Some high-performance OATS.
\r\n\tThorough research and analyses of most upfront neuroimaging techniques and various in vivo applications of specific mapping of normal function and under diseases are the purpose of this book. This book would hopefully capture the interests of colleagues interested in neuroimaging principles and applications, research and developments, as well as disease diagnosis and treatment, and could help convey the methodological and developmental perspectives of brain function in the medical application and research field.
\r\n\t
An open-area test site (OATS) is one of the key facilities in EMC. An OATS is a basic facility to measure the antenna factor [1] and the “benchmark” for semi-anechoic chambers over (30–1000) MHz [2].
When we make radiated disturbance measurements, both antennas and anechoic chambers are required. An OATS is the best choice for measuring the antenna factor over 30 MHz to 1000 MHz. We use antennas to “capture” the invisible electric fields (E-fields) or magnetic fields (M-fields) by converting the fields into voltage signals, which can be measured by an EMI receiver. However, the converting capabilities are different for different antennas. Thus, we need a parameter to “eliminate” this difference; thus, the antenna factor is defined as the ratio between the magnitude of E-field and the voltage induced on the load (usually 50 Ω) connected into the feed of an antenna [1]. One of the major activities for a calibration laboratory is to measure their value. In order to get a very accurate value for the antenna factor over 30 to 1000 MHz, OATS is the best choice since it can provide the most accurate value for broadband EMC antennas over this frequency band.
An OATS is the “benchmark” for semi-anechoic chambers over (30–1000) MHz. An EMC chamber can isolate the radiated emission by an EUT from others by a shielding enclosure outside the antenna and EUT. Usually, absorbers are lined inside to reduce the reflections along the four sides of an EUT and from the ceiling. However, it is very difficult to reduce totally due to the limits of technology and cost. Therefore, a quality verification process called validation is needed. The basic idea of this validation procedure is to measure the site attenuation (SA) between a pair of broadband antennas in the EMC chamber over (30–1000) MHz and compared with some standard value, which is defined as antenna pair reference site attenuation,
Due to the significance of OATS in the EMC area, it is worthwhile reviewing the technologies of design, construction and validation of a high-performance OATS for the following:
the antenna users
calibration Labs
potential builders of high-performance OATS
certificate examinants
revisions of international standards CISPR 16–1-6, CISPR 16–1-5, ANSI C63.5, and so on.
The start of the art on the OATS is introduced in Section 2; a detailed case study is provided on the design, construction, and validation in Section 3. An uncertainty example in measuring
The theoretical model of an OATS is listed in Figure 1. The electromagnetic fields (EM fields) radiated by a transmit antenna (Tx) are reflected only by the ground plane (GP), and the GP is infinitely large, consisting of a perfect electric conductor. Then, there are no other reflections. The receive antenna (Rx) received the EM fields radiated directly from the Tx and the reflections from the ground plane. Any other reflections are regarded as uncertainty sources. This means the better the ground plane size, the better agreement with an ideal OATS. Standard CISPR 16–1-5 states the minimum should be 30 m by 20 m. The most common ground plane size is 60 m by 30 m. The flatness of the metal ground plane is also important, especially above 700 MHz [6].
The theoretical model of an OATS—Half-space model.
There are basically two types of structures on the large ground plane, as shown in Figure 2. In the solid type, the metal ground plane is placed directly on the surface of the concrete layer. This structure makes sure the metal ground plane can hold heavy EUT. In order to adjust the flatness of the ground plane, some space is designed between the metal ground plane and the concrete layer, which is called the floating type.
The structures of the ground plane. (a) Solid type and (b) floating type.
The impedance transition between the edge of the ground plane and the soil is also important. Usually, stainless wire mesh is designed for this purpose. Figure 3 shows some typical designs for the stainless wire mesh. In (a), the wire mesh is mounted vertically down to the soil. In (b), a wire-mesh belt is mounted around the edge of the metal ground plane. In (c), the belt is replaced by some triangular mire mesh. Simulation shows design (c) agrees best with the theoretical mode in Figure 1, under the same ground plane size and wire mesh area.
The design of stainless wire mesh (showing the corner of an OATS). (a) Inverted L-shape (vertical cut), (b) belt (bird view), and (c) triangular (bird view).
