)) showed large and significant improvements for trained spoken words over therapy versus standard care (11%, Cohen’s d=1.12). Gains were largely maintained at 12 and 24 weeks. There were no therapy effects on the second standardised co-primary outcome (Comprehensive Aphasia Test: Spoken Words and Sentences). Change on ACT trained words was associated with volume of pretherapy right hemisphere white matter and post-therapy grey matter tissue density changes in bilateral temporal lobes.ConclusionsIndividuals with chronic aphasia can improve their spoken word comprehension many years after stroke. Results contribute to hemispheric debates implicating the right hemisphere in therapy-driven language recovery. Listen-In will soon be available on GooglePlay.Trial registration numberNCT02540889.

We investigated spatio-chromatic contrast sensitivity in both younger and older color-normal observers. We tested how the adapting light level affected the contrast sensitivity and whether there was a differential age-related change in sensitivity. Contrast sensitivity was measured along three directions in colour space (achromatic, red-green, yellowish-violet), at background luminance levels from 0.02 to 2000 cd/m2, and different stimuli sizes using 4AFC method on a high dynamic range display. 20 observers with a mean age of 33 y.o.a. and 20 older observers with mean age of 65 participated in the study. Within each session, observers were fully adapted to the fixed background luminance. Our main findings are: (1) Contrast sensitivity increases with background luminance up to around 200 cd/m2, then either declines in case of achromatic contrast sensitivity, or remains constant in case of chromatic contrast sensitivity; (2) The sensitivity of the younger age group is higher than that for the older age group by 0.3 log units on average. Only for the achromatic contrast sensitivity, the old age group shows a relatively larger decline in sensitivity for medium to high spatial frequencies at high photopic light levels; (3) Peak frequency, peak sensitivity and cut-off frequency of contrast sensitivity functions show decreasing trends with age and the rate of this decrease is dependent on mean luminance. The data is being modeled to predict contrast sensitivity as a function of age, luminance level, spatial frequency, and stimulus size.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. They allow cell-surface receptors that stimulate IP3 formation to evoke rapid Ca2+ release from the endoplasmic reticulum (ER). IP3Rs initiate local and global cytosolic Ca2+ signals, they deliver Ca2+ selectively to other organelles including mitochondria and lysosomes, and, by depleting the ER of Ca2+, they control store-operated Ca2+ entry (SOCE). We consider two areas where recent work highlights the importance of liaisons between IP3Rs and other intracellular membranes. Interactions between IP3Rs and lysosomes illustrate striking parallels with the relationships between IP3Rs and mitochondria. In each case, the ER concentrates Ca2+ from the cytosol and then delivers it through IP3Rs to a low-affinity Ca2+-uptake system in a juxtaposed organelle. Evidence that only immobile IP3Rs parked alongside the sites where SOCE occurs suggests a mechanism whereby local depletion of the ER may activate SOCE without compromising other Ca2+-dependent ER functions.

The United Kingdom has been severely affected by the coronavirus disease 2019 (COVID-19) pandemic. As the National Health Service (NHS) has urgently prioritized management of this outbreak, the UK clinical oncology community has had to adapt rapidly to maintain cancer services and training. These unprecedented times have altered countless aspects of cancer care, education, and research, providing a legacy that will extend well beyond the pandemic that catalyzed them. This editorial focuses on 3 key themes that distinguish the United Kingdom from many other countries.

Hip arthroscopic surgery for femoroacetabular impingement and periacetabular osteotomy (PAO) for dysplasia is the most commonly used contemporary treatment for these conditions and has been shown to provide pain relief and restore function. What is less understood and perhaps of more interest to health economists, is the role of these procedures in preserving the hip joint and avoiding hip arthroplasty. The aim of this systematic review was to determine whether hip joint preservation surgery, indeed, preserves the hip joint by looking at conversion rates to total hip arthroplasty (THA). Two separate searches were undertaken, using PRISMA guidelines and utilizing PubMed and Open Athens search engines, identifying manuscripts that looked at conversion to THA following either hip arthroscopy (HA) or PAO. When considering HA, we found 64 eligible papers. Out of these studies, there were 59 430 hips with 5627 undergoing conversion to THA (9.47% [95% CI 9.23–9.71%]) with a mean conversion time of 24.42 months. Regarding PAO, there were 46 eligible papers including 4862 patients who underwent PAO with subsequent conversion to THA in 404 patients (8.31% [95% CI 7.54–9.12%]). with a mean conversion time of 70.11 months. Certain features were associated with increased conversion rates, including increasing age, worsening arthritis and joint space

Background A considerable portion of the HIV pandemic is composed of people under antiretroviral therapy, many of whom get a late diagnosis. Patients starting antiretroviral therapy (ART) at a very advanced stage of HIV disease attain a low recovery of CD4 T cells. Factors associated with poor recovery are incompletely described. This study aimed at finding variables associated with CD4 T cell recovery in late-presenting HIV patients. Methods We studied a cohort of HIV+ patients initiating ART with very low basal CD4 T cell counts. We defined immune recovery as the net increase in circulating CD4 T cell counts after one year on ART. We analyzed diverse routine laboratory determinations at different times using Least Absolute Shrinkage and Selection Operator (LASSO), adaptive LASSO and Conditional Inference Random Forest. Results CD4/CD8 ratio, % CD4 T cells and CD8 T cell counts at different times were the main recovery correlates, validated by all approaches. Unexpectedly, basal hematocrit was a consistent predictor. Additionally, week 24 creatinine had a high lasso coefficient, and alkaline phosphatase had a high conditional inference random forest coefficients, although neither was verified by other tests. Conclusions CD4 T cell proportions are associated with CD4 T cell recovery, independently of cell counts. Inflammation-related variables could also affect reconstitution. These accessible variables may reflect underlying mechanisms and could improve the follow up of patients starting ART with an advanced HIV infection.

Immune checkpoint inhibitors have revolutionized cancer therapy, but not all cancers respond to the currently available drugs, and even within cancers considered responsive to such modality, response rates range between 15 and 40%, depending on the cancer type, the line of treatment, and yet unknown clinical/molecular factors. Coordinated expression of checkpoint proteins was shown to occur on T cells, probably allowing fine-tuning of the signal transmitted to the cell. We performed a bioinformatic analysis of the expression of putative checkpoint mRNAs at the cancer side of the immunological synapse from the bladder cancer tumorgenome atlas (TCGA) database. Fifteen mRNAs, corresponding to both coinhibitory and costimulatory checkpoints, were shown to be expressed above a designated threshold. Of these, seven mRNAs were found to be coexpressed: CD277, PD-1L, CD48, CD86, galectin-9, TNFRSF14 (HVEM), and CD40. The expression of 2 of these mRNAs-BTN3A1 (CD277) and TNFRSF14 (HVEM)-was positively correlated with overall survival in the TCGA database. All these seven mRNA share putative binding sites of a few transcription factors (TFs). Of these, the expression of the TF BACH-2 was positively correlated with the expression of checkpoint mRNAs from the network. This suggests a joint transcriptional regulation on the expression of checkpoint mRNAs at the bladder tumor side of the immunological synapse.

Fetal growth depends on placental function, which requires energy from mitochondria. Here we investigated whether mitochondrial function in the placenta relates to the growth of the lightest and heaviest fetuses of each sex within the litter of mice. Placentas from the lightest and heaviest fetuses were taken to evaluate placenta morphology (stereology), mitochondrial energetics (high-resolution respirometry), mitochondrial regulators, nutrient transporters, hormone handling, and signaling pathways (qPCR and Western blotting). We found that mitochondrial complex I and II oxygen consumption rate was greater for placentas supporting the lightest female fetuses, although placental complex I abundance of the lightest females and complexes III and V of the lightest males were decreased compared to their heaviest counterparts. Expression of mitochondrial biogenesis (Nrf1) and fission (Drp1 and Fis1) genes was lower in the placenta from the lightest females, whilst biogenesis-related gene Tfam was greater in the placenta of the lightest male fetuses. In addition, placental morphology and steroidogenic gene (Cyp17a1 and Cyp11a1) expression were aberrant for the lightest females, but glucose transporter (Slc2a1) expression was lower in only the lightest males versus their heaviest counterparts. Differences in intra-litter placental phenotype were related to changes in the expression of hormone-responsive (androgen receptor) and metabolic signaling (AMPK, AKT, and PPARγ) pathways. Thus, in normal mouse pregnancy, placental structure, function, and mitochondrial phenotype are differentially responsive to the growth of the female and male fetus. This study may inform the design of sex-specific therapies for placental insufficiency and fetal growth abnormalities with life-long benefits for the offspring.

