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The legibility of the letters in the Latin alphabet has been measured numerous times since the beginning of experimental psychology. To identify the theoretical mechanisms attributed to letter identification, we report a comprehensive review of literature, spanning more than a century. This review revealed that identification accuracy has frequently been attributed to a subset of three common sources: perceivability, bias, and simi- larity. However, simultaneous estimates of these values have rarely (if ever) been performed. We present the results of two new experiments which allow for the simultaneous estimation of these factors, and examine how the shape of a visual mask impacts each of them, as inferred through a new statistical model. Results showed that the shape and identity of the mask impacted the inferred perceivability, bias, and similarity space of a letter set, but that there were aspects of similarity that were robust to the choice of mask. The results illustrate how the psychological concepts of perceivability, bias, and similarity can be estimated simultaneously, and how each make powerful contributions to visual letter identification.

Live-cell imaging using fluorescence microscopy enables researchers to study cellular processes in unprecedented detail. These techniques are becoming increasingly popular among microbiologists. The emergence of microfluidics and deep learning has significantly increased the amount of quantitative data that can be extracted from such experiments. However, these techniques require highly specialized expertise and equipment, making them inaccessible to many biologists. Here we present a guide for microbiologists, with a basic understanding of microfluidics, to construct a custom-made live-cell interrogation system that is capable of recording and analyzing thousands of bacterial cell-cycles per experiment. The requirements for different microbiological applications are varied, and experiments often demand a high level of versatility and custom-designed capabilities. This work is intended as a guide for the design and engineering of microfluidic master molds and how to build polydimethylsiloxane chips. Furthermore, we show how state-of-the-art deep-learning techniques can be used to design image processing algorithms that allow for the rapid extraction of highly quantitative information from large populations of individual bacterial cells.

Genome maintenance comprises a group of complex and interrelated processes crucial for preserving and safeguarding genetic information within all organisms. Key aspects of genome maintenance involve DNA replication, transcription, recombination, and repair. Improper regulation of these processes could cause genetic changes, potentially leading to antibiotic resistance in bacterial populations. Due to the complexity of these processes, ensemble averaging studies may not provide the level of detail required to capture the full spectrum of molecular behaviors and dynamics of each individual biomolecule. Therefore, researchers have increasingly turned to single-molecule approaches, as these techniques allow for the direct observation and manipulation of individual biomolecules, and offer a level of detail that is unattainable with traditional ensemble methods. In this review, we provide an overview of recent in vitro and in vivo single-molecule imaging approaches employed to study the complex processes involved in prokaryotic genome maintenance. We will first highlight the principles of imaging techniques such as total internal reflection fluorescence microscopy and atomic force microscopy, primarily used for in vitro studies, and highly inclined and laminated optical sheet and super-resolution microscopy, mainly employed in in vivo studies. We then demonstrate how applying these single-molecule techniques has enabled the direct visualization of biological processes such as replication, transcription, DNA repair, and recombination in real time. Finally, we will showcase the results obtained from super-resolution microscopy approaches, which have provided unprecedented insights into the spatial organization of different biomolecules within bacterial organisms., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Co-published by Nanjing University and American Chemical Society.)

Background: Low-frequency variants play an important role in breast cancer (BC) susceptibility. Gene-based methods can increase power by combining multiple variants in the same gene and help identify target genes., Methods: We evaluated the potential of gene-based aggregation in the Breast Cancer Association Consortium cohorts including 83,471 cases and 59,199 controls. Low-frequency variants were aggregated for individual genes' coding and regulatory regions. Association results in European ancestry samples were compared to single-marker association results in the same cohort. Gene-based associations were also combined in meta-analysis across individuals with European, Asian, African, and Latin American and Hispanic ancestry., Results: In European ancestry samples, 14 genes were significantly associated (q < 0.05) with BC. Of those, two genes, FMNL3 (P = 6.11 × 10 -6 ) and AC058822.1 (P = 1.47 × 10 -4 ), represent new associations. High FMNL3 expression has previously been linked to poor prognosis in several other cancers. Meta-analysis of samples with diverse ancestry discovered further associations including established candidate genes ESR1 and CBLB. Furthermore, literature review and database query found further support for a biologically plausible link with cancer for genes CBLB, FMNL3, FGFR2, LSP1, MAP3K1, and SRGAP2C., Conclusions: Using extended gene-based aggregation tests including coding and regulatory variation, we report identification of plausible target genes for previously identified single-marker associations with BC as well as the discovery of novel genes implicated in BC development. Including multi ancestral cohorts in this study enabled the identification of otherwise missed disease associations as ESR1 (P = 1.31 × 10 -5 ), demonstrating the importance of diversifying study cohorts., (© 2023. The Author(s).)