Figure 4 shows the surface current induced. A pair of dipole antennas are resonated at 30 MHz and VP; the dipole antennas are separated by 20 m (R = 20 m) both the Tx and the Rx are 1.3 m above the perfect electric conductor (PEC) ground plane. The height of the isosceles triangles is 10 m, and the bottom width is 5 m. The inner area (25 m by 15 m) of the ground plane is simulated with physical optics (PO), while others are simulated with Method of Moment (MoM). The simulation model is set up in free space; in other words, no consideration is taken into account on the effect of the grounding of wire mesh into the soil. Figure 4 shows that the surface current fluctuated rapidly near the edge of the ground plane or the tips of the mesh [7].
The comparison of triangular wire mesh. (a) No wire mesh and (b) triangular wire mesh.
The deviation
“30 m × 20 R20 m”: no wire mesh, as shown in Figure 4(a), GP is 30 m by 20 m, both dipole antennas are separated by 20 m;
“30 m × 20 Trimesh R20 m”: triangular wire mesh as shown in Figure 4(b) is introduced based on ①; The meanings for other legends are similar.
The deviation of finite-size GP at VP (R = 20 m).
As shown in [7], a GP of 30 m by 20 m is not qualified for measurements at R = 20 m, whose deviation is larger than 1.5 dB. However, it can be reduced to 1.2 dB at VP by introducing some triangular wire mesh shown in Figure 4(b). If the GP is increased to 40 m by 30 m, the deviation is less than 1 dB. Less deviation at 30 m separation can be achieved by increasing the GP to 60 m by 40 m.
Further simulation shows that the triangular wire mesh is also effective for HP at R = 20 m, as shown in Figure 6.
The deviation of finite-size GP at HP (R = 20 m).
The performance of a practical OATS should be as close as possible to the half-space model shown in Figure 1. The IEC standard CISPR 16–1-5:2014 provides a detailed method to validate this, which is briefly summarized as in Eq. (1).
where
For a reference test site (REFTS),
Lab. | Dimensions | Structure | CR Locations | ||
---|---|---|---|---|---|
Metal GP | Stainless wire mesh | ||||
NIM (China) | 60 m x 40 m x 10 mm | 10 m Triangular | Floating type | Under | 0.26 dB (HP) 0.34 dB (VP) |
NPL (UK) | 60 m x 30 m x 8 mm | Inverted L-type | Solid type | Above GP, Wooden, 25 m far away | 0.3 dB (HP) |
NIST (USA) | 60 m x 30 m x 8 mm | Triangular | Solid type | N/A | N/A |
Liberty Lab. (USA) | 60 m x 30 m x 8 mm | Rectangular10 m width | Floating type | Half under | 0.5 dB (HP) |
ETS-LINDGREN (USA) | 60 m x 30 m x 8 mm | Strip | Solid type | Under | N/A |
NIMJ (Japan) | 45 m x 30 m x 16 mm | Rectangular | Floating type | Above GP | N/A |
KRISS (Korea) | 30 m x 30 m x 2 mm | N/A | Solid type | N/A | N/A |
CENAM (Mexico) | 60 m x 30 m | N/A | N/A | N/A | 0.5 (HP) |
ARC (Austria) | 20 m x 17 m | Rectangular10 m width | Solid type | Under GP, with 50 cm roof above GP | N/A |
Some high-performance OATS.
Due to the limited knowledge of the author, the data could be wrong. N/A, not available.
Figure 7 shows a photo of NIM OATS taken in 2014. It is located in NIM’s Changpin Campus. The white part is the reflective GP, and usually, there are a motorized mast and a manual mast for holding the antennas to be measured. The masts are made of low permittivity dielectric materials. The motor of the motorized mast is located under the metal GP to avoid unwanted reflections. Obviously, these setups will imitate the theoretical model shown in Figure 1.
Photo of NIM OATS.
BTW, in this photo, there are two more masts on the upper right corner of the GP, which is for backup, and it would better be moved away. There is also a beautiful lake in the north of the OATS. Some sand cypress trees are planted around to keep the soil wet and as fences to prevent the entrance of unintentional persons and trucks.
The structure of the OATS is illustrated in Figure 8.
The structure illustration of NIM OATS.
The whole system is designed carefully. The metal GP will suffer more than 80 degrees temperature variations seriously during four seasons in Beijing. In order to cope with this critical problem, the large metal GP is placed “freely” on the top 2501 pieces (41 by 61) of the ceramics plate. The metal GP can move freely in the horizontal plane but is kept fixed in the vertical direction. The ceramics plates are mounted on adjustable bolts, which are mounted on the steel frames. These steel frames are mounted on the box-layered concrete structure. The whole concrete structure is located on 138 pile foundations, which are over 20 m deep into the rock layer, to maintain a very stable foundation.