Funder: Funding details are provided in the Supplementary Material, Polygenic risk scores (PRS) for epithelial ovarian cancer (EOC) have the potential to improve risk stratification. Joint estimation of Single Nucleotide Polymorphism (SNP) effects in models could improve predictive performance over standard approaches of PRS construction. Here, we implemented computationally-efficient, penalized, logistic regression models (lasso, elastic net, stepwise) to individual level genotype data and a Bayesian framework with continuous shrinkage, “select and shrink for summary statistics” (S4), to summary level data for epithelial non-mucinous ovarian cancer risk prediction. We developed the models in a dataset consisting of 23,564 non-mucinous EOC cases and 40,138 controls participating in the Ovarian Cancer Association Consortium (OCAC) and validated the best models in three populations of different ancestries: prospective data from 198,101 women of European ancestry; 7,669 women of East Asian ancestry; 1,072 women of African ancestry, and in 18,915 BRCA1 and 12,337 BRCA2 pathogenic variant carriers of European ancestry. In the external validation data, the model with the strongest association for non-mucinous EOC risk derived from the OCAC model development data was the S4 model (27,240 SNPs) with odds ratios (OR) of 1.38(95%CI:1.28–1.48,AUC:0.588) per unit standard deviation, in women of European ancestry; 1.14(95%CI:1.08–1.19,AUC:0.538) in women of East Asian ancestry; 1.38(95%CI:1.21-1.58,AUC:0.593) in women of African ancestry; hazard ratios of 1.37(95%CI:1.30–1.44,AUC:0.592) in BRCA1 pathogenic variant carriers and 1.51(95%CI:1.36-1.67,AUC:0.624) in BRCA2 pathogenic variant carriers. Incorporation of the S4 PRS in risk prediction models for ovarian cancer may have clinical utility in ovarian cancer prevention programs.

Funder: Stanley Medical Research Institute, There have been no new drugs for the treatment of schizophrenia in several decades and treatment resistance represents a major unmet clinical need. The drugs that exist are based on serendipitous clinical observations rather than an evidence-based understanding of disease pathophysiology. In the present review, we address these bottlenecks by integrating common, rare, and expression-related schizophrenia risk genes with knowledge of the druggability of the human genome as a whole. We highlight novel drug repurposing opportunities, clinical trial candidates which are supported by genetic evidence, and unexplored therapeutic opportunities in the lesser-known regions of the schizophrenia genome. By identifying translational gaps and opportunities across the schizophrenia disease space, we discuss a framework for translating increasingly well-powered genetic association studies into personalized treatments for schizophrenia and initiating the vital task of characterizing clinically relevant drug targets in underexplored regions of the human genome.

Variations in neuronal connectivity occur widely in nervous systems from invertebrates to mammals. Yet, it is unclear how neuronal variability originates, to what extent and at what time scales it exists, and what functional consequences it might carry. To assess inter- and intraindividual neuronal variability, it would be ideal to analyze the same identified neuron across different brain hemispheres and individuals. Here, using genetic labeling and electron microscopy connectomics, we show that an identified inhibitory olfactory local interneuron, TC-LN, exhibits extraordinary variability in its glomerular innervation patterns. Moreover, TC-LN’s innervation of the VL2a glomerulus, which processes food signals and modulates mating behavior, is sexually dimorphic, is influenced by female’s courtship experience, and correlates with food intake in mated females. Mating also affects output connectivity of TC-LN to specific local interneurons. We propose that mating-associated variability of TC-LNs regulates how food odor is interpreted by an inhibitory network to modulate feeding.

Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272, Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265, People with schizophrenia show higher risk for abdominal obesity than the general population, which could contribute to excess mortality. However, it is unclear whether this is driven by alterations in abdominal fat partitioning. Here, we test the hypothesis that individuals with schizophrenia show a higher proportion of visceral to total body fat measured using magnetic resonance imaging (MRI). We recruited 38 participants with schizophrenia and 38 healthy controls matched on age, sex, ethnicity, and body mass index. We found no significant differences in body fat distribution between groups, suggesting that increased abdominal obesity in schizophrenia is not associated with altered fat distribution.

Background and Objective CADASIL is the most common monogenic form of stroke and early onset dementia. We determined the prevalence of vascular cognitive impairment (VCI) in a cohort of CADASIL patients, and investigated which factors were associated with VCI risk, including clinical, genetic and MRI parameters. Methods Cognition was assessed in genetically confirmed CADASIL patients (n = 176) and healthy controls (n= 265) (mean(SD) age 50.95(11.35) v 52.37(7.93) years), using the Brief Memory and Executive Test (BMET) and the Montreal Cognitive Assessment (MoCA). VCI was defined according to previously validated cut-offs. We determined the prevalence of VCI and its associations with clinical risk factors, mutation location (EGFr 1-6 versus EGFr 7-34), and MRI markers of small vessel disease. Results VCI was more common in CADASIL than controls; 39.8 v 10.2% on BMET 47.7% v 19.6% of MOCA. CADASIL patients had worse performance across all cognitive domains. History of stroke was associated with VCI on the BMET (OR 2.12, 95% CI [1.05, 4.27] p = 0.04) and on the MoCA (OR 2.55 [1.21, 5.41] p = 0.01), after controlling for age and sex. There was no association of VCI with mutation site. Lacune count was the only MRI parameter independently associated with VCI on the BMET (OR: 1.63, 95% CI [1.10, 2.41], p = 0.014), after controlling for other MRI parameters. These associations persisted after controlling for education in the sensitivity analyses. Conclusions VCI is present in almost half of CADASIL patients with a mean age of 50. Stroke and lacune count on MRI were both independent predictors of VCI on the BMET.

The interaction of descending neocortical outputs and subcortical premotor circuits is critical for shaping skilled movements. Two broad classes of motor cortical output projection neurons provide input to many subcortical motor areas: pyramidal tract (PT) neurons, which project throughout the neuraxis, and intratelencephalic (IT) neurons, which project within the cortex and subcortical striatum. It is unclear whether these classes are functionally in series or whether each class carries distinct components of descending motor control signals. Here, we combine large-scale neural recordings across all layers of motor cortex with cell type–specific perturbations to study cortically dependent mouse motor behaviors: kinematically variable manipulation of a joystick and a kinematically precise reach-to-grasp. We find that striatum-projecting IT neuron activity preferentially represents amplitude, whereas pons-projecting PT neurons preferentially represent the variable direction of forelimb movements. Thus, separable components of descending motor cortical commands are distributed across motor cortical projection cell classes.

We read with interest the article from Braungart and colleagues, discussing management of young females with benign ovarian tumors [1]. This is important and timely, and we agree with the need for clarity in management of this under-investigated group. As the authors note, published evidence to support decisions is limited, so in certain areas, guidance may have to rely on expert opinion. The authors emphasize the importance of multidisciplinary team (MDT) discussion to optimise management and risk-stratification [1]. They highlight the importance and safety of ovarian-sparing-surgery in benign tumors, to preserve fertility and avoid long-term effects including premature ovarian failure [1].

Introduction During the course of the COVID-19 pandemic, there have been suggestions that various techniques could be employed to improve the fit and, therefore, the effectiveness of face masks. It is well recognized that improving fit tends to improve mask effectiveness, but whether these fit modifiers are reliable remains unexplored. In this study, we assess a range of common “fit hacks” to determine their ability to improve mask performance. Methods Between July and September 2020, qualitative fit testing was performed in an indoor living space. We used quantitative fit testing to assess the fit of both surgical masks and KN95 masks, with and without ‘fit hacks’, on four participants. Seven fit hacks were evaluated to assess impact on fit. Additionally, one participant applied each fit hack multiple times to assess how reliable hacks were when reapplied. A convenience of four participants took part in the study, three females and one male with a head circumference range of 54 to 60 centimetres. Results and discussion The use of pantyhose, tape, and rubber bands were effective for most participants. A pantyhose overlayer was observed to be the most effective hack. High degrees of variation were noted between participants. However, little variation was noted within participants, with hacks generally showing similar benefit each time they were applied on a single participant. An inspection of the fit hacks once applied showed that individual facial features may have a significant impact on fit, especially the nose bridge. Conclusions Fit hacks can be used to effectively improve the fit of surgical and KN95 masks, enhancing the protection provided to the wearer. However, many of the most effective hacks are very uncomfortable and unlikely to be tolerated for extended periods of time. The development of effective fit-improvement solutions remains a critical issue in need of further development.

Background: Flagellate dermatitis (FD) is a rare skin rash, which may occur following the administration of antineoplastic agents. It has been reported following the administration of bleomycin, docetaxel, trastuzumab, cisplatin, bendamustine and doxorubicin. We provide a summary of the epidemiology, aetiology, pathophysiology, and distribution of chemotherapy-induced FD. Methods: PubMed was searched using ((flagellat*) AND (Dermat*)) OR ((Flagellat*) AND (Erythema)). The search yielded 206 publications, out of which 54 individual case reports were identified which fulfilled our inclusion criteria. Statistical analysis was performed where appropriate. Results: Female patients were slightly more likely to develop FD compared to males. In the majority of cases FD appeared on the upper and lower limbs and pruritus was an accompanying feature in 51% of cases. Most cases developed after the first cycle of chemotherapy and females were statistically more likely to present within the first 72 hr (p

ObjectivesThe aim of this study was to assess the association of antenatal maternal dietary patterns (DPs) and other health aspects with infant temperament in a large multiethnic cohort, taking maternal personality and prenatal stress into account.Design and methodsUsing data from 3968 children born in 2009/2010 and their mothers from the Growing Up in New Zealand cohort, infant temperament was assessed at 9 months using the Infant Behavior Questionnaire-Revised Very Short Form. Maternal antenatal diet and other health aspects were assessed antenatally. Maternal DPs (n=4) were derived using principal components analysis based on food intake reported on a 44-item food frequency questionnaire. Path analyses investigated factors associated with infant temperament, namely maternal personality, prenatal maternal stress, DPs and other health aspects, including potential inter-relations and mediating effects.ResultsWomen who scored higher in the fusion DP (standardised beta (β)=0.05; 95% CI 0.02 to 0.09) and healthy DP (β=0.05; 95% CI 0.02 to 0.09), who exercised more (β=0.04; 95% CI 0.01 to 0.07), and who drank less alcohol (β=−0.05; 95% CI −0.08 to –0.02) were more likely to have infants with an overall less difficult temperament. Sex-specific differences were found in the associations between maternal DP and infant temperament. Maternal personality and prenatal stress were significantly associated with all dimensions of infant temperament. The strongest predictors for a more difficult temperament were prenatal stress (β=0.12; 95% CI 0.08 to 0.15) and the personality dimensions neuroticism (β=0.10; 95% CI 0.07 to 0.14) and extraversion (β=−0.09; 95% CI −0.12 to –0.06).ConclusionsAssociations of antenatal maternal diet and health aspects with infant temperament were statistically significant but small. While they should not be overinterpreted as being deterministic, the findings of this study support the link between maternal modifiable health-related behaviours and infant temperament outcomes.

