Single-molecule mapping of replisome progression

Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription and whether the progression of sister replisomes is coordinated are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a new method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single molecule readout of long DNA molecules. Using Replicon-seq, we have investigated replisome movement along chromatin. We found that sister replisomes progress with remarkable consistency from the origin of replication but function autonomously. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and facilitating efficient termination. Replicon-seq provides an unprecedented means of defining replisome movement across the genome.

CARTOGRAPHY AND MAPPING FOR PRECISION HEALTH: REVIEW OF ESSENTIAL ELEMENTS FOR DISEASE MAPPING

A well-developed healthcare system, decent access to clean water and sanitation, and programmes to eliminate poverty and build modern infrastructure are essential components to create healthier Malaysia's population. Non-communicable diseases currently account for most of the mortality and morbidity, although communicable diseases such as dengue fever, avian flu and covid-19 still pose a threat. The World Health Organization (WHO) identified COVID-19 is a rare pneumonia disease that originated in Wuhan, on January 12, 2020, before it became an outbreak in all countries including Malaysia. The requirement of a precise mapping and Cartography for the accurate disease mapping and data management are crucial due to a precise map gives higher resolution of the data and for more specific data analysis, interpretation and decision making process. In Malaysia, there no specific report on precise mapping for health applications, and it is therefore this paper is to identify the potential criteria and factors needed for precise health mapping applications. A precise health mapping is essential to create a precise risk map towards the surveillance and signal detection, predicting future risk, targeted interventions, and understanding disease phenomena.

Elucidating Macular Structure-Function Correlations in Glaucoma

Correlation between structural data from optical coherence tomography (OCT) and functional data from the visual field (VF) may be suboptimal because of poor mapping of OCT measurement locations to VF test stimuli. We tested the hypothesis that stronger structure-function correlations in the macula can be achieved with fundus-tracking perimetery, by precisely mapping OCT measurements to VF sensitivity at the same location. The conventional 64 superpixel (3°x3°) OCT grid was mapped to VF sensitivities averaged in 40 corresponding VF units with standard automated perimetry (conventional mapped approach, CMA) in 38 glaucoma patients and 10 healthy subjects. Similarly, a 144 superpixel (2°x2°) OCT grid was mapped to each of the 68 VF locations with fundus-tracking perimetry (localized mapped approach, LMA). For each approach, the correlation between sensitivity at each VF unit and OCT superpixel was computed and the maximum value used to generate vector maps. CMA yielded significantly higher structure-function correlations compared to LMA. Only 20% of the vectors with CMA and <5% with LMA were within corresponding mapped OCT superpixels, while most were directed towards loci with structural damage. Measurement variability and patterns of glaucomatous damage are more likely to affect the correlations rather than precise mapping of VF stimuli.

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

Neuronal firing patterns are the result of inputs converging onto single cells. Identifying these inputs, anatomically and functionally, is essential to understand how neurons integrate information. Single-cell electroporation of helper genes and subsequent local injection of recombinant rabies viruses enable precise mapping of inputs to individual cells in superficial layers of the intact cortex. However, access to neurons in deeper structures requires more invasive procedures, including removal of overlying tissue. We have developed a method that through a combination of virus injections allows us to target ≤4 hippocampal cells 48% of the time and a single cell 16% of the time in wildtype mice without the use of electroporation or tissue aspiration. We identify local and distant monosynaptic inputs that can be functionally characterised in vivo. By expanding the toolbox for monosynaptic circuit tracing, this method will help further our understanding of neuronal integration at the level of single cells.

The Home-Based Sleep Laboratory

Sleep disturbances are prevalent in neurodegenerative diseases in general, and in Parkinson’s disease (PD) in particular. Recent evidence points to the clinical value of sleep in disease progression and improving quality of life. Therefore, monitoring sleep quality in an ongoing manner at the convenience of one’s home has the potential to improve clinical research and to contribute to significantly better personalized treatment. Further, precise mapping of sleep patterns of each patient can contribute to a better understanding of the disease, its progression and the appropriate medical treatment. Here we review selective, state-of-the-art, home-based devices for assessing sleep and sleep related disorders. We highlight the large potential as well as the main challenges. In particular, we discuss medical validity, standardization and regulatory concerns that currently impede widespread clinical adoption of existing devices. Finally, we propose a roadmap with the technological and scientific steps that are required to impact PD research and treatment.

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.