As shown in Figure 8, the equipment room (ER) is located under the bottom of antenna masts, which can make sure the shortest cable routing from antenna masts to the ER. The control room is located under the GP, but near the entrance, to make sure a short way for operators to make measurements.
The dimesion of the GP and stainless triangular wire mesh are shown in Figure 9.
The grounding of the triangular wire mesh. (a) Triangular wire mesh and (b) tip of the triangular wire mesh.
As shown in Figures 4–6, the triangular is very useful for reducing the reflections from the edge of the GP. Figure 9(a) is the photo for the stainless triangular wire mesh, and Figure 9(b) shows the tips of the triangular wire mesh connected each other and being grounded to the soil with resistance less than 1 Ω.
Lots of precise “measurements” are carried out for this OATS. The deviation
The deviation of NIM’s OATS at HP.
The deviation of NIM’s OATS at VP.
The deviation is less than 0.26 dB for HP and 0.34 dB for VP. Obviously, it meets the requirements for both CALTS and REFT, though the uncertainty
In order to investigate the results at other frequencies, sweeping measurements between a pair of broadband calculable dipole antennas separated by 10 m and HP are shown in Figure 12. The difference between
Deviation of NIM’s OATS over sweeping frequencies, HP,
The setup of the standard site method (SSM) is shown in Figure 13. The Rx is swept from 1 m to 4 m in height to get the minimum SIL (and this is defined as site attenuation-SA). The horizontal separation
The setup of the SSM.
After measuring three SAs for three pairs of antennas (#1 vs. #2, #1 vs. #3, and #2 vs. #3), respectively, the free-space antenna factor
Where
Where
where
It is very hard to deduce an analysis equation for measuring antenna factor. Usually, a hybrid model (both analytical and dark box model) is adopted, as shown in Eq. (7) as an example,
The meaning of the other symbols is shown in Table 2. The evaluated standard uncertainty is shown in Table 2, too. The combined standard uncertainty can be calculated with Eq. (8), assuming the above uncertainty sources are independent.
No. | Symbol | Source of uncertainty or quantity ( | Value (dB) | Probability | Divisor | Sensitivity | Standard uncertainty ( |
---|---|---|---|---|---|---|---|
1 | Uncertainty from measuring S21 with VNA | 0.25 | Normal | 2 | 0.11 | ||
2 | Impedance mismatch between transmit and receive antennas | 0.28 | U-shaped | 0.17 | |||
3 | Cable loss variation due to temperature variation | 0.2 | Rectangular | 0.10 | |||
4 | Uncertainty from through calibration | 0.2 | Rectangular | 0.10 | |||
5 | Ambient noise | 0.1 | Normal | 2 | 0.04 | ||
6 | Antenna positing error | 0.26 | U-shaped | 0.16 | |||
7 | Imperfection of the OATS and masts | 0.6 | Rectangular | 0.30 | |||
8 | Repeatability of the measurement system | 0.3 | Normal | 2 | 0.13 | ||
9 | Symmetry of the antenna under measurement | 0.8 | Rectangular | 0.40 | |||
10 | Cross-polarization | 0 | Rectangular | 0.00 | |||
11 | Deviation from the free-space antenna factor | 0.4 | Rectangular | 1 | 0.23 | ||
12 | Combined standard uncertainty, | 0.68 | |||||
13 | Expanded uncertainty, | 1.4 |
Measurement uncertainty budget for
Relacing the value in Table 2, there will be,
The expanded uncertainty can be calculated with Eq. (4) by assuming a normal distribution since there are many numbers, and their values are similar.
Taking
This is the expanded uncertainty in measuring the free-space antenna factor of a biconical antenna with the standard site method, as shown in Table 2.
Key technologies on the design, construction and validation of a high-performance OATS have been provided, based on the author’s many years of experience. Some famous OATS in the world regarding their structure, the dimensions of the ground plane (GP), the location of the control room, and performance have been summarized. A detailed case study is provided on NIM’s high-performance OATS. A measurement uncertainty example has been provided in measuring the free-space antenna factor of biconical antennas.
Dr. Alexander KRIZ and Dr. Katsumi Fujii provide many details on their OATS. There are many persons involved in the design, construction, and validation of NIM’s OATS. Special acknowledgments are due to TDK, Mr. Gao X. X, Dr. Cui X. H., Dr. Song Z. F., Mr. Hong L., and Ban H.