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI), Funder: Mayo Foundation for Medical Education and Research (Mayo Foundation); doi: https://doi.org/10.13039/100007048, Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases, yet is responsible for a disproportionately high percentage of breast cancer mortalities. Thus, there is an urgent need to identify novel biomarkers and therapeutic targets based on the molecular events driving TNBC pathobiology. Estrogen receptor beta (ER��) is known to elicit anti-cancer effects in TNBC, however its mechanisms of action remain elusive. Here, we report the expression profiles of ER�� and its association with clinicopathological features and patient outcomes in the largest cohort of TNBC to date. In this cohort, ER�� was expressed in approximately 18% of TNBCs, and expression of ER�� was associated with favorable clinicopathological features, but correlated with different overall survival outcomes according to menopausal status. Mechanistically, ER�� formed a co-repressor complex involving enhancer of zeste homologue 2/polycomb repressive complex 2 (EZH2/PRC2) that functioned to suppress oncogenic NF��B/RELA (p65) activity. Importantly, p65 was shown to be required for formation of this complex and for ER��-mediated suppression of TNBC. Our findings indicate that ER��+ tumors exhibit different characteristics compared to ER��- tumors and demonstrate that ER�� functions as a molecular switch for EZH2, repurposing it for tumor suppressive activities and repression of oncogenic p65 signaling.

Introduction: Prediagnostic features of Parkinson’s Disease are well described but prediagnostic Progressive Supranuclear Palsy (PSP) is less understood. The diagnosis of PSP is delayed by an average of three years after symptom onset. Understanding the changes that occur in the prediagnostic period will aid earlier diagnosis, clarify the natural history, and may aid the design of early disease-modifying therapy trials. We set out to identify motor and cognitive markers of prediagnostic PSP, with Parkinson’s disease as a comparator condition, in a large prospective cohort. Methods: Baseline UK Biobank data from 502,504 individuals were collected between 2006 and 2010. Subsequent PSP and Parkinson’s disease cases were identified from primary and secondary care electronic health records’ diagnostic coding data and death registry, with 5,404 matched controls. Results: 176 PSP cases (time to diagnosis 7.8±2.8 years) and 2,526 Parkinson’s disease cases (time to diagnosis 7.8±2.9 years) were identified. At baseline, those later diagnosed with PSP had slower reaction times, weaker hand grip, lower fluid intelligence, prospective memory, self-rated health scores and digit recall than controls. Reaction times were correlated with time to diagnosis. The PSP group had higher mortality than both Parkinson’s disease and control groups. Conclusions: Motor slowing, cognitive dysfunction, and postural instability are clinical diagnostic features of PSP that are typically symptomatic three years before diagnosis. Objective markers of these features were evident on average 7.8 years before diagnosis. Our findings suggest the existence of a long prediagnostic phase in PSP, with subtle changes in motor and cognitive function.

Funder: Medimmune; Id: http://dx.doi.org/10.13039/501100004628, Funder: Infinitus (China) Ltd., Wound biofilms represent a particularly challenging problem in modern medicine. They are increasingly antibiotic resistant and can prevent the healing of chronic wounds. However, current treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review, we present new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity, and biocompatibility. We briefly discuss existing methods for biofilm detection and focus on novel, sensor‐based approaches that show promise for early, accurate detection of biofilm formation on wound sites and that can be translated to point‐of‐care settings. We then discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound‐induced microbubbles and nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods.

The peptide hormone GDF15 is a potent anorectic factor. Raised circulating levels of GDF15 are seen in a large number of pathological disorders and are associated invariably with adverse clinical outcomes. GDF15 signalling is mediated through GFRAL receptor in the hindbrain. A greater understanding of the mechanisms that drive expression of GDF15 within different tissues has led to the GDF15-GFRAL axis being considered a route through which somatic distress is signalled to the brain. We highlight key recent insights into the role of GDF15 in appetitive behaviour and reflect on the therapeutic potential of the system for disorders of energy balance.

The composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes by characterizing EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3‐L1 adipocytes. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal markers in comparison with their lean counterpart. Moreover, proteomic analysis revealed that those EVs reflect the metabolic state of the parent cell in that the majority of proteins upregulated relate to fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway. In addition, hepatocytes‐secreted EVs influenced lipolysis and insulin sensitivity in recipient 3T3‐L1 adipocytes. Untargeted metabolomic analysis detected alterations in different adipocyte metabolic pathways in cells treated with hepatic EVs. In summary, our work showed that steatosis has a significant impact in the amount and composition of EVs secreted by hepatocytes. Moreover, our data point to the involvement of hepatic‐EVs in the development of pathologies associated with MetS.

The foundations for language acquisition are laid in infancy. A key feature of infant-directed speech (IDS) is that the slowest modulations of its amplitude envelope (~2 Hz) contain more energy than in adult-directed speech. These slow modulations may provide a cross-language rhythmic scaffold for the neural tracking of speech in infancy. To investigate relations between early neural processing of speech and language acquisition in English, the BabyRhythm project followed 113 infants during infancy and toddlerhood. The neural predictor of language development reported here was the cortical tracking of slow, rhythmic audiovisual stimuli, processing of which is known to differ in older children with dyslexia. To find out how such stimuli are tracked early in development, infants were presented with videos of a woman repeating the syllable “Ta” twice per second, and a ball bouncing on a drum to create a 2Hz beat. At the ages of six and nine months, infants exhibited a significant peak in EEG power at 2Hz when listening to these stimuli, indicating that the infant brain was responding to these stimuli at the expected frequency. Time-frequency analysis showed increased inter-trial EEG phase coherence at 2Hz, suggesting that the increase in oscillatory power was driven by the stimuli. There were no differences in how the speech and non-speech stimuli were tracked. These results indicate that the infant brain can track the rhythm of slow auditory stimuli. They lay the foundation for future investigation of how individual differences in tracking might relate to later language acquisition.

Background We aimed to quantify the association between chronic musculoskeletal pain and all-cause mortality, and to investigate the extent to which this association is mediated by physical activity, smoking status, alcohol consumption, and opioid use. Methods For this population-based cohort study, we used data from UK Biobank, UK between baseline visit (2006–2010) to 18th December 2020. We assessed the associations between chronic musculoskeletal pain and all-cause mortality using a Cox proportional hazards model. We performed causal mediation analyses to examine the proportion of the association between chronic musculoskeletal pain and all-cause mortality. Findings Of the 384,367 included participants, a total of 187,269 participants reported chronic musculoskeletal pain. Higher number of pain sites was associated with increased risk of all-cause mortality compared to having no pain (e.g., four sites vs no site of pain, Hazard Ratio [HR] 1.46, 95% Confidence Interval [CI] 1.35 to 1.57). The multiple mediator analyses showed that the mediating proportions of all four mediators ranged from 53.4% to 122.6%: among participants with two or more pain sites, the effect estimate reduced substantially, for example, HR reduced from 1.25 (95% CI: 1.21 to 1.30; two pain sites) to 1.07 (95% CI: 1.01 to 1.11; two pain sites). Interpretation We found that higher number of pain sites was associated with increased risk of all-cause mortality compared to having no pain, and at least half of the association of chronic musculoskeletal pain with increased all-cause mortality may be accounted for by four mediators. Funding Twins Research Australia.

Objectives: Coronary and cardiac calcification are frequent incidental findings on non-gated thoracic computed tomography (CT). However, radiologist opinions and practices regarding the reporting of incidental calcification are poorly understood. Methods: UK radiologists were invited to complete this online survey, organised by the British Society of Cardiovascular Imaging (BSCI). Questions included anonymous information on subspecialty, level of training and reporting practices for incidental coronary artery, aortic valve, mitral and thoracic aorta calcification. Results: The survey was completed by 200 respondents: 10% trainees and 90% consultants. Calcification was not reported by 11% for the coronary arteries, 22% for the aortic valve, 35% for the mitral valve and 37% for the thoracic aorta. Those who did not subspecialise in cardiac imaging were less likely to report coronary artery calcification (p = 0.005), aortic valve calcification (p = 0.001) or mitral valve calcification (p = 0.008), but there was no difference in the reporting of thoracic aorta calcification. Those who did not subspecialise in cardiac imaging were also less likely to provide management recommendations for coronary artery calcification (p < 0.001) or recommend echocardiography for aortic valve calcification (p < 0.001), but there was no difference for mitral valve or thoracic aorta recommendations. Conclusion: Incidental coronary artery, valvular and aorta calcification are frequently not reported on thoracic CT and there are differences in reporting practices based on subspeciality. Advances in knowledge: On routine thoracic CT, 11% of radiologists do not report coronary artery calcification. Radiologist reporting practices vary depending on subspeciality but not level of training.

