Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.
Highly infectious illness caused by pathogens is endemic especially in developing nations where there is limited laboratory infrastructure and trained personnel. Rapid point-of-care (POC) serological assays with minimal sample manipulation and low cost are desired in clinical practice. In this study, we report an automated POC system for Ebola RNA detection with RNA-guided RNA endonuclease Cas13a, utilizing its collateral RNA degradation after its activation. After automated microfluidic mixing and hybridization, nonspecific cleavage products of Cas13a are immediately measured by a custom integrated fluorometer which is small in size and convenient for in-field diagnosis. Within 5 min, a detection limit of 20 pfu/mL (5.45 × 107 copies/mL) of purified Ebola RNA is achieved. This isothermal and fully solution-based diagnostic method is rapid, amplification-free, simple, and sensitive, thus establishing a key technology toward a useful POC diagnostic platform.
SUMMARY Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, and not by DNA methylation, histone deacetylation or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres become more accessible to telomere-associated proteins and accumulate DDR signals. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery.
Since late December 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-negative rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathology and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomography will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.
During protein synthesis, mRNA and tRNA are moved through the ribosome by the process of translocation. The small diameter of the mRNA entrance tunnel only permits unstructured mRNA to pass through. However, there are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here, we show using single molecule F€ orster resonance energy transfer and small angle X-ray scattering experiments a new global conformational state of the ribosome. In the presence of the frameshift inducing dnaX hairpin, the ribosomal subunits are driven into a hyper-rotated state and the L1 stalk is predominantly in an open conformation. This previously unobserved conformational state provides structural insight into the helicase activity of the ribosome and may have important implications for understanding the mechanism of reading frame maintenance.
Phosphomannomutase/phosphoglucomutase contributes to infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. NMR spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. 13C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These are attributable to the phosphorylation state and possibly to conformational interconversions. S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose-1-phosphate substrate by impairment of kcat. Addition of the glucose-1,6-bisphosphate intermediate accelerated this reaction by 2 – 3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft, while preserving the secondary structure in solution. Diminished peak heights and faster 15N relaxation are suggestive of line broadening and millisecond fluctuations within four loops that can contact phosphosugars. 15N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1 to 3, and with a different principal axis of diffusion. This adds to the crystallographic evidence for domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of intermediate, substrate binding, and product release.
A novel micro- and nanofluidic device stacked with magnetic beads has been developed to efficiently trap, concentrate, and retrieve Escherichia coli (E. coli) from the bacterial suspension and pig plasma. The small voids between the magnetic beads are used to physically isolate the bacteria in the device. We used computational fluid dynamics, three-dimensional (3D) tomography technology, and machine learning to probe and explain the bead stacking in a small 3D space with various flow rates. A combination of beads with different sizes is utilized to achieve a high capture efficiency (∼86%) with a flow rate of 50 μL/min. Leveraging the high deformability of this device, an E. coli sample can be retrieved from the designated bacterial suspension by applying a higher flow rate followed by rapid magnetic separation. This unique function is also utilized to concentrate E. coli cells from the original bacterial suspension. An on-chip concentration factor of ∼11× is achieved by inputting 1300 μL of the E. coli sample and then concentrating it in 100 μL of buffer. Importantly, this multiplexed, miniaturized, inexpensive, and transparent device is easy to fabricate and operate, making it ideal for pathogen separation in both laboratory and point-of-care settings.
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