The author declares no conflict of interest.
Full name | Symbol | |
---|---|---|
OATS | open-area test site | / |
CALTS | calibration test site | |
REFTS | reference test site | |
GP | ground plane | |
PEC | perfect electric conductor | |
CR | control room | |
ER | equipment room | |
CDA | calculable dipole antenna | / |
MoM | Method of Moments | / |
NIM | National Institute of Metrology | / |
SA | site attenuation | / |
NSA | normalized site attenuation | / |
HP | horizontal polarization | / |
VP | vertical polarization | / |
SIL | site insertion loss | / |
/ | calculated site insertion loss | |
/ | measured site insertion loss | |
/ | free-space antenna factor |
p53 is a multifunctional transcription factor that induces cell cycle arrest, DNA repair, and apoptosis, thereby suppressing cell cancerization [1, 2]. It is referred to as a guardian of the genome that determines cell fate. When p53 is activated by various stress factors, it searches for and binds to target DNA sequences and regulates the expression of downstream genes. p53 is composed of an N-terminal (NT) domain, core domain, linker, tetramerization (Tet) domain, and C-terminal (CT) domain. The core and Tet domains possess specifically folded structures, while other domains are intrinsically disordered [3, 4, 5]. p53 forms a tetramer via Tet domains [5]. Core and CT domains are involved in its binding to DNA sequences in a specific and nonspecific manner, respectively [6]. Fifty percent of gene mutations in tumor cells were found in p53, and many of the identified mutations were located in structured domains, which inhibited target DNA binding [3]. Comprehensive mutagenesis analysis supports the correlation between the structured domains of p53 and its function [7]. Since p53 possesses common properties frequently observed in DNA-binding proteins, including oligomerization, disordered regions, and multiple DNA-binding domains [8], it is used as a model protein in the target search study described below [9, 10, 11].
\nThe target DNAs for p53 were ~ 20 bp, while the genomic DNA was ~109 bp. Accordingly, p53 was required to search for small targets efficiently from within vast lengths of non-target DNAs. This is known as a target search problem for sequence-specific DNA-binding proteins. To solve this problem, a facilitated diffusion mechanism has been proposed for DNA-binding proteins. The facilitated diffusion is the integration of three-dimensional (3D) diffusion in solution, one-dimensional (1D) diffusion along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs (Figure 1a). In 3D diffusion, p53 diffuses in solution, altering the search sites on genomic DNA. In 1D sliding, it moves along the DNA, while maintaining continuous contact. In addition, p53 hops or jumps along DNA (within 100 bp of jump). Intersegmental transfer enables p53 to move from one DNA to another without dissociation. Theoretical studies suggest that the integration of multiple search dynamics, while not requiring all dynamics, can facilitate the target search [14, 15, 16, 17]. The facilitation factor depends on various physical parameters, such as diffusion coefficient along DNA, residence time on DNA, dissociation time in solution, and frequency of transfer and jump.
\nTarget search dynamics of DNA-binding proteins and visualization of p53 dynamics on DNA by single-molecule fluorescence microscopy. (a) Schematic diagram of four target search dynamics. (b) Schematic diagram of single-molecule fluorescence microscope and flow cell. In the flow cell, one end of the DNA is tethered to the surface and it is stretched by buffer flow. p53 molecule labeled to a fluorescence dye is illuminated by TIRF and the fluorescence is detected by EM-CCD through an objective lens. Panels (a) and (b) are adapted from ref. [
How does p53 solve the target search problem using facilitated diffusion? How is the target search and binding of p53 regulated? In this chapter, I explain the facilitated diffusion and regulation of p53 based on recently accumulated single-molecule data.
\nSingle-molecule fluorescence microscopy enables the differentiation and characterization of individual search dynamics of DNA-binding proteins, including p53, as reported previously [18, 19, 20, 21, 22, 23, 24]. In general, the system combines a fluorescence microscope and a flow cell (Figure 1b). In the flow cell, one end of the DNA is tethered to the surface, and it is stretched by buffer flow. Several methods have been proposed for tethering DNAs [18, 25, 26, 27, 28, 29]. For example, a DNA garden is a simple method for producing DNA arrays, in which neutravidin molecules are printed in a line on polymer-coated coverslips, and biotinylated DNAs are tethered to the printed neutravidin [29]. p53 molecules labeled with a fluorescence dye are introduced into the flow cell using a syringe pump. The fluorescent p53 bound to DNA is selectively illuminated by total internal reflection fluorescence (TIRF). p53 molecules on DNA are detected as fluorescent spots on the sequential images of an electron-multiplying charge-coupled device (EM-CCD). The positions of molecules were tracked using an appropriate analysis program to visualize the search dynamics of p53.