The Inflammatory bowel diseases (IBD), include Crohn’s Disease (CD) and Ulcerative Colitis (UC), are characterised by chronic relapsing inflammation of the gastrointestinal tract. The accepted disease aetiology is the homeostatic relationship between intestinal bacteria and intestinal immunity breaks down, resulting in chronic relapsing inflammation that can irreversibly destroy the intestinal mucosa. Contributing factors for disease are thought to be host genetic factors, environmental factors and the host gut microbiome. However, disease heterogeneity and the complex characteristics of disease have made it difficult to precisely define disease causation. The aim of this thesis was to investigate characteristics of the gut microbiome at diagnosis of pediatric IBD in an attempt to describe features that may explain disease causation. Treatment naïve children with gastrointestinal symptoms undergoing investigation by colonoscopy were recruited. Prior to colonoscopy, faecal samples were collected. During colonoscopy, mucosal washings and biopsies were collected at multiple sites along the large intestine. Participants were characterised as IBD or with a Functional Gastrointestinal Disorder (FGID) using standard guidelines. Faecal samples were also collected from healthy children (HC) with no gastrointestinal symptoms. Microbial composition was investigated by 16S Ribosomal subunit (16S rRNA) analysis and whole genomic sequencing (WGS). Initial findings were that multiple sampling (mucosal washings, biopsies and feces) was more informative than a single sample and this approach was used in subsequent investigations. Initial comparisons of CD and UC showed greater variability in the gut community structure in CD. Although both UC and CD vary from FGID and HC, with UC microbial profiles more closely resembling FGID. Bacteria that accounted for most difference between inflamed and non-inflamed sites were Bacteroides, Akkermansia, Faecalibacterium, Eschricia, Odoribacter and Parabacteroides. Overall, the microbial gene functions and pathways between disease and non-disease groups, and between sample types, were similar. Combined analysis of 16S rRNA and WGS indicated some overall changes may be associated with inflammation, however individual patients appear to have unique microbial characteristics associated with disease. Therefore, patients with similar disease phenotypes may have different microbial drivers of disease. The outcomes of this thesis suggests that a personalised approach to investigating and treating disease may be warranted.

Obstructive sleep apnoea (OSA) pathogenesis is multifactorial with contributions from anatomical and non-anatomical endotypes. Current anatomical-orientated therapies are often inadequate or poorly tolerated with no pharmacotherapies available for OSA. Recent research shows that a combination of noradrenergic and anti-muscarinic agents increases upper-airway muscle activity (key non-anatomical endotype) and reduces OSA severity. Thus, my thesis aimed to investigate alternate therapies for OSA including novel pharmacotherapies targeted towards non-anatomical OSA endotypes as well as combining with existing anatomical approaches based on OSA endotype characterisation. Study 1 investigated the effects of the noradrenergic agent atomoxetine combined with 2 different anti-muscarinics (solifenacin or biperiden) with different receptor-selectivity profiles. Previous studies combined atomoxetine with the antimuscarinic oxybutynin which has broad receptor-selectivity. The goal was to gain mechanistic insight into specific antimuscarinic receptor subtypes for OSA pharmacotherapy which may also have a better side-effect profile versus oxybutynin. The different anti-muscarinics plus atomoxetine improved upper airway function and perceived next-day sleepiness in people with OSA albeit to a lesser extent compared to oxybutynin. This suggests broad or at least M2 muscarinic receptor selectivity may be important in mediating the efficacy of this drug combination for OSA pharmacotherapy. Previous studies with noradrenergic and antimuscarinic agents have been short term (≤1 week) and have not included different doses. Accordingly, in study 2, I investigated longer term (1-month) safety, tolerability, and efficacy of different doses of atomoxetine plus oxybutynin (ato-oxy) versus placebo. 1-month of ato-oxy was generally well-tolerated with a side effect profile consistent with the known profile of each agent alone. An 80/5 mg dosage combination of ato-oxy reduced key OSA severity metrics by ~50%. In study 3 I aimed to investigate if OSA endotype characterisation can be used to inform targeted therapy to resolve OSA in the clinically relevant group of patients who have an incomplete therapeutic response to oral appliance alone (~50% of patients). In these individuals, I systematically added existing anatomical therapies and emerging non-anatomical therapies (i.e., ato-oxy) according to OSA endotype characterisation. OSA was controlled in 50% of participants with addition of other existing anatomical interventions. Almost all the remaining participants were fully treated with the addition of non-anatomical pharmacotherapies. These novel findings provide important insight for the development of novel pharmacotherapy and combination therapy approaches informed by underlying physiological mechanisms for future treatment and management of OSA.

Low back pain is common and burdensome. The economic burden of low back pain in Australia includes total costs that exceed A$8 billion per year, costs which are projected to increase by 60% over the next 10 years. Less is known about the personal burden of low back pain. The first-line treatment consistently recommended for people with low back pain is patient education and advice. Regardless of the duration of low back pain, clinicians should provide advice to remain active, education on the benign nature of low back pain, and reassurance about the absence of a serious medical condition. Little guidance is available on how best this can be achieved. New treatments are urgently required to stem the rising costs of low back pain. Two proposed strategies are repurposing medicines, such as sleep medicines and media campaigns to target unhelp behaviours and beliefs. The overarching aim of this thesis was to investigate the personal burden of low back pain, evaluate attitudes toward education and advice for low back pain and explore contemporary options for managing low back pain. The methods used included qualitative content analysis, an observational study, development and evaluation of a new measurement tool, a systematic review and a randomised controlled trial. People with and without low back pain, online and in-person were recruited to participate across the five studies. The findings from each study are presented in individual chapters. The evidence in this thesis provides a scientific basis for understanding the personal burden of low back pain. The results describe how evaluating attitude toward education and advice could enable clinicians to tailor the patient education and advice they provide. Specific messages of reassurance rather than information about staying active should be prioritised. The Attitude toward Education and advice for Low back pain Questionnaire (AxEL-Q) is a valid and reliable tool to provide clinicians with an insight into attitudes toward education and advice at the outset of a clinical encounter. Sleep medicines should be further investigated before being endorsed to reduce pain intensity in people with acute low back pain. Social media and digital health interventions such as conversational agents provide options for supplementing low back pain management in the future.

CRISPR/Cas9-based gene editing is no doubt among the most intensively studied topics in bio-related fields in the recent decade, and certain new programmable Cas nucleases have been exploited recently for the development of a variety of biological tools far beyond gene editing, particularly for biosensor development. Although CRISPR/Cas-based biosensing has brought about a revolution in the area of nucleic acid diagnostic with their superior performances, its advantages were challenged when attempting to expand towards a broader range of non-nucleic acid targets. The currently reported methods for non-nucleic acid targets has successfully demonstrated the versality of CRISPR/Cas components in combining with other biosensing elements, however, combining these elements without proper optimization or controllable bioreaction environments could also bring additional variable factors into the system, hence potentially leading to compromised sensitivity or overall increase of system complexity. In this project, two novel CRISPR/Cas12a-based biosensing systems have been developed to realise simple, sensitive and universal non-nucleic acid detections. Both of these systems are established on a standard 96-well plate format, similar to the widely used ELISA diagnostic approach. The first system utilised an aptamer as its recognition molecule for rapid detection of two small proteins with fM-level sensitivity within 1.5 hours. In the second system, antibody was used as a recognition molecule, allowing to draw on a huge pool of commercially available antibodies to support its universality. This system exhibits ultra-high sensitivity down to 1 fg/mL (aM-level) for the detection of two protein targets. With these two successfully developed CRISPR/Cas12a-based non-nucleic acid biosensing systems, the universality and feasibility to deal with two practical bio-detection scenarios was investigated. The antibody-based system with minor modifications was directly used as a ready-to-use sensitivity enhancer onto a commercial IFN-γ ELISA kit, which resulted in a 2-log increase in sensitivity without changing its original protocol. Then, a similar modification strategy was used to re-direct the antibody-based system to detect whole pathogenic microorganism, Cryptosporidium parvum oocyst. Without the need for specialised instruments, the results show a successful detection of this pathogen with single oocyst sensitivity and capable of applying in challenging environmental samples. All these results serve as successful demonstration of the great potential in CRISPR/Cas-based biosensing technology to achieve affordable, translatable, and deployable solutions for various clinical, industrial and research diagnostic needs.