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5'-, but not 3'-ends of biotinylated DNA., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Randomised controlled trials (RCTs) inform prescription guidelines, but stringent eligibility criteria exclude individuals with vulnerable characteristics, which we define as comorbidities, concomitant medication use, and vulnerabilities due to age. Poor external validity can result in inadequate treatment decision information. Our first aim was to quantify the extent of exclusion of individuals with vulnerable characteristics from RCTs for all prescription drugs. Our second aim was to quantify the prevalence of individuals with vulnerable characteristics from population electronic health records who are actively prescribed such drugs. In tandem, these two aims will allow us to assess the representativeness between RCT and real-world populations and identify vulnerable populations potentially at risk of inadequate treatment decision information. When a vulnerable population is highly excluded from RCTs but has a high prevalence of individuals actively being prescribed the same medication, there is likely to be a gap in treatment decision information. Our third aim was to investigate the use of real-world evidence in contributing towards quantifying missing treatment risk or benefit through an observational study., Methods: We extracted RCTs from ClinicalTrials.gov from its inception to April 28, 2021, and primary care records from the Clinical Practice Research Datalink Gold database from Jan 1, 1998, to Dec 31, 2020. We referred to the British National Formulary to classify prescription drugs into drug categories. We conducted descriptive analyses and quantified RCT exclusion and prevalence of individuals with vulnerable characteristics for comparison to identify populations without treatment decision information. Exclusion and prevalence were assessed separately for different age groups, individual clinical specialities, and for quantities of concomitant conditions by clinical specialities, where multimorbidity was defined as having two or more clinical specialties, and medications prescribed, where polypharmacy was defined as having five or more medications prescribed. Population trends of individuals with multimorbidity or polypharmacy were assessed separately by age group. We conducted an observational cohort study to validate the use of real-world evidence in contributing towards quantifying treatment risk or benefit for patients with dementia on anti-dementia drugs with and without a contraindicated clinical speciality. To do so, we identified the clinical specialities that anti-dementia drug RCTs highly excluded yet had corresponding high prevalence in the real-world population, forming the groups with highest risk of having scarce treatment decision information. Cox regression was used to assess if the risk of mortality outcomes differs between both groups., Findings: 43 895 RCTs from ClinicalTrials.gov and 5 685 738 million individuals from primary care records were used. We considered 989 unique drugs and 286 conditions across 13 drug-category cohorts. For the descriptive analyses, the median RCT exclusion proportion across 13 drug categories was 81·5% (IQR 76·7-85·5) for adolescents (aged <18 years), 26·3% (IQR 21·0-29·5) for individuals older than 60 years, 40·5% (IQR 33·7-43·0) for individuals older than 70 years, and 52·9% (IQR 47·1-56·0) for individuals older than 80 years. Multimorbidity had a median exclusion proportion of 91·1% (IQR 88·9-91·8) and median prevalence of 41·0% (IQR 34·9-46·0). Concomitant medication use had a median exclusion proportion of 52·5% (IQR 50·0-53·7) and a median prevalence of 94·3% (IQR 84·3-97·2), and polypharmacy had a median prevalence of 47·7% (IQR 38·0-56·1). Population trends show increasing multimorbidity with age and consistently high polypharmacy across age groups. Populations with cardiovascular or otorhinolaryngological comorbidities had the highest risk of having scarce treatment decision information. For the observational study, populations with cardiovascular or psychiatric comorbidities had highest risk of having scarce treatment decision information. Patients with dementia with an anti-dementia prescription and contraindicated cardiovascular condition had a higher risk of mortality (hazard ratio [HR] 1·20 [95% CI 1·13-1·28 ; p<0·0001]) compared with patients with dementia without a contraindicated cardiovascular condition. Patients with dementia with comorbid delirium (HR 1·25 [95% CI 1·06-1·48]; p<0·0088), intellectual disability (HR 2·72 [95% CI 1·53-4·81]; p=0·0006), and schizophrenia and schizotypal delusional disorders (HR 1·36 [95% CI 1·02-1·82]; p=0·036) had a higher risk of mortality compared with patients with dementia without these conditions., Interpretation: Overly stringent RCT exclusion criteria do not appropriately account for the heterogeneity of vulnerable characteristics observed in real-world populations. Treatment decision information is scarce for such individuals, which might affect health outcomes. We discuss the challenges facing the inclusivity of such individuals and highlight the strength of real-world evidence as an integrative solution in complementing RCTs and increasing the completeness of evidence-based medicine assessments in evaluating the effectiveness of treatment decisions., Funding: Wellcome Trust, National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre, NIHR Great Ormond Street Hospital Biomedical Research Centre, Academy of Medical Sciences, and the University College London Overseas Research Scholarship., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Reduced provision of protein translation machinery promotes healthy aging in a number of animal models. In humans, however, inborn impairments in translation machinery are a known cause of several developmental disorders, collectively termed ribosomopathies. Here, we use casual inference approaches in genetic epidemiology to investigate whether adult, tissue-specific biogenesis of translation machinery drives human aging. We assess naturally occurring variation in the expression of genes encoding subunits specific to the two RNA polymerases (Pols) that transcribe ribosomal and transfer RNAs, namely Pol I and III, and the variation in expression of ribosomal protein (RP) genes, using Mendelian randomization. We find each causally associated with human longevity (β = -0.15 ± 0.047, P = 9.6 × 10 -4 , q = 0.015; β = -0.13 ± 0.040, P = 1.4 × 10 -3 , q = 0.023; β = -0.048 ± 0.016, P = 3.5 × 10 -3 , q = 0.056, respectively), and this does not appear to be mediated by altered susceptibility to a single disease. We find that reduced expression of Pol III, RPs, or Pol I promotes longevity from different organs, namely visceral adipose, liver, and skeletal muscle, echoing the tissue specificity of ribosomopathies. Our study shows the utility of leveraging genetic variation in expression to elucidate how essential cellular processes impact human aging. The findings extend the evolutionary conservation of protein synthesis as a critical process that drives animal aging to include humans., (© 2022 Javidnia et al.; Published by Cold Spring Harbor Laboratory Press.)