\nIn 2008, 1D sliding of p53 along DNA was observed for the first time using single-molecule fluorescence microscopy [30]. This observation was consistent with a reported indirect evidence that p53 dissociated rapidly from short DNA in the absence of blocks at ends by sliding off from DNA [31]. In 2011, a study of p53 mutants deleting either of two DNA-binding proteins revealed that p53 can slide along DNA using disordered CT domains [32]. This is consistent with the fact that a designed peptide targeting CT domains suppressed the 1D sliding of p53 [33]. Furthermore, 1D sliding of p53 was supported by molecular dynamics simulations [34, 35]. In 2012, it was shown that 1D sliding dynamics of p53 depends slightly on DNA sequence, suggesting that p53 feels the energy landscape based on DNA sequence through interactions between core domains and DNA [36]. In 2015, a detailed analysis of 1D sliding dynamics demonstrated that p53 possesses two sliding modes on non-target DNA [37, 38]. In the fast mode, it interacts with DNA loosely using CT domains. In contrast, in the slow mode, it binds tightly to DNA using core and CT domains (Figure 2a). In 2017, the disordered linker was identified to trigger the switch between the two modes (Figure 2a) [41]. In 2016, the target recognition process of p53 was characterized in detail [42]. The results demonstrated that target recognition occurs mainly via 1D sliding. The target recognition of p53 was quite low (the successful recognition probability was 7%), but it was enhanced two-fold upon a post-translational modification. Accordingly, 1D sliding is considered as one of the important dynamics in the target search and binding of p53.
\nTarget search dynamics of p53. (a) Schematic diagram of two modes for 1D sliding p53 along DNA. p53 is composed of the NT (purple), Core (green), linker (black), Tet (yellow), and CT (pink) domains. The switch between two modes is triggered by the linker. (b) Typical single-molecule data showing intersegmental transfer of p53 between crisscrossing DNAs. (c) Schematic diagram of intersegmental transfer of p53 between two DNAs. p53 uses CT domains (pink) for the transfer. (d) Typical single-molecule data showing jumping of p53 along DNA (white traces). Arrows denote the jumping events. (e) Schematic diagram of encounter complex formation of p53 and conversion from the encounter complex to long-lived complex. Panel (b) is adapted from ref. [
In 2018, intersegmental transfer of p53 was examined using ensemble kinetic and single-molecule fluorescence measurements [39]. After the solutions of p53 bound to fluorescently labeled DNA and non-labeled DNA were mixed, the transfer reaction of p53 was monitored between the two DNAs. The observed reactions included the dissociation of p53 from one DNA and its transfer to the other. Actually, as the concentration of non-labeled DNA increased, the observed rate constant increased, suggesting intersegmental transfer. The rate constant of the transfer was ~108 M−1 s−1, which is close to the diffusion limit. Furthermore, single-molecule tracking of p53 on crisscrossed DNAs demonstrated that p53 moves along the first DNA and then moves along the second DNA through the transfer at the intersection (Figure 2b). A study of p53 mutants deleting either of two DNA-binding domains identified that p53 binds to the first DNA and then to the second DNA using disordered CT domains at the same time; it then releases the first DNA, resulting in a transfer between the two DNAs (Figure 2c). This mechanism is supported by molecular dynamics simulations of p53 [43].
\nIn 2020, the hopping/jumping of p53 on DNA was investigated [40]. Hopping/jumping was expected to occur at a time scale that is faster than the time resolution of the microscope (ex. 33 ms). To detect these events, the time resolution of the microscope was improved to 500 μs by optimizing the fluorescence excitation based on critical angle TIRF illumination and by utilizing the time delay integration mode of the EM-CCD [40]. Using the sub-millisecond-resolved microscope, jumping events of p53 along DNA were directly detected (arrows in Figure 2d). The jump frequency of p53 was ~6 s−1, and the jump time was 2.2 ms. Based on the study of p53 mutants deleting either of two DNA-binding domains, disordered CT domains were identified to be indispensable for the jumping of p53 along DNA [13]. Furthermore, 1D diffusion along DNA was enhanced upon increasing the salt concentration, suggesting that p53 moves along DNA by hopping DNA-binding domains. Thus, it was revealed that p53 possesses hopping and jumping dynamics along DNA.
