Gene machines

Abstract:

BACKGROUND:
The novel coronavirus SARS-CoV-2, which causes the COVID-19 disease, has resulted in a global pandemic. Since its emergence in December 2019, the virus has infected millions of people, caused the deaths of hundreds of thousands, and resulted in incalculable social and economic damage. Understanding the infectivity and transmission dynamics of the virus is essential to determine how best to reduce mortality while ensuring minimal social restrictions on the lives of the general population. Anecdotal evidence is available, but detailed studies have not yet revealed whether infection with the virus results in immunity.
OBJECTIVE:
The objective of this study was to use mathematical modeling to investigate the reinfection frequency of COVID-19.
METHODS:
We have used the SIR (Susceptible, Infected, Recovered) framework and random processing based on empirical SARS-CoV-2 infection and fatality data from different regions to calculate the number of reinfections that would be expected to occur if no immunity to the disease occurred.
RESULTS:
Our model predicts that cases of reinfection should have been observed by now if primary SARS-CoV-2 infection did not protect individuals from subsequent exposure in the short term; however, no such cases have been documented.
CONCLUSIONS:
This work concludes that infection with SARS-CoV-2 provides short-term immunity to reinfection and therefore offers useful insight for serological testing strategies, lockdown easing, and vaccine development.

Abstract:

The fluorescent nitrogen-vacancy (NV) defect in diamond has remarkable photophysical properties, including high photostability which allows stable fluorescence emission for hours; as a result, there has been much interest in using nanodiamonds (NDs) for applications in quantum optics and biological imaging. Such applications have been limited by the heterogeneity of NDs and our limited understanding of NV photophysics in NDs, which is partially due to the lack of sensitive and high-throughput methods for photophysical analysis of NDs. Here, we report a systematic analysis of NDs using two-color wide-field epifluorescence imaging coupled to high-throughput single-particle detection of single NVs in NDs with sizes down to 5-10 nm. By using fluorescence intensity ratios, we observe directly the charge conversion of single NV center (NV- or NV0) and measure the lifetimes of different NV charge states in NDs. We also show that we can use changes in pH to control the main NV charge states in a direct and reversible fashion, a discovery that paves the way for performing pH nanosensing with a non-photobleachable probe.

Abstract:

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.

Abstract:

Cotranscriptional RNA folding plays important roles in gene regulation steps such as splicing, transcription termination, and translation initiation. Progression of our understanding of cotranscriptional RNA folding mechanisms is still retarded by the lacking of experimental tools to study the kinetics of cotranscriptional RNA folding properly. In this chapter, we describe fluorescence resonance energy transfer (FRET) assay that enables the study of RNA cotranscriptional folding at the single-molecule level.

Abstract:

ABSTRACT

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli . The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.