Importance: Essential tremor (ET) is one of the most common movement disorders, affecting 5% of the general population older than 65 years. Common variants are thought to contribute toward susceptibility to ET, but no variants have been robustly identified., Objective: To identify common genetic factors associated with risk of ET., Design, Setting, and Participants: Case-control genome-wide association study. Inverse-variance meta-analysis was used to combine cohorts. Multicenter samples collected from European populations were collected from January 2010 to September 2019 as part of an ongoing study. Included patients were clinically diagnosed with or reported having ET. Control individuals were not diagnosed with or reported to have ET. Of 485 250 individuals, data for 483 054 passed data quality control and were used., Main Outcomes and Measures: Genotypes of common variants associated with risk of ET., Results: Of the 483 054 individuals included, there were 7177 with ET (3693 [51.46%] female; mean [SD] age, 62.66 [15.12] years), and 475 877 control individuals (253 785 [53.33%] female; mean [SD] age, 56.40 [17.6] years). Five independent genome-wide significant loci and were identified and were associated with approximately 18% of ET heritability. Functional analyses found significant enrichment in the cerebellar hemisphere, cerebellum, and axonogenesis pathways. Genetic correlation (r), which measures the degree of genetic overlap, revealed significant common variant overlap with Parkinson disease (r, 0.28; P = 2.38 × 10-8) and depression (r, 0.12; P = 9.78 × 10-4). A separate fine-mapping of transcriptome-wide association hits identified genes such as BACE2, LRRN2, DHRS13, and LINC00323 in disease-relevant brain regions, such as the cerebellum., Conclusions and Relevance: The results of this genome-wide association study suggest that a portion of ET heritability can be explained by common genetic variation and can help identify new common genetic risk factors for ET.