\nIn 2016, 3D diffusion of p53 was characterized using ensemble kinetic measurements [42]. Association rate constants for target and non-target DNAs were determined to be ~109 M−1 s−1, comparable to the diffusion limits. The difference in affinity for target and non-target DNAs was attributed to the dissociation rate constants. In 2020, the association process of p53 with non-target DNA was further investigated at the single-molecule level using a sub-millisecond resolved fluorescence microscope [40]. Kymographs demonstrated that short-lived traces of p53 with an average residence time of 2.8 ms were detected in addition to long-lived traces moving along DNA. The short-lived complex was interpreted as an encounter complex. Disordered CT domains of p53 were identified to participate in the transient complex formation and in the conversion from the transient complex to the long-lived complex [13] (Figure 2e). The long-lived complex was further stabilized by core domains [13].
\nOverall, single-molecule fluorescence microscopy revealed that p53 possesses all four search dynamics proposed theoretically: 3D diffusion, 1D sliding, hopping/jumping, and intersegmental transfer. The unique structure of p53, which is a tetramer of two DNA-binding domains, enables these search dynamics. This is the first study to examine all search possibilities for a single model protein.
\nTarget search and binding of p53 might be regulated by a liquid-like assembly of p53 molecules. In a liquid–liquid phase separation (LLPS), p53 molecules, which disperse in the bulk phase, assemble and form a condensed phase called liquid droplets. In the droplet phase, p53 can move fluidly while maintaining a high concentration. This fluid property in the condensed phase differs from the solid aggregation that causes malfunction of p53. Early
In 2020, this possibility was extensively examined using
Liquid droplet formation of p53 regulates its function. (a) Time course of a typical fusion event of three p53 droplets into a single droplet using DIC microscopy. (b) DIC and fluorescence images of the droplets of Alexa488-labeled p53 and non-labeled p53. Scale bars in panels (a) and (b) represent 10 μm. (c) Schematic diagram of p53 conformation in the droplet. p53 is composed of the NT (purple), Core (orange), Tet (yellow), and CT (red) domains. In the droplets, the NT and CT domains interact electrostatically. Arrows denote the structural changes on the different domains of p53 that are induced by the intermolecular interactions in a droplet. The dimer structure is displayed for clarity. (d) Functional switch model of p53. The panels (a)-(d) are adapted from ref. [
The structural properties of p53 in solution and in droplet form were investigated using fluorescence resonance energy transfer (FRET) between two fluorophores labeled at two residues of p53. Since FRET depends on the distance between the two fluorophores, it was used to measure the conformational changes. The distance between the core domains of p53 was slightly longer in the droplets, while the distance between the CT domains became slightly shorter (Figure 3c). Accordingly, p53 adopted a new tertiary structure, forming interactions with the adjacent molecules in the droplets.
\nDoes p53 maintain binding to the target DNA after experiencing the droplet formation? The reactions of p53 binding to the target DNA were similar before and after the droplet formation. These results indicate that droplet formation of p53 is reversible, and p53 dispersed in solution from the droplets retains its DNA binding ability.
\nDroplet formation of p53 was found to be regulated by molecular crowding, endogenous molecules, and post-translational modification. Molecular crowding agents, mimicking the cellular crowding condition, promoted droplet formation. In contrast, ssDNA, dsDNA, and ATP suppressed it. The p53 mutant mimicking post-translational phosphorylation did not form droplets. Based on these results, a functional switch model was proposed (Figure 3d). Under normal cell conditions, the compartmentalization of p53 into the droplets suppresses its function as a transcriptional regulator. Under stress conditions, the activation of p53, triggered by posttranslational phosphorylation, releases p53 from the droplets and promotes target search and binding.
\nIn this section, the current model of p53 is described in terms of target search and regulation. p53 functions as a transcription factor that responds to various emergency situations in cells. Under normal cell conditions, p53 turns off through the following mechanisms. First, the copy number of p53 is maintained at a low level, allowing dimers with a low affinity to target DNAs in an oligomeric state [48, 49]. Second, post-translational modifications for activating p53 are not added, for example, suppressing the target recognition of p53 [42]. Third, p53 is stored in liquid droplets [47]. These actions of p53 prevent its malfunction under normal conditions.