Background: More than two thirds of patients with advanced breast cancer (ABC) have estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2−) tumors. In most countries, current standard-of-care first-line treatments include an aromatase inhibitor or fulvestrant, a selective ER degrader, combined with cyclin-dependent kinase 4/6 inhibitors. Concurrent use of luteinizing hormone-releasing hormone agonists is recommended for men and premenopausal women with ABC. Nevertheless, almost all ABCs eventually become resistant to endocrine therapy and the disease is incurable in these cases. New therapies are needed to combat endocrine therapy resistance, maintain patient health-related quality-of-life, and delay the need for chemotherapy. AZD9833 (camizestrant) is a highly potent, next-generation selective ER degrader and pure ER antagonist that has demonstrated anticancer properties across a range of preclinical models, including those with ER-activating mutations (Scott et al, AACR 2020). A Phase I dose-escalation and expansion study (SERENA-1) has demonstrated that AZD9833 is well tolerated and has a promising antitumor activity when administered alone or in combination with the cyclin-dependent kinase 4/6 inhibitor palbociclib (Baird et al, SABCS 2020). SERENA-4 (NCT04711252) is a randomized, multicenter, double-blind, Phase III trial to evaluate the safety and efficacy of AZD9833 in combination with palbociclib for patients with ER+/HER2− ABC who have not received systemic treatment in the advanced disease setting. Methods: SERENA-4 will enroll 1342 patients with de novo or recurrent ER+/HER2− ABC who have not previously received systemic treatment for their locoregionally recurrent or metastatic disease. Patients with recurrent disease must have received adjuvant aromatase inhibitor or tamoxifen therapy for at least 24 months without relapse. Patients will be randomized 1:1 to receive oral treatment with either (a) AZD9833 75 mg once daily, palbociclib 125 mg once daily for 21 days followed by 7 days off treatment and a placebo for anastrozole 1 mg once daily or (b) anastrozole 1 mg once daily, palbociclib (same as active arm) and a placebo for AZD9833 75 mg once daily. Men and premenopausal women will also receive a luteinizing hormone-releasing hormone agonist. The primary endpoint is progression-free survival (PFS; up to 5 years). Secondary endpoints include overall survival (up to 8 years), second PFS, time to chemotherapy, objective response rate, and changes in health-related quality-of-life measures. Enrollment began in January 2021. As of 02 July 2021, the number of open sites is 57 across 15 countries. Acknowledgments: We thank Julia Mawer, PhD, of Oxford PharmaGenesis, Oxford, UK, for providing medical writing support funded by AstraZeneca Funding: The SERENA-4 trial is funded and overseen by AstraZeneca. © 2021 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2021 ASCO Annual Meeting. All rights reserved. Citation Format: Fabrice André, Seock-Ah Im, Patrick Neven, Richard D Baird, Johannes Ettl, Matthew P Goetz, Erika Hamilton, Hiroji Iwata, Zefei Jiang, Anil Abraham Joy, Vincent Haddad, Andrew Walding, Manuel Selvi Miralles, Cynthia Huang Bartlett, Antonio Llombart-Cussac. SERENA-4: A Phase III comparison of AZD9833 (camizestrant) plus palbociclib, versus anastrozole plus palbociclib, for patients with ER-positive/HER2-negative advanced breast cancer who have not previously received systemic treatment for advanced disease [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-11-06.

NAD+ has long been an important molecule in cell metabolism for its redox activity in processes such as glycolysis, ATP production and lipid oxidation. In recent years NAD+ has also been recognised as an important substrate for enzymes including PARPs, sirtuins and ADP-ribosylases such as CD38 and CD157. As a result, NAD+ has been hailed as a key molecule in maintaining cell survival, and its concentrations directly impact key cellular processes such as cell metabolism, mitochondrial biogenesis, DNA damage repair, Ca2+signalling and lifespan. Given the wide array of functions mediated by NAD+, changing NAD+concentrations are often indicative of metabolic flux, with NAD+ levels consistently shown to decrease with ageing and metabolic disease, whereas exercise and caloric restriction increase cellular NAD+. The alteration of NAD+ levels via genetic manipulation, administration of precursors or inhibition of NAD+ consumers have all been shown to improve overall metabolic health and physiology across a variety of tissues and species, with increases to cellular NAD+levels increasing healthspan, extending lifespan and reversing the adverse impacts induced by high-fat feeding. The family of NMNAT enzymes involved in the final steps of NAD+biosynthesis present a unique opportunity for intervention in increasing NAD+ levels that is yet to be widely explored in the literature. Distinct subcellular localisation of NAD+ pools and NMNAT enzymes also suggest distinct functions for NAD+ in discrete intracellular organelles and sites. This thesis aimed to investigate the overexpression of NMNAT1 – the nuclear isomer of the NMNAT enzyme family and its impact on whole-body metabolism and ageing. Both whole-body and muscle-specific overexpression of NMNAT1 resulted in a significant reduction in skeletal muscle mass. Despite the marked decrease in muscle mass, NMNAT1 transgenic mice showed improved glucose tolerance, increased insulin sensitivity and uncompromised endurance capacity when challenged with exercise. These changes were also observed despite a paradoxical increase in skeletal muscle lipid content in NMNAT1 transgenic mice. NMNAT1 whole-body overexpression also showed some protective effects with regards to age-induced impairments to glucose tolerance and loss of muscle mass. These results indicate great potential for NMNAT1 in regulating whole-body metabolism in metabolically unfavourable conditions

Objectives: To investigate the relationship between magnetization transfer (MT) imaging and tissue macromolecules in high-grade serous ovarian cancer (HGSOC) and whether MT ratio (MTR) changes following neoadjuvant chemotherapy (NACT). Methods: This was a prospective observational study. 12 HGSOC patients were imaged before treatment. MTR was compared to quantified tissue histology and immunohistochemistry. For a subset of patients (n = 5), MT imaging was repeated after NACT. The Shapiro–Wilk test was used to assess for normality of data and Spearman’s rank-order or Pearson’s correlation tests were then used to compare MTR with tissue quantifications. The Wilcoxon signed-rank test was used to assess for changes in MTR after treatment. Results: Treatment-naïve tumour MTR was 21.9 ± 3.1% (mean ± S.D.). MTR had a positive correlation with cellularity, rho = 0.56 (p < 0.05) and a negative correlation with tumour volume, ρ = −0.72 (p = 0.01). MTR did not correlate with the extracellular proteins, collagen IV or laminin (p = 0.40 and p = 0.90). For those patients imaged before and after NACT, an increase in MTR was observed in each case with mean MTR 20.6 ± 3.1% (median 21.1) pre-treatment and 25.6 ± 3.4% (median 26.5) post-treatment (p = 0.06). Conclusion: In treatment-naïve HGSOC, MTR is associated with cellularity, possibly reflecting intracellular macromolecular concentration. MT may also detect the HGSOC response to NACT, however larger studies are required to validate this finding. Advances in knowledge: MTR in HGSOC is influenced by cellularity. This may be applied to assess for cell changes following treatment.

There are few detailed studies on the pathways through the human brainstem and even fewer on those through the pons. This thesis aims to address this lack of fine detail, and used ultra-high-field magnetic resonance imaging (MRI) of human and macaque brains to identify and characterise fibre tracts connecting cortical and spinal areas as they traverse through brainstem and thalamic structures. The material in this thesis is based on a unique dataset of ultra-high-field (7 Tesla – Duke and 11, 7 Tesla – Johns Hopkins) MRI scans on postmortem specimens, on which deterministic tractography has been applied based on high-angular-resolution diffusion imaging (HARDI) and subsequently higher order tensor glyph models. The first results section of the thesis (Chapter 3) maps the descending fibre bundles associated with movement. From the motor cortical areas, the fibres of the internal capsule are traced through the crus cerebri, basilar pons and pyramids in three dimensions to reveal their organisation into functional and topographic subdivisions. While human cortico-pontine, -bulbar and -spinal tracts were traditionally considered to be dispersed, or a “melange”, I show here a much more discrete and defined organisation of these descending fibre bundles. Nine descending fibre bundles are identified and their anatomical location and terminations are described. A hitherto unknown pathway at the midline of the pons has been discovered and named herein as the Stria Pontis which connects the neocortex to the pontine tegmentum. Ten transverse fibre bundles connecting the pontine nuclei to the cerebellum are also identified. The second results section (Chapter 4) analyses the sensory pathways; the dorsal column - medial lemniscus pathway, the spinothalamic tract, the spinal trigeminal tract and the trigeminothalamic tracts. The third results section (Chapter 5) analyses the dentato-rubro-thalamic tract. The mapping identifies the superior cerebellar peduncle, the patterning of the fibres within the superior cerebellar decussation, the patterning of the fibres within the red nucleus and finally the projection of the dentato-rubro-thalamic tract from the red nucleus to the ventral lateral nucleus of the thalamus. Finally, I characterised 117 already known anatomical parts, areas and structures of the brainstem and thalamus in 3D.

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodelling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. The canonical Wnt/beta-catenin signaling pathway is a potent regulator of bone formation. We sought to examine whether Wnt-targeted agents could be of therapeutic benefit to rebuild lost bone and improve bone strength in MM patients. Utilising the 5TGM1eGFP murine model of myeloma, this thesis first demonstrated that a novel anti-LRP6 antibody, which potentiates LRP6-mediated Wnt1 class signaling, prevented myeloma-induced bone disease primarily through reduced bone resorption. The greatest protection against myeloma-induced bone loss however was shown when anti-LRP6 was used in combination with the bone anabolic agent, anti-DKK1, in the 5TGM1eGFP murine model of myeloma; compared to anti-LRP6 single treatment approaches. Importantly, stimulation of the Wnt/beta-catenin signaling pathway through these therapeutic strategies does not exacerbate tumour growth, which was a concern raised previously in the literature. Moreover, this thesis established in vivo murine models of bone repair following myeloma-induced bone destruction utilising the 5T2MM and Vk*Myc cell lines. Improving upon bone repair methods published recently, this thesis provides the first evidence that anti-SOST antibody treatment could repair osteolytic lesions within the 5T2MM model of myeloma. Importantly, this thesis is the first to characterise the skeletal destruction and osteolytic lesions associated with the Vk*Myc murine model of myeloma. Results arising from this thesis will define novel therapeutic strategies that could be beneficial to address the skeletal complications associated with MM. Murine models of bone repair established in this investigation will provide opportunities for the field to explore the potential for these Wnt-targeted agents to repair bone following cancer-induced bone destruction.