Background: People with liver disease are at increased risk of developing cardiovascular disease (CVD), however, there has yet been an investigation of incidence burden, risk, and premature mortality across a wide range of liver conditions and cardiovascular outcomes., Methods: We employed population-wide electronic health records (EHRs; from 1998 to 2020) consisting of almost 4 million adults to assess regional variations in disease burden of five liver conditions, alcoholic liver disease (ALD), autoimmune liver disease, chronic hepatitis B infection (HBV), chronic hepatitis C infection (HCV) and NAFLD, in England. We analysed regional differences in incidence rates for 17 manifestations of CVD in people with or without liver disease. The associations between biomarkers and comorbidities and risk of CVD in patients with liver disease were estimated using Cox models. For each liver condition, we estimated excess years of life lost (YLL) attributable to CVD (i.e., difference in YLL between people with or without CVD)., Results: The age-standardised incidence rate for any liver disease was 114.5 per 100,000 person years. The highest incidence was observed in NAFLD (85.5), followed by ALD (24.7), HCV (6.0), HBV (4.1) and autoimmune liver disease (3.7). Regionally, the North West and North East regions consistently exhibited high incidence burden. Age-specific incidence rate analyses revealed that the peak incidence for liver disease of non-viral aetiology is reached in individuals aged 50-59 years. Patients with liver disease had a two-fold higher incidence burden of CVD (2634.6 per 100,000 persons) compared to individuals without liver disease (1339.7 per 100,000 persons). When comparing across liver diseases, atrial fibrillation was the most common initial CVD presentation while hypertrophic cardiomyopathy was the least common. We noted strong positive associations between body mass index and current smoking and risk of CVD. Patients who also had diabetes, hypertension, proteinuric kidney disease, chronic kidney disease, diverticular disease and gastro-oesophageal reflex disorders had a higher risk of CVD, as do patients with low albumin, raised C-reactive protein and raised International Normalized Ratio levels. All types of CVD were associated with shorter life expectancies. When evaluating excess YLLs by age of CVD onset and by liver disease type, differences in YLLs, when comparing across CVD types, were more pronounced at younger ages., Conclusions: We developed a public online app ( https://lailab.shinyapps.io/cvd&#95;in&#95;liver&#95;disease/ ) to showcase results interactively. We provide a blueprint that revealed previously underappreciated clinical factors related to the risk of CVD, which differed in the magnitude of effects across liver diseases. We found significant geographical variations in the burden of liver disease and CVD, highlighting the need to devise local solutions. Targeted policies and regional initiatives addressing underserved communities might help improve equity of access to CVD screening and treatment., (© 2021. The Author(s).)