\nUnder cellular stress, p53 is activated by post-translational modifications [1, 2, 50, 51, 52, 53, 54, 55, 56] and by a change in its oligomeric state from dimers to tetramers, with a high affinity for target DNAs [48, 57, 58, 59]. Phosphorylation of the CT domain of p53 triggers its release from the droplets, allowing it to engage in target search [47]. The increase in the copy number of p53 also facilitates the target search [60]. As explained above, p53 utilizes facilitated diffusion combining four search dynamics. Using 3D diffusion, p53 associates randomly with nonspecific sites of DNA, followed by dissociation. Until p53 associates with the target sequence by chance, it repeats such association and dissociation motions. If the search motion of p53 is limited to 3D diffusion, it would be a time-consuming endeavor. After p53 associates with the nonspecific site of DNA by 3D diffusion, it can search for the target sequence along DNA near the bound site using 1D sliding and hopping/jumping. The search distance of p53 per association event is estimated to be 700 bp [40], corresponding to approximately 35-fold facilitation of the target search.
\nIn cells, genomic DNAs are covered by many DNA-binding proteins, including histones and other nucleoid proteins. These DNA-binding proteins may act as obstacles in the target search of p53. For example, when the sliding p53 collides with other DNA-binding proteins on DNA, it may not be able to bypass these obstacles due to steric hindrance, thereby limiting the search distance on DNA. However, p53 possesses two bypass mechanisms: the jumping along DNA [40] and the intersegmental transfer between two DNAs [39]. Using these motions, it can overcome such obstacles and continue its search for targets in cells. Overall, the search and regulation strategies of p53 could satisfy various cellular requirements.
\nThe target search and binding of p53 and its regulation have been characterized using single-molecule fluorescence microscopy and relevant biophysical methods. The accumulated data demonstrate that p53 searches for target DNAs utilizing four search dynamics: 3D diffusion in solution, 1D sliding along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs (Figure 4). Especially, hopping/jumping and intersegmental transfer between two DNAs are required to bypass obstacles bound to DNA. It was reported that other DNA-binding protein with a disordered DNA-binding domain bypasses obstacles through obstacle-unbound region of DNA [24]. Since p53 possesses a similar disordered DNA-binding domain, it is not surprising that p53 possesses this bypass mechanism. Target search and binding are regulated by copy number, post-translational modifications, and liquid droplet formation. Considering that p53 can interact with many partner proteins, the partner proteins may affect the target search. Complexity in the target search and regulation of p53 would enable a response to various emergency situations in cells and be required to satisfy various cellular requirements.
\nSchematic diagram of target search of p53. Pink and gray circles are p53 and obstacle bound to DNA, respectively. Four search mechanisms are illustrated.
I thank the corroborators for their helpful discussions on our studies of target search by p53.
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Chemotherapy has improved, but many drugs still do not reach the tumor site at effective doses and are often associated with high systemic toxicity and poor pharmacokinetics. Moreover, for many malignancies, diagnosis is obtainable only in metastatic stages of development, reducing the overall effectiveness of treatment. The choice of available treatments depends on tumor characteristics such as biomarkers, tumor size, metastatic disease, ligands, and antigen or endocrine receptor expression. Combined with surgical resection, chemotherapy and radiation remain the first line of treatment for patients with cancer. Even with these treatments, however, cancer continues to have high fatality rates and current therapeutic modalities have yet to significantly improve the often dismal prognosis of this disease. Nanotechnology is a highly focused approach, which may provide more effective and less toxic treatment when compared to chemotherapy. This area of research has emerged as cancer treatment in the form of new drugs and has reached promising results in preclinical and clinical trials proving its value as a potential tumor therapy.",book:{id:"5431",slug:"breast-cancer-from-biology-to-medicine",title:"Breast Cancer",fullTitle:"Breast Cancer - From Biology to Medicine"},signatures:"Márcia Rocha, Natalia Chaves and Sônia Báo",authors:[{id:"147895",title:"Dr.",name:"Sônia Nair",middleName:null,surname:"Báo",slug:"sonia-nair-bao",fullName:"Sônia