Both smoking and low back pain (LBP) are common conditions worldwide. Although tobacco smoking has enormous negative health and economic impacts, over 1.1 billion people worldwide continue to smoke. In this thesis, we built a correlation and revealed the detailed mechanisms between cigarette smoking and low back pain, especially intervertebral disc (IVD) degeneration. A prospective observational study was conducted to evaluate the clinical outcomes in 56 discectomy patients who are smokers, former smokers, or never smokers. Tightly controlled amounts of cigarette smoke were delivered to the airways of mice and the development of spinal pain and IVD degeneration was assessed by micro-CT, differentiation assay, proteomic analysis, and histology. These models showed hallmark features of mast cell (MC) activation. In vivo and in vitro studies evaluated the role of MCs tryptase in IVDD pathogenesis. MCs tryptase potential targeting for nucleus pulposus cells was examined using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq). The MCs tryptase regulated RNA stability, and binding to target genes were assessed by RNA stability assay and RNA immunoprecipitation assays. Current smoking status showed poor ODI benefit compared with never smokers when discectomy was conducted. The in vivo study showed smoking exposure can induce disc degeneration in mouse models. Smoking exposure can induce mast cell activation and tryptase release inside the disc tissues. Tryptase-beta promotes nucleus pulposus (NP) degeneration via N6-Methyladenosine (m6A)-mediated upregulation of DIX Domain Containing 1 (DIXDC1), which is an m6A “writer” protein, METTL14, regulated. The upregulated DIXDC1 was revealed to impact matrix degradation and NP cell senescence. Overall, this thesis provides us with knowledge of the smoking related IVD degeneration, providing us with the mechanical basis for the development of potential therapeutic methods.

3D bioprinting is an emerging technology in the tissue engineering field to treat or replace damaged body tissues and organs. Bioink is the printable material that is required for 3D bioprinting of structures that may contain cells and biological components. Designing a bioink remains a challenge as it needs to be viscous enough for printing, however it needs to be compatible with cells and biological components. The aim of this study is to develop a conducting bioink for 3D bioprinting applications. Thus, I need to develop a viscous mixture based on not only cell growth media to support the cells but also conductive components to provide conductivity. I designed the bioink based on nanocellulose, due to its excellent properties such as its highly elongated structure and biocompatibility which can provide a desirable structure for 3D bioprinting. Moreover, nanocellulose is obtained from renewable sources. In the first chapter of this thesis, I explored if it is possible to stabilize the bioink via photopolymerization considering the fact that it needs to contain cell growth media to keep the cells alive. This investigation showed that photopolymerization of bioinks is not completely controllable in the presence of cell culture media as the media components can interfere with photopolymerization processes through different pathways such as radical chain transfer and radical scavenging effects. Therefore, in the second chapter, I explored a combination of photocrosslinking and ionic crosslinking to obtain suitable viscous bioinks for 3D printing. A bioink based on nanocellulose and poly (ethylene glycol) was developed and optimized for 3D bioprinting by adjusting the physical crosslinking of nanocellulose with CaCl2 and chemical crosslinking of poly (ethylene glycol) under visible light irradiation. The study of physical, mechanical, rheological, and cellular interactions of the designed bioink revealed that the developed bioink has a high potential for various regenerative medicine applications. In the last chapter, I investigated if it is possible to blend conducting material into the bioink and achieve alignment in the material to improve its directional conductivity. This feature would have the potential for future applications in anisotropic tissue engineering such as neural and cardiac regeneration.

Cytomegalovirus (CMV) breakthrough despite therapy with ganciclovir remains a problem, particularly for solid organ transplant (SOT) patients. This could be owing to inadequate ganciclovir blood concentrations, and it is unclear whether monitoring of this is of clinical use. The first study aim was to verify the validity of ganciclovir blood concentration monitoring with daily area-under-curve (AUC24) as a predictive marker for breakthrough CMV. Systematic review of 610 SOT recipients across 11 studies evaluated the association between ganciclovir AUC24 and CMV incidence. Despite dose adjustments for creatinine clearance (CrCL), AUC24 for patients varied from 28-53.7 µg·h/mL. The incidence of CMV infection ranged from 0-50%, while disease ranged from 0-3.1%. One study showed a reduced risk of viraemia when AUC24 was 40-50 µg·h/mL. This formed the basis for a prospective 2-year study on 50 kidney transplant recipients monitored for ganciclovir AUC24 and CMV infection and genotypic resistance markers. Doses given to patients with CrCL

Type 2 diabetes is a major global health issue. It is projected that by 2045, 783 million people worldwide will be living with diabetes, making it one of the leading contributors to premature death globally. Approximately 30 to 40% of individuals with diabetes develop chronic kidney disease (CKD), making diabetes the leading cause of CKD worldwide. Despite glucose, blood pressure and lipid lowering, and treatment with renin-angiotensin system (RAS) blockade, the risk of cardiovascular events, kidney failure and death remains high for millions of people with diabetes and CKD worldwide. Originally developed to lower blood glucose, sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to have favourable effects on multiple metabolic risk factors including blood pressure, glucose, body weight and albuminuria. Large, randomized trials, including those reported herein, have demonstrated the capacity of these agents to reduce the risk of cardiovascular events, kidney failure and extend survival in increasingly diverse populations, including those without diabetes. This doctorate aims to evaluate the efficacy and safety of SGLT2 inhibitors in people with type 2 diabetes and CKD. Chapter 1 introduces the doctorate, summarizing the epidemiology of type 2 diabetes and CKD, as well as traditional approaches to improving outcomes in this population and the potential role for SGLT2 inhibition in people with diabetes. Chapter 2 provides an overview of SGLT2 inhibitors, practical considerations and evidence for their use in people with type 2 diabetes from cardiovascular outcome trials. Chapters 3 to 5 assess the efficacy and safety of the SGLT2 inhibitor, canagliflozin, across different levels of kidney function, defined by estimated glomerular filtration rate (eGFR) and albuminuria, using data from the CANVAS Program. Whilst the relative effects of canagliflozin on cardiovascular and kidney outcomes are consistent across different levels of eGFR and albuminuria, absolute risk reductions are largest for individuals with severely increased albuminuria. Further, the Kidney Disease Improving Global Outcomes classification of CKD, which combines eGFR and albuminuria to risk stratify individuals, can accurately identify those who are likely to derive the greatest absolute benefits with treatment. In Chapter 6, the results of a systematic review and meta-analysis are presented, which demonstrate that for people with type 2 diabetes, SGLT2 inhibitors substantially reduce the risk of the most important patient-centred kidney outcome – the need for dialysis, kidney transplantation or death due to kidney disease – and provide protection against acute kidney injury. The results of a meta-analysis are presented in Chapter 7, indicating that the benefits of SGLT2 inhibitors are similar with and without metformin, which is widely recommended as first-line glucose lowering therapy in type 2 diabetes. In Chapter 8, an individual participant data meta-analysis demonstrated that SGLT2 inhibitors reduce the risk of serious hyperkalaemia in people with type 2 diabetes at high cardiovascular risk and/or with CKD, which may enable better use of RAS blockade and mineralocorticoid receptor antagonists to improve cardiorenal outcomes. Chapter 9 explores questions about this class of agent that remain to be answered by ongoing randomized trials, and how SGLT2 inhibitors and other kidney protective therapies might be used for people with CKD in the future. Taken together, the findings of this doctorate provide compelling evidence that SGLT2 inhibitors should be routinely offered to individual with type 2 diabetes to safely reduce the risk of major kidney outcomes and cardiovascular events.

Background: Simplified and affordable HCV diagnostic pathways that incorporate innovative collection and testing methods are required to globally scale up HCV elimination efforts. This is especially applicable among highly marginalised populations. Aims: The aim of this research was to evaluate the performance of emerging diagnostic technologies with novel sample types such as dried blood spots (DBS) and finger-stick samples in clinical and decentralised settings. The specific aims include 1) Assessing performance of the Aptima HCV Quant Dx real-time assay for HCV RNA detection using DBS (from a laboratory and cohort collected population), 2) Evaluating performance of HCV core antigen (HCVcAg) detection in plasma and DBS, and 3) Performing a systematic review of point-of-care and DBS HCV RNA testing. Methods: In Chapter Two, HCV RNA detection from retrospective paired plasma and laboratory spotted DBS was compared on the Aptima assay. The same evaluation was performed in Chapter Three, however paired finger-stick capillary DBS and venepuncture collected samples obtained through the LiveRlife study (observational cohort study) were tested. In Chapter Four, detection of HCVcAg from plasma and DBS samples (LiveRlife study) was evaluated on the ARCHITECT-i2000R analyser. For Chapters Two, Three and Four data were analysed for assay sensitivity, specificity, bias and agreement and participant characteristics (Chapters Three and Four only) using statistical methods including paired T-test, Deming correlation, Bland-Altman, data distribution and summary statistics. In Chapter Five, bibliographic databases and conference presentations were searched for eligible studies. Meta-analysis was used to pool estimates. Key Findings: Laboratory spotted and clinically collected DBS samples tested on the Aptima assay displayed good sensitivity, specificity, and correlation to plasma for the detection of HCV RNA. HCVcAg testing in plasma may be an affordable tool for determining active HCV infection with the potential to reflex test from a positive anti-HCV antibody result. HCVcAg DBS testing showed lower sensitivity but may be suitable for screening high-risk populations in countries with medium to high (1.5-3.5% and >3.5%) HCV prevalence. Results from our systematic review showed excellent diagnostic performance among assays that detect HCV RNA from finger-stick whole blood and DBS samples. However, the proportion of invalid results following Xpert HCV Viral Load Finger-stick testing highlights the importance of operator training and quality assurance programs. Conclusion: Novel collection and testing methods, including DBS and finger-stick point-of-care are suitable diagnostic alternatives to standard pathology testing. A targeted testing approach that considers the setting and population could facilitate enhanced HCV testing, linkage to care and treatment.

