Interplay of dynamic genome organization and biomolecular condensates.

After 60 years of chromatin investigation, our understanding of chromatin organization has evolved from static chromatin fibers to dynamic nuclear compartmentalization. Chromatin is embedded in a heterogeneous nucleoplasm in which molecules are grouped into distinct compartments, partitioning nuclear space through phase separation. Human genome organization affects transcription which controls euchromatin formation by excluding inactive chromatin. Chromatin condensates have been described as either liquid-like or solid-like. In this short review, we discuss the dynamic nature of chromatin from the perspective of biomolecular condensates and highlight new live-cell synthetic tools to probe and manipulate chromatin organization and associated condensates.

Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA.

The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods.

Advances and challenges in CRISPR-based real-time imaging of dynamic genome organization.

Nuclear chromosome compaction is non-random and dynamic. The spatial distance among genomic elements instantly modulates transcription. Visualization of the genome organization in the cell nucleus is essential to understand nuclear function. In addition to cell type-dependent organization, high-resolution 3D imaging shows heterogeneous compaction of chromatin organization among the same cell type. Questions remain to be answered if these structural variations were the snapshots of dynamic organization at different time points and if they are functionally different. Live-cell imaging has provided unique insights into dynamic genome organization at short (milliseconds) and long (hours) time scales. The recent development of CRISPR-based imaging opened windows for studying dynamic chromatin organization in single cells in real time. Here we highlight these CRISPR-based imaging techniques and discuss their advances and challenges as a powerful live-cell imaging method that poses high potential to generate paradigm-shifting discoveries and reveal functional implications of dynamic chromatin organization.

Single-chromosome dynamics reveals locus-dependent dynamics and chromosome territory orientation.

Dynamic chromatin organization instantly influences DNA accessibility through modulating local macromolecular density and interactions, driving changes in transcription activities. Chromatin dynamics have been reported to be locally confined but contribute to coherent chromatin motion across the entire nucleus. However, the regulation of dynamics, nuclear orientation and compaction of subregions along a single chromosome are not well-understood. We used CRISPR-based real-time single-particle tracking and polymer models to characterize the dynamics of specific genomic loci and determine compaction levels of large human chromosomal domains. Our studies showed that chromosome compaction changed during interphase and that compactions of two arms on chromosome 19 were different. The dynamics of genomic loci were subdiffusive and dependent on chromosome regions and transcription states. Surprisingly, the correlation between locus-dependent nuclear localization and mobility was negligible. Strong tethering interactions detected at the pericentromeric region implies local condensation or associations with organelles within local nuclear microenvironments, such as chromatin-nuclear body association. Based on our findings, we propose a 'guided radial model' for the nuclear orientation of the long arm of chromosome 19.

Exploration of Preventable Hospitalizations for Colorectal Cancer with the National Cancer Control Program in Taiwan.

BACKGROUND: Causing more than 40,000 deaths each year, cancer is one of the leading causes of mortality and preventable hospitalizations (PH) in Taiwan. To reduce the incidence and severity of cancer, the National Cancer Control Program (NCCP) includes screening for various types of cancer. A cohort study was conducted to explore the long-term trends in PH/person-years following NCCP intervention from 1997 to 2013. METHODS: Trend analysis was carried out for long-term hospitalization. The Poisson regression model was used to compare PH/person-years before (1997-2004) and after intervention (2005-2013), and to explore the impact of policy intervention. RESULTS: The policy response reduced 26% for the risk of hospitalization; in terms of comorbidity, each additional point increased the risk of hospitalization by 2.15 times. The risk of hospitalization doubled for each 10-year increase but was not statistically significant. Trend analysis validates changes in the number of hospitalizations/person-years in 2005. CONCLUSIONS: PH is adopted as an indicator for monitoring primary care quality, providing governments with a useful reference for which to gauge the adequacy, accessibility, and quality of health care. Differences in PH rates between rural and urban areas can also be used as a reference for achieving equitable distribution of medical resources.

Cell cycle- and genomic distance-dependent dynamics of a discrete chromosomal region.

In contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics during interphase are less understood. We deployed a CRISPR-based DNA imaging system to track the dynamics of genomic loci situated kilobases to megabases apart on a single chromosome. Two distinct modes of dynamics were resolved: local movements as well as ones that might reflect translational movements of the entire domain within the nucleoplasmic space. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S phase. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle dependence suggest the existence of a hitherto unrecognized compaction-relaxation dynamic of the interphase chromosome fiber, operating concurrently with changes in the extent of overall movements of loci in the 4D genome.

CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging.

Clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA scaffolds have been adapted to carry multiple binding sites for fluorescent proteins to enhance brightness for live cell imaging of genomic loci. However, many of these modifications result in guide RNA instability and thus produce lower genome-labeling efficiency than anticipated. Here we introduce CRISPR-Sirius, based on octet arrays of aptamers conferring both enhanced guide RNA stability and brightness, and provide initial biological applications of this platform.

The impact of inappropriate antibiotics on bacteremia patients in a community hospital in Taiwan: an emphasis on the impact of referral information for cases from a hospital affiliated nursing home.

BACKGROUND: Evidence for the impact of inappropriate antimicrobial therapy on bacteremia is mainly from studies in medical centers. We investigated the impact of inappropriate antimicrobial therapy on bacteremia in a community hospital. In particular, patients from the hospital's affiliated nursing home were sent to the hospital with adequate referral information. METHODS: We performed a retrospective study to collect data of patients with bacteremia in a community hospital in Taiwan from 2005 to 2007. RESULTS: A total of 222 patients with blood stream infection were diagnosed, of whom 104 patients (46.8%) died. The rate of initial inappropriate antibiotic prescriptions was high (59%). Multivariate analysis revealed that patients with initial inappropriate antibiotics, patients with ventilator support and patients requiring ICU care were the independent predictors for inhospital mortality. Patients referred from the hospital-affiliated nursing home and patients with normal WBC counts had better survival outcome. More than 80% cases infected with methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis received initial inappropriate antimicrobial therapy. With the longer delay to administer appropriate antibiotic, a trend of higher mortality rates was observed. CONCLUSIONS: Bacteremia patients from a hospital-affiliated nursing home had a better prognosis, which may have been due to the adequate referral information. Clinicians should be aware of the commonly ignored drug resistant pathogens, and efforts should be made to avoid delaying the administration of appropriate antibiotic therapy.