Nair Báo"},{id:"190527",title:"MSc.",name:"Natalia",middleName:null,surname:"Chaves",slug:"natalia-chaves",fullName:"Natalia Chaves"},{id:"190529",title:"MSc.",name:"Marcia",middleName:null,surname:"Oliveira Da Rocha",slug:"marcia-oliveira-da-rocha",fullName:"Marcia Oliveira Da Rocha"}]},{id:"23376",doi:"10.5772/21984",title:"Heterogeneity of Phenotype in Breast Cancer Cell Lines",slug:"heterogeneity-of-phenotype-in-breast-cancer-cell-lines",totalDownloads:2888,totalCrossrefCites:0,totalDimensionsCites:10,abstract:null,book:{id:"329",slug:"breast-cancer-carcinogenesis-cell-growth-and-signalling-pathways",title:"Breast Cancer",fullTitle:"Breast Cancer - Carcinogenesis, Cell Growth and Signalling Pathways"},signatures:"Bruce C. 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In 2015, an estimated 231,840 cases of invasive carcinoma were diagnosed, and over 40,000 deaths were caused by breast cancer which accounts for almost 7% of all cancer mortality each year. In 2015, 60,290 cases of in situ breast cancer were diagnosed, representing over 14% of all new cancer cases among women and men. The steep increase in diagnosis of early‐stage breast cancer over the past 10 years is believed to be a result of more frequent mammography. However, since over half of these in situ lesions will not progress to invasive breast cancer, controversies have arisen about approaches to treatment and prevention of progression of early‐stage in situ breast cancer. Understanding the mechanisms of transition of normal breast to in situ pre‐neoplastic lesions and invasive breast cancer is currently a major focus of breast cancer research with implications for preventive and clinical management of breast cancer. In this review, we give an overview of current knowledge on the molecular and pathological changes that occur during early‐stage progression of breast cancer and describe some of the current models that are used to study this process.",book:{id:"5431",slug:"breast-cancer-from-biology-to-medicine",title:"Breast Cancer",fullTitle:"Breast Cancer - From Biology to Medicine"},signatures:"William Kietzman, Anna T. 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Fentiman",authors:[{id:"119147",title:"Prof.",name:"Ian",middleName:null,surname:"Fentiman",slug:"ian-fentiman",fullName:"Ian Fentiman"}]},{id:"52969",title:"Histopathological Characteristics: Clinical Course of Breast Cancer Subtypes Depending on the ER(+) (−)/PR(+) (−) Receptor Status",slug:"histopathological-characteristics-clinical-course-of-breast-cancer-subtypes-depending-on-the-er-pr-r",totalDownloads:1867,totalCrossrefCites:0,totalDimensionsCites:1,abstract:"Breast cancer patients were divided into separate groups, which were the estrogen receptor (ER)+/progesterone receptor (PR)+ HER2−, the ER or PR+ HER2−, the ER+/PR+ HER2+, the ER or PR+ HER2+, the ER−/PR− HER2−, and the ER−/PR− HER2+ groups. Patients with the ER/PR(+)/HER2− subtype breast cancers show better clinical prognosis compared to the hormone-negative, triple-negative (TN), and HER2+ subtypes. TN, HER2+ tumors in postmenopausal women were of higher grade, showing lymph node and lymphovascular invasion with poor prognosis in all case series. However, the ER+/PR−/HER2+ subgroup had the lowest survival rates in 2- and 5-year follow-ups. Comparison between the ER+PR+HER2+ and ER+PR−HER2− subgroups showed that HER2− status is an indicator of improved prognosis in long-term follow-up. Single hormone receptor (HR)(+) status, particularly HER2(−) cases, was in between the favorable and poor survival subgroups. The ER−, PR−, and HER2+ properties were found to be risk factors for frequent recurrences. In this chapter, breast cancer subtypes are compared with each other. Results from different studies highlight the importance of ER/PR/HER2 receptor variations in the choice of treatment and prognosis of breast cancer.",book:{id:"5431",slug:"breast-cancer-from-biology-to-medicine",title:"Breast Cancer",fullTitle:"Breast Cancer - From Biology to Medicine"},signatures:"Nilufer Bulut",authors:[{id:"189255",title:"Associate Prof.",name:"Nilufer",middleName:null,surname:"Bulut",slug:"nilufer-bulut",fullName:"Nilufer Bulut"}]},{id:"42313",title:"HER2-Driven Carcinogenesis: New Mouse Models for Novel Immunotherapies",slug:"her2-driven-carcinogenesis-new-mouse-models-for-novel-immunotherapies",totalDownloads:4463,totalCrossrefCites:2,totalDimensionsCites:2,abstract:null,book:{id:"2883",slug:"oncogene-and-cancer-from-bench-to-clinic",title:"Oncogene and Cancer",fullTitle:"Oncogene and Cancer - From Bench to Clinic"},signatures:"Cristina Marchini, Lucia Pietrella, Cristina Kalogris, Chiara Garulli, Federico Gabrielli, Elena Quaglino, Manuela Iezzi, Serenella M. 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