Introduction: Weight loss through behavioural weight management interventions can have important health benefits for people with obesity. However, to maximise the health benefits, weight lost must be maintained. Evidence suggests that behavioural weight loss interventions do not exacerbate inequalities in the short-term. However, no study has yet considered whether inequalities exist in long-term weight change following intervention. We aimed to investigate if there are inequalities in weight change following weight loss intervention. Methods: We conducted a cohort analysis of data from the Weight Loss Referrals for Adults in Primary Care (WRAP) trial (N=1267). WRAP randomised participants to receive a brief intervention information booklet or vouchers for 12-weeks or 52-weeks of WW (formerly Weight Watchers) and followed them for 5-years. Multiple linear regression estimated the association between exposures (indicators of inequality) and outcome (change in weight between 1- and 5-years). Each model was adjusted for intervention group, baseline weight, weight change between baseline and 1-year, research centre, and source of the 5-year weight data. Results: Of the 1267 participants in WRAP, 708 had weight change data available. Mean weight change between 1- and 5-years was +3.30 kg (SD 9.10 kg). A 1 year difference in age at baseline was associated with weight change of 0.11kg ((95%CI 0.06, 0.16), p

Heart transplantation remains the gold standard treatment for end stage heart failure, however its use remains limited by a paucity of available donor hearts, and its efficacy by primary graft failure (PGF), the most common cause of mortality following heart transplantation. Research in the area of myocardial preservation is aimed at reducing the incidence and severity of this morbid complication. This thesis presents a series of experimental studies investigating a novel cardiac allograft preservation strategy, the use of Hi1a, an ASIC1a inhibitor as a pharmacological conditioning agent. This is followed by a large animal model aimed at investigating the effect of antemortem heparin administration on allograft recovery following donation after circulatory death donation (DCD). The final three chapters describe the investigation and results of studies aimed at further elucidating the unique pathophysiology of donation and preservation of DCD allografts with a view to increasing their utilisation as well as decreasing rates of PGD in this cohort. Isolated working rat hearts were arrested with Celsior and subjected to prolonged hypothermic storage. Treatment with Celsior supplemented with 10nM Hi1a, an ASIC1a inhibitor, resulted in significantly improved recovery of aortic flow and cardiac output. Furthermore, the cardioprotection afforded by Hi1a as a single supplement was equivalent to that of our previously identified pharmacological conditioning agents, glyceryl trinitrate (GTN) and zoniporide. Improved functional recovery afforded by Hi1a in this model appeared to be associated with upregulation of pAkt. This agent was then investigated in a post-conditioning model of ischemia reperfusion. Treatment with Celsior supplemented with 10 nM Hi1a resulted in significant improvement in recovery of aortic flow, cardiac output and pulse pressure following a twelve-minute period of in-situ warm ischemia and 60 minutes of subsequent ex situ reperfusion. Additionally, combination of Hi1a with the clinical conditioning agents GTN and erythropoietin (EPO) resulted in significantly improved recovery of aortic flow, compared to Hi1a supplementation alone. The background and methodology for a porcine study investigating the benefits of antemortem heparin in DCD cardiac donation was then described. Unfortunately, the studies outlined in their chapter could not be completed due to the COVID-19 pandemic. The next three chapters identify and investigate developments in our understanding of the unique pathophysiology of DCD allograft donation and preservation. A limited clinical series found that withdrawal of life support (WLS) in DCD donors was at times associated with significant hyperactivation of platelet function, an effect which was suppressed through the administration of the GPIIb/IIIa receptor inhibitor, tirofiban. Detailed analysis of clinical data and outcomes from our DCD cohort revealed an associated between prolonged asystolic warm ischemic time (aWIT) and increased rates of severe primary graft dysfunction (sPGD). Counterintuitively, a longer time from WLS to cessation of circulation was associated with a lower rate of sPGD. Finally, we demonstrated that the adoption of pressure-controlled ex situ perfusion targets was associated with a significant reduced in the prevalence of sPGD in DCD heart transplant recipients, from 36% to 4%. This thesis presents a novel pharmacological conditioning agent for allograft preservation, with shows promise for application in human heart transplantation. In addition, our understanding of the unique pathophysiology of the donation and preservation of DCD allografts has been further through the assessment of platelet activation following WLS, associations between patterns of deterioration following WLS and sPGD, and assessment of novel ex situ perfusion strategies and their association with a reduction in rates of sPGD in our clinical DCD heart transplant recipients. In combination, these findings have the potential to further increase the number of cardiac allografts available for transplantation as well as to improve early post-transplant outcomes.

Prostate cancer (PCa) is the most common cancer in men worldwide with higher prevalence in the developed countries. Current screening and diagnostic approaches cannot accurately detect PCa in the early stage and stratify the risk groups to guide clinicians for the best treatment option. Extracellular vesicles (EVs) secreted from all kinds of cells contain proteins, nucleic acids and metabolites and play an important role in intercellular communication. EVs hold promise for PCa early diagnosis and progression monitoring. The goals of my PhD study were to: 1) evaluate the EVs isolated from PCa cell lines with immunoaffinity and aldehyde magnetic beads capturing, and investigate the performance of self-conjugated ApoB beads for lipoprotein quantification; 2) investigate the characteristics of plasma-derived EVs with commercial isolation kits combined with magnetic beads for clinical assignment; 3) discover novel biomarkers in a panel of PCa cell lines, and then validate these identified potential markers in plasma and urine of PCa patients for predicting the presence of “clinically significant cancer” and the prognostic risk groups. My findings indicate that magnetic beads can pull down EVs easily and immunoaffinity beads approach, based on biochemical composition by using common EV markers, is obviously better than the aldehyde beads for EV isolation and quantification with flow cytometry. Furthermore, the study of combination of three commercial kits with magnetic beads capturing established a systematic assessment of the EV quality, demonstrating that beads combining with size exclusion chromatography are superior to that combining with precipitation kit for EV isolation. Moreover, my proteomics analysis of PCa cell lines identified a total of 20 new EV protein biomarkers such as Kindlin3, LAMB1, Histone H4 et al. These EV markers are associated with cancer invasion, migration, regulation, tumour microenvironment and metastasis. My validation results show the unique expression patterns of these markers, in EVs of plasma and urine samples from different stages of PCa patients, are promising and demonstrate the potential value for PCa diagnosis and prognosis. In summary, my PhD studies have established unique EV isolation methods for clinical EV application and discovered a panel of novel EV biomarkers for PCa diagnosis and stage stratification, holding promise for personalised medicine.

Secreted by pancreatic β-cells, insulin is the major anabolic hormone, regulating the metabolism of fats, proteins, and carbohydrates. Defects in insulin production or action can lead to diabetes characterized by derangements in glucose handling and metabolic disease. Diabetes affects 420 million people worldwide, increasing morbidity, mortality and placing a burden on healthcare of nations. There is a need for rapid and accurate monitoring of insulin levels to optimize diabetes management and facilitate early diagnosis of insulin related chronic diseases. Conventional strategies such as HPLC, MALDI-TOF, ELISA, etc. used for insulin detection are not suitable for point-of-care testing (POCT) as they are expensive, and require sample preparation, sophisticated instruments, and skilled personnel. Our goal was to develop techniques to allow POCT for insulin in real time. In this study we developed two lateral flow assays (LFAs) based POCT platforms using aptamers as the biorecognition molecules for colorimetric and fluorescent detection of insulin. A range of conditions were tested such as concentrations of aptamers, reporter molecules used, volume of sample required, and assay time to obtain quantify insulin levels using a standard LFA reader. The colorimetric LFAs had linear detection range of 0.01-1 ng.mL-1 and LOD of 0.01 ng.mL-1. The fluorescent LFAs exhibited a linear detection range of 0-4 ng.mL-1 and 0.1 ng.mL-1 LOD. Various signal amplification strategies were incorporated, ie., gold-silver amplification technique and rolling circular amplification (RCA) to further enhance the signal. The developed colorimetric LFAs were successfully used for insulin quantification in rat blood, human blood, and human saliva samples. Although insulin levels were quantified within 12 min, some issues arose such as coagulation, need for dilution, and non-uniform flow through the test strips. Further work is required to optimize blood handling to progress an insulin POCT in real time. Future work could develop a multiplexed strip for detection of different analytes such as HbA1c, glucose, and C-peptide for better management of diabetes, along with a smartphone reader App. This research goes some way to addressing the challenge of providing a reliable and rapid approach for highly sensitive and specific detection of insulin for POCT applications.

