Chua, J. | Search bioRxiv

bioRxiv2022

Neuroimaging within the Dominantly Inherited Alzheimer's Network (DIAN): PET and MRI

The Dominantly Inherited Alzheimer Network (DIAN) Observational Study is an international collaboration studying autosomal dominant Alzheimer disease (ADAD). This rare form of Alzheimer disease (AD) is caused by mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), or amyloid precursor protein (APP) genes. As individuals from these families have a 50% chance of inheriting the familial mutation, this provides researchers with a well-matched cohort of carriers vs non-carriers for case-control studies. An important trait of ADAD is that the age at symptom onset is highly predictable and consistent for each specific mutation, allowing researchers to estimate an individuals point in their disease time course prior to symptom onset. Although ADAD represents only a small proportion (approximately 0.1%) of all AD cases, studying this form of AD allows researchers to investigate preclinical AD and the progression of changes that occur within the brain prior to AD symptom onset. Furthermore, the young age at symptom onset (typically 30-60 years) means age-related comorbidities are much less prevalent than in sporadic AD, thereby allowing AD pathophysiology to be studied independent of these confounds. A major goal of the DIAN Observational Study is to create a global resource for AD researchers. To that end, the current manuscript provides an overview of the DIAN magnetic resonance imaging (MRI) and positron emission tomography (PET) protocols and highlights the key imaging results of this study to date.

neuroscience↗

bioRxiv2020

Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multi-level regulation. Compared to established knockout methodologies, CRISPRi approaches enable rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both non-template and template strand targeting. Since nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust, and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacteria interactions, and inter-species communication. IMPORTANCEEnterococcus faecalis causes multidrug resistant life-threatening infections, and is often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labour-intensive allelic exchange methods. Built upon streptococcal dCas9, we developed an easily-modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

microbiology↗