Background: Children, teenagers and young adults who survived cancer are prone to developing late effects. The burden of late effects across a large number of conditions, in-patient hospitalisation and critical care admissions have not been described using a population-based dataset. We aim to systematically quantify the cumulative burden of late effects across all cancer subtypes, treatment modalities and chemotherapy drug classes., Methods: We employed primary care records linked to hospitals, the death registry and cancer registry from 1998-2020. CTYA survivors were 25 years or younger at the time of cancer diagnosis had survived ≥5 years post-diagnosis. Year-of-birth and sex-matched community controls were used for comparison. We considered nine treatment types, nine chemotherapy classes and 183 physical and mental health late effects. Cumulative burden was estimated using mean cumulative count, which considers recurring events. Multivariable logistic regression was used to investigate the association between treatment exposures and late effects. Excess years of life lost (YLL) attributable to late effects were estimated., Findings: Among 4,063 patients diagnosed with cancer, 3,466 survived ≥ 5 years (85%); 13,517 matched controls were identified. The cumulative burden of late effects at age 35 was the highest in survivors of leukaemia (23.52 per individual [95% CI:19.85-29.33]) and lowest in survivors of germ cell tumours (CI:6.04 [5.32-6.91]). In controls, the cumulative burden was 3.99 (CI:3.93-4.08) at age 35 years. When survivors reach age 45, the cumulative burden for immunological conditions and infections was the highest (3.27 [CI:3.01-3.58]), followed by cardiovascular conditions (3.08 [CI:1.98-3.29]). Survivors who received chemotherapy and radiotherapy had the highest disease burden compared to those who received surgery only. These patients also had the highest burden of hospitalisation (by age 45: 10.43 [CI:8.27-11.95]). Survivors who received antimetabolite chemotherapy had the highest disease and hospitalisation burden, while the lowest burden is observed in those receiving antitumour antibiotics. Regression analyses revealed that survivors who received only surgery had lower odds of developing cardiovascular (adjusted odds ratio 0.73 [CI:0.56-0.94]), haematological (aOR 0.51 [CI:0.37-0.70]), immunology and infection (aOR 0.84 [CI:0.71-0.99]) and renal (aOR 0.51 [CI:0.39-0.66]) late effects. By contrast, the opposite trend was observed in survivors who received chemo-radiotherapy. High antimetabolite chemotherapy cumulative dose was associated with increased risks of subsequent cancer (aOR 2.32 [CI:1.06-4.84]), metastatic cancer (aOR 4.44 [CI:1.29-11.66]) and renal (aOR 3.48 [CI:1.36-7.86]) conditions. Patients who received radiation dose of ≥50 Gy experienced higher risks of developing metastatic cancer (aOR 5.51 [CI:2.21-11.86]), cancer (aOR 3.77 [CI:2.22-6.34]), haematological (aOR 3.43 [CI:1.54-6.83]) and neurological (aOR 3.24 [CI:1.78-5.66]) conditions. Similar trends were observed in survivors who received more than three teletherapy fields. Cumulative burden analyses on 183 conditions separately revealed varying dominance of different late effects across cancer types, socioeconomic deprivation and treatment modalities. Late effects are associated with excess YLL (i.e., the difference in YLL between survivors with or without late effects), which was the most pronounced among survivors with haematological comorbidities., Interpretation: To our knowledge, this is the first study to dissect and quantify the importance of late morbidities on subsequent survival using linked electronic health records from multiple settings. The burden of late effects is heterogeneous, as is the risk of premature mortality associated with late effects. We provide an extensive knowledgebase to help inform treatment decisions at the point of diagnosis, future interventional trials and late-effects screening centred on the holistic needs of this vulnerable population., Competing Interests: None declared., (© 2021 The Author(s).)