Background: Ocular surface characteristics in children have not been as well investigated as in adults. Children’s digital device usage is rapidly increasing, and smartphones are the most commonly used device. The ocular surface impacts are unknown in children. In adults, use of digital devices induces ocular symptoms, adversely impacts blinking, and disrupts ocular surface homeostasis. Although blinking is integral to a healthy ocular surface, this is yet to be characterised in children, including the effects of digital devices. This thesis aims to characterise the ocular surface of children including blinking and to examine the impact of smartphone use on ocular surface homeostasis in children. Methods: The literature on ocular surface (symptoms, clinical indices, tear film function, blinking) of healthy children was reviewed and a meta-analysis of tear film stability and tear secretion was conducted. Cross-sectional studies of healthy school-aged children were conducted to examine the utility of commonly used adult-validated dry eye symptom questionnaires (SANDE, OSDI, NRS, OCI, DEQ-5, IOSS), and to characterise ocular surface clinical indices. This included blinking which was measured in situ using a novel eye tracking headset (Pupil Labs GmbH, Germany). The impact of one hour smartphone gaming on the ocular surface (including symptoms and blink parameters) of this paediatric population was examined with an intervention study. Blinking was also examined in situ under different conditions and tasks (reading from hard copy and on digital devices, conversation, walking) using the eye tracking headset in healthy adults. Repeatability of blink measurements (blink rate and interblink interval) in adults using the eye tracking headset was determined. Results: Ocular symptoms, tear film function and blinking were sparsely reported in children. The pooled mean by tear stability measurement methods in the meta-analysis were higher than previously reported in healthy adults while the pooled mean for tear secretion by methods were within the expected normal range for adults. Six existing dry eye questionnaires could be successfully used in paediatric eye care, and their repeatability was mostly comparable to that reported previously in adults. More time and assistance were at times required for younger children and specific terms such as ‘gritty’ and ‘foreign body sensation’ were not always well understood by younger children. The DEQ-5 and IOSS are recommended for use in younger age children. Blinking was associated with greater tear volume and worse meibomian gland expressibility but not digital device use, age, sex, or symptoms in children. One hour smartphone gaming led to increased symptoms of dryness, discomfort, and tiredness but did not impact tear film function. Blinking was rapidly reduced by a third within the first minute of gaming and this effect remained unchanged throughout one hour of gaming (p

Inhibiting the androgen receptor (AR), a ligand-activated transcription factor, with androgen deprivation therapy is a standard-of-care treatment for metastatic prostate cancer. Paradoxically, activation of AR can also inhibit the growth of prostate cancer in some patients and experimental systems, but the mechanisms underlying this phenomenon are poorly understood. This study exploited a potent synthetic androgen, methyltestosterone (MeT), to investigate AR agonist-induced growth inhibition. MeT strongly inhibited growth of prostate cancer cells expressing AR, but not AR-negative models. Genes and pathways regulated by MeT were highly analogous to those regulated by DHT, although MeT induced a quantitatively greater androgenic response in prostate cancer cells. MeT potently downregulated DNA methyltransferases, leading to global DNA hypomethylation. These epigenomic changes were associated with dysregulation of transposable element expression, including upregulation of endogenous retrovirus (ERV) transcripts after sustained MeT treatment. Increased ERV expression led to accumulation of double-stranded RNA and a “viral mimicry” response characterized by activation of IFN signaling, upregulation of MHC class I molecules, and enhanced recognition of murine prostate cancer cells by CD8+ T cells. Positive associations between AR activity and ERVs/antiviral pathways were evident in patient transcriptomic data, supporting the clinical relevance of our findings. Collectively, our study reveals that the potent androgen MeT can increase the immunogenicity of prostate cancer cells via a viral mimicry response, a finding that has potential implications for the development of strategies to sensitize this cancer type to immunotherapies. Significance: Our study demonstrates that potent androgen stimulation of prostate cancer cells can elicit a viral mimicry response, resulting in enhanced IFN signaling. This finding may have implications for the development of strategies to sensitize prostate cancer to immunotherapies.

Among emerging technologies developed to interface neuronal signaling, engineering electrodes at the nanoscale would yield more precise biodevices opening to progress in neural circuit investigations and to new therapeutic potential. Despite remarkable progress in miniature electronics for less invasive neurostimulation, most nano-enabled, optically triggered interfaces are demonstrated in cultured cells, which precludes the studies of natural neural circuits. We exploit here free-standing silicon-based nanoscale photodiodes to optically modulate single, identified neurons in mammalian spinal cord explants. With near-infrared light stimulation, we show that activating single excitatory or inhibitory neurons differently affects sensory circuits processing in the dorsal horn. We successfully functionalize nano-photodiodes to target single molecules, such as glutamate AMPA receptor subunits, thus enabling light activation of specific synaptic pathways. We conclude that nano-enabled neural interfaces can modulate selected sensory networks with low invasiveness. The use of nanoscale photodiodes can thus provide original perspective in linking neural activity to specific behavioral outcome. We thank M. Gigante, G. Cellot, S. Mortal, and M. Lazzarino for help with setting up the laser stimulation setup; the group of P. Heppenstall for help with the AAV production; M. Giugliano for MATLAB code optimization; R. Owen for suggestions in antibody nPD characterization; and S. Usmani for help with initial experiments. This project was funded by HFSP program grant RGP0004/2019 (to L.B., B.T., and L.F.).

Nanocatalytic tumor therapies involve established strategies to increase the concentration of endogenous oxygen species (ROS) H2O2 to cytotoxic levels. These strategies are based on increasing the ROS levels through stimuli from drugs, the action of ROS-producing agents, and nanoparticulate catalysis. However, these techniques frequently are indiscriminatory, being cytotoxic to diseased cells and normal cells alike, leading to significant unwanted side-effects. The present work reports a new paradigm strategy based upon the catalytic action of a cell-discriminative, ROS-mediating, autophagy-suppressive nanoparticle, which is CePO4·H2O (rhabdophane). CePO4·H2O nanoparticles were synthesised using CeNO3·6H2O precipitated in an aqueous solution of sodium tripolyphosphate (STPP) at room temperature. The nanoparticles were well crystallised, equiaxed (~10-35 nm), of positive surface charge, and of general valence ratio 〖"Ce" 〗_"0.8" ^"3+" 〖"Ce" 〗_"0.2" ^"4+" 〖"PO" 〗_"4.1" . Materials characterisation involved particuological (hydrodynamic particle size, surface area, zeta potential), mineralogical (X-ray diffraction, laser Raman microspectroscopy), chemical (X-ray photoelectron spectroscopy), structural (Fourier transform infrared spectroscopy), and microstructural (transmission electron microscopy) analyses. Biological characterisation involved examination of the effects on HT-1080 fibrosarcoma cells and MRC-5 normal fibroblasts in terms of cellular interactions (cell viability by MTT assay), cellular uptake and trafficking (confocal laser scanning microscopy, biological transmission electron microscopy, flow cytometry), ROS generation (confocal laser scanning microscopy, flow cytometry), apoptosis (annexin V-FITC assay), gene expression (q-RT-PRC), and protein expression (western blot analyses). The key observations and conclusions from the biological evaluation are as follows: Discriminative Cytotoxicity: CePO4·H2O nanoparticles are the first to exhibit discriminative cytotoxicity: At 24 h, fibrosarcoma HT-1080 cell viability is ~10% but MRC-5 normal cell viability is ~45%. Discriminative Uptake: CePO4·H2O nanoparticles are the first, without the use of a targeting ligand, to be internalized readily by cancer cells but scarcely by normal cells. Self-Targeting: CePO4·H2O nanoparticles are trafficked toward the mitochondrial environment and possibly the converse trafficking. Mitochondrial Starvation: The preceding proximity between CePO4·H2O nanoparticles and cancer cell leads to increased phosphate concentration in the cellular environment, the concentration gradient of which effectively starves the mitochondria, leading to mitochondrial stress and dysfunction. Discriminative ROS Generation: CePO4·H2O nanoparticles are the first to demonstrate elevated cellular ROS in cancer cells by multiple mechanisms while normal cells exhibit only a low level of such elevation. Autophagy Suppression: CePO4·H2O nanoparticles suppress autophagy, thereby increasing cellular stress and suppressing cancer cell survival, thus offering a complement to mitochondrial starvation. Redox Switching: CePO4·H2O nanoparticles are the first nonmetallic nanoparticles to balance redox switching through simple electronic charge compensation rather than more complex ionic charge compensation. Biocompatibility: As hydrated phosphates, CePO4·H2O nanoparticles are more biocompatible than metals or oxides, suggesting greater feasibility of renal clearance. These advantages derive from the key role of the redox and defect equilibria arising from the oxidation reaction Ce3+ → Ce4+ + e′, which is induced by the acidic pH environment of the cancer call versus the stability of the Ce3+ valence in the basic pH environment of the normal cell. The former both elevates the ROS level and disrupts the electron transfer chain. Ultimately, the suppression of the proliferation of cancer cells derives from the cross-talk involving cellular ROS elevation, autophagy suppression, and their mitochondrial control.