Background: Patients with liver disease have complex haemostasis and due to such contraindications, landmark randomised controlled trials investigating antithrombotic medicines have often excluded these patients. As a result, there has been limited consensus on the safety, efficacy and monitoring practices of anticoagulant and antiplatelet therapy in patients with liver disease. This study aims to investigate prescribing prevalence, adherence, persistence and impact of adherence on bleeding and stroke risk in people with and without liver disease taking anticoagulants and antiplatelets., Methods: We employed a population-based cohort consisting of person-level linked records from primary care, secondary care and the death registry. The cohort consisted of 3,929,596 adults aged ≥ 30 years during the study period of 1998 to 2020 and registered with an NHS general practitioner in England. The primary outcome was prescribing prevalence, adherence to and persistence with anticoagulant and antiplatelet therapy comparing patients with and without liver disease. Risk factors for non-adherence and non-persistence were analysed using multivariable logistic regression and Cox regression. Impact of adherence on bleeding and ischaemic stroke was assessed., Findings: Among patients with any of the six liver diseases (ALD, autoimmune liver disease, cirrhosis, HBV, HCV and NAFLD), we identified 4,237 individuals with incident atrial fibrillation (indication for anticoagulants) and 4,929 individuals with incident myocardial infarction, transient ischaemic attack, unstable angina or peripheral arterial disease (indication for antiplatelets). Among patients without liver disease, 321,510 and 386,643 individuals were identified as having indications for anticoagulant and antiplatelet therapy, respectively. Among drug-naïve individuals, prescribing prevalence was lower in patients with liver disease compared with individuals without liver disease: anticoagulants (20.6% [806/3,921] vs. 33.5% [103,222/307,877]) and antiplatelets (56.2% [2,207/3,927] vs. 71.1% [249,258/350,803]). Primary non-adherence rates (stopping after one prescription) were higher in patients with liver disease, compared with those without liver disease: anticoagulants (7.9% [64/806] vs. 4.7% [4,841/103,222]) and antiplatelets (6.2% [137/2,207] vs. 4.4% [10,993/249,258]). Among individuals who were not primary non-adherent and had at least 12 months of follow-up, patients with liver disease however had a higher one-year adherence rate: anticoagulants (33.1% [208/628] vs. 29.4% [26,615/90,569]) and antiplatelets (40.9% [743/1,818] vs. 34.4% [76,834/223,154]). Likelihood of non-adherence was lower in apixaban and rivaroxaban (relative to warfarin) and lower in clopidogrel (relative to aspirin). Increased comorbidity burden (by CHA 2 DS 2 VASc score) was associated with decreased risk of non-adherence and non-persistence with anticoagulants. Overall rates of 'non-adherent, non-persistent' were highest in warfarin (compared with apixaban and rivaroxaban) and aspirin (compared with clopidogrel or dipyridamole) in patients with and without liver disease. Among patients without liver disease, not taking antithrombotic medications for >3 months was associated with a higher risk of stroke, however, adherence to these medications was also associated with a small increase in risk of bleeding. Patients with liver disease (when compared with those without liver disease) had higher risks of stroke, especially when they stopped taking antiplatelets for >3 months. Patients with liver disease who were adherent to antiplatelets, however, had a higher risk of bleeding compared with patients without liver disease., Interpretation: Use of antithrombotic medicines in patients with and without liver disease is suboptimal with heterogeneity across medicines. As patients with liver disease are excluded from major randomised trials for these drugs, our results provide real-world evidence that may inform medicine optimisation strategies. We outline challenges and opportunities for tackling non-adherence, which begins with understanding patients' views of medicines to help them make informed decisions about appropriate use., Funding: AGL is supported by funding from the Wellcome Trust (204841/Z/16/Z), National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre (BRC714/HI/RW/101440), NIHR Great Ormond Street Hospital Biomedical Research Centre (19RX02), the Health Data Research UK Better Care Catalyst Award (CFC0125) and the Academy of Medical Sciences (SBF006\1084). The funders have no role in the writing of the manuscript or the decision to submit it for publication., Competing Interests: None declared., (© 2021 The Author(s).)

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Febrile infection-related epilepsy syndrome (FIRES) is a devastating epilepsy characterized by new-onset refractory status epilepticus with a prior febrile infection. We performed exome sequencing in 50 individuals with FIRES, including 27 patient-parent trios and 23 single probands, none of whom had pathogenic variants in established genes for epilepsies or neurodevelopmental disorders. We also performed HLA sequencing in 29 individuals with FIRES and 529 controls, which failed to identify prominent HLA alleles. The genetic architecture of FIRES is substantially different from other developmental and epileptic encephalopathies, and the underlying etiology remains elusive, requiring novel approaches to identify the underlying causative factors., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Objective: Impaired lysosomal degradation of α-synuclein and other cellular constituents may play an important role in Parkinson's disease (PD). Rare genetic variants in the glucocerebrosidase (GBA) gene were consistently associated with PD. Here we examine the association between rare variants in lysosomal candidate genes and PD., Methods: We investigated the association between PD and rare genetic variants in 23 lysosomal candidate genes in 4096 patients with PD and an equal number of controls using pooled targeted next-generation DNA sequencing. Genewise association of rare variants in cases or controls was analyzed using the optimized sequence kernel association test with Bonferroni correction for the 23 tested genes., Results: We confirm the association of rare variants in GBA with PD and report novel associations for rare variants in ATP13A2, LAMP1, TMEM175, and VPS13C., Conclusion: Rare variants in selected lysosomal genes, first and foremost GBA, are associated with PD. Rare variants in ATP13A2 and VPC13C previously linked to monogenic PD and more common variants in TMEM175 and VPS13C previously linked to sporadic PD in genome-wide association studies are associated with PD. © 2020 International Parkinson and Movement Disorder Society., (© 2020 International Parkinson and Movement Disorder Society.)

Many strategies have been developed to manipulate DNA molecules and investigate protein-DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a diverse range of single-molecule approaches., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019. Published by Elsevier Inc.)

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein-DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9-guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

DNA replication, or the copying of DNA, is a fundamental process to all life. The system of proteins that carries out replication, the replisome, encounters many roadblocks on its way. An inability of the replisome to properly overcome these roadblocks will negatively affect genomic integrity which in turn can lead to disease. Over the past decades, efforts by many researchers using a broad array of approaches have revealed roles for many different proteins during the initial response of the replisome upon encountering roadblocks. Here, we revisit what is known about DNA replication and the effect of roadblocks during DNA replication across different organisms. We also address how advances in single-molecule techniques have changed our view of the replisome from a highly stable machine with behavior dictated by deterministic principles to a dynamic system that is controlled by stochastic processes. We propose that these dynamics will play crucial roles in roadblock bypass. Further single-molecule studies of this bypass will, therefore, be essential to facilitate the in-depth investigation of multi-protein complexes that is necessary to understand complicated collisions on the DNA.

We performed a genome-wide association study in 1,194 controls and 150 patients with anti-N-methyl-D-aspartate receptor (anti-NMDAR, n = 96) or anti-leucine-rich glioma-inactivated1 (anti-LGI1, n = 54) autoimmune encephalitis. Anti-LGI1 encephalitis was highly associated with 27 single-nucleotide polymorphisms (SNPs) in the HLA-II region (leading SNP rs2858870 p = 1.22 × 10 -17 , OR = 13.66 [7.50-24.87]). Potential associations, below genome-wide significance, were found with rs72961463 close to the doublecortin-like kinase 2 gene (DCLK2) and rs62110161 in a cluster of zinc-finger genes. HLA allele imputation identified association of anti-LGI1 encephalitis with HLA-II haplotypes encompassing DRB1*07:01, DQA1*02:01 and DQB1*02:02 (p < 2.2 × 10 -16 ) and anti-NMDAR encephalitis with HLA-I allele B*07:02 (p = 0.039). No shared genetic risk factors between encephalitides were identified. Ann Neurol 2018;83:863-869., (© 2018 American Neurological Association.)

A 1-year study measured the effect of administration of an inactivated human immunodeficiency virus type 1 (HIV-1) immunogen in incomplete Freund's adjuvant (IFA) on HIV-1-specific immunity and viral burden in asymptomatic HIV-1-infected patients. A total of 103 patients were enrolled in this double-blind, randomized study comparing immunogen in adjuvant with adjuvant alone. This study was conducted at nine medical centers throughout the United States. Significant differences in cell-mediated immune responses to HIV-1 and p24 core antigen were observed between the treated and control groups (P < .01). Compared with controls, treated patients as a group had a significantly lower rate of increase in viral DNA as determined by quantitative HIV-1 DNA-polymerase chain reaction (P = .016). Significant differences in the rate of percent CD4 cell decline were also observed between the 2 groups (P = .024). These data suggest that augmentation of HIV-1-specific immunity via immunotherapy may alter the rate of increase of HIV-1 DNA in peripheral blood mononuclear cells and stabilize the percent of CD4 cell decline in relatively healthy HIV-1-infected persons.