Sarcopenia is the decline in muscle strength and mass attributed to aging. The pathogenesis of sarcopenia may be triggered by oxidative stress and uric acid (UA) has strong antioxidant properties. The aim of this study was to investigate the relationship between UA and sarcopenia in community-dwelling adults of West China using the baseline data of West China Health and Aging Trend (WCHAT) study.A cross-sectional study.4236 adults aged 50 years or older in communities of west China were enrolled in this study. We applied Asian Working Group for Sarcopenia (AWGS) 2019 criteria to define sarcopenia. Muscle mass was measured using skeletal muscle index (SMI) based on bioimpedance analysis (BIA). Handgrip strength (HGS) and gait speed (GS) were recorded, respectively. Different variables like anthropometry measures, life styles, chronic disease and blood test were collected. General linear model was done to investigate the relationship between UA and HGS/GS/SMI, adjusting age, ethnic groups, sleeping quality, education level, cognitive function, smoking history, drinking history, ADL score, and chronic disease.Participants were grouped according to UA quartiles by gender. After adjusting for potential confounders, a negative association between serum UA levels and sarcopenia was shown both in men and women. And a significant association between serum UA levels and HGS in women was shown as an inverted J shape. Besides, a positive association between the UA quartiles and SMI was observed, irrespective of gender.Our results showed that higher uric acid levels were significantly correlated with higher muscle mass and grip strength among Chinese adults aged over 50. Higher UA serum levels might slow down the progression of sarcopenia. Sarcopenia was an age-dependent loss of muscle mass and function which was common among older adults, leading to disability, loss of independence and death . The preAs the final product of purine metabolism, UA is generated in the xanthine/hypoxanthine reactions and other potentially deleterious prooxidant molecules are produced as a by-product of this reaction. As a result of this, UA has been treated as a reliable marker of oxidative stress . UA is aUnfortunately, there are few studies on the association of serum UA levels and sarcopenia in west China. We speculated that serum UA was negatively associated with sarcopenia and higher UA was associated with better grip strength or muscle mass, particularly in old adults. To explore this hypothesis, we grouped the participants according to UA quartiles by gender. Then we performed our study to determine the relationship between sarcopenia and UA in a large group of multi-ethnic residents enrolled in the West-China Health and Aging Trend Study (WCHAT). Specifically, we also investigated the relationship between UA and skeletal muscle index (SMI), grip strength (HGS) and gait speed (GS).n = 67), participants without blood uric acid test (n = 57), participants with kidney disease (n = 87), participants with mental disease (n = 5) and participants with tumor (n = 24) were excluded. Finally, 4260 participants were included and were grouped according to UA quartiles in our study  to test the muscle strength. Tests were performed on two independent occasions using the dominant hand and the largest value was recorded. Cutoffs of grip strength was defined as 28 kg in men and 18 kg in women. The physical function was estimated using gait speed (GS) through a 4-m walking test. The walking time was recorded using a kind of infrared sensor and the acceleration phase was strictly excluded. The participants were asked to perform the test by walking at a normal pace. Subjects stood at the starting point and upon the starting command, walked forward at a normal pace to the 4-m line. During the test, subjects wore common shoes, could use mobility aids, but could not be assisted. There were no time limits to the assessments and subjects could stop and rest if necessary. Sitting down was prohibited. The participants performed 2 trials, and the results were averaged to the nearest 0.01 m/s. The cutoff of gait speed was defined as less than 1.0 m/s.We defined sarcopenia according to the AWGS 2019 , 12 whicInformation regarding age, gender, ethnic groups, education level, smoking history and alcohol consumption history was gathered. Blood samples were drawn from the vein in the morning after a minimum of 8 h of fasting. Blood handling and collection was carried out under strictly standardized conditions. UA level were measured using the same standard.Cognitive status was assessed using a 10-item Short Portable Mental Status Questionnaire (SPMSQ)  and the #, < 253.85umol/l, 253.85 ≤ Q2# < 293.8umol/l, 293.8 ≤ Q3# < 340.4umol/l, 340.4umol/l ≤ Q4#; In male, Q1* < 319.2umol/l, 319.2 ≤ Q2* < 372.25umol/l, 372.25 ≤ Q3* < 423.525umol/l, 423.525umol/l ≤ Q4*). For continuous variables, one-way ANOVA was used to detect differences across groups for the continuous variables, and Fisher’s Least Significant Difference (LSD) post hoc analysis was used to determine the difference between every two groups. For the categorical variables, the chi-squared test was used to detect the difference across groups. When significant difference was identified across groups, column proportions tests (z-tests) with Bonferroni correction were performed to determine the difference between every two groups. During most testing, p < 0.05 was considered statistically significant, however, p-values were corrected for z-tests with the Bonferroni correction . We also performed UA subgroup analyses according to gender. The relationship between UA and sarcopenia was estimated by deriving odds ratios (ORs) and 95% confidence intervals (CIs) from multivariate logistic regression models. General linear model was done to investigate the relationship between UA and HGS/GS/SMI, adjusting age, ethnic groups, GDS score, sleeping quality, education level, cognitive function, smoking history, drinking history, ADL score, and chronic disease. A value of P < 0.05 (two-side) was considered to be statistically significant.The data analyses were performed using SPSS version 20.0 . The categorical data was presented as counts (percentages), and the normal distributed continuous data was presented as mean (standard deviation [SD]). We grouped the participants to different quartile boundaries in men and women, separately . Participants was grouped according to quartiles of UA in male and female, separately. Table #,Q3#  and Q4# in female. Q1 was regarded as the baseline category. In adjusted model which adjusted age, ethnic groups, GDS score, sleeping quality, education level, cognitive function, smoking history, drinking history, ADL score, and chronic disease, sarcopenia was still significantly associated with Q3*  and Q4*  group in male. Besides, in adjusted model, sarcopenia was significantly associated with UA in female with a dosage effect .Table #, Q3#  and Q4# group with a dosage effect. But UA level was negatively associated with GS in female in the Q4 group (β-0.039). Besides, the serum UA levels showed an inverted J-shaped relationship with HGS in female . While in male, the UA levels was positively associated with SMI in Q3* and Q4* group. And UA levels was positively associated with HGS in the Q4* group in male .The results of general linear models for the relationship between UA levels and SMI/GS/HGS in male and female were presented in Table We found that UA was negatively associated with sarcopenia after adjusted confounding factors. Besides, serum UA levels shared a significant inverted J-shaped curve relationship with HGS in female and only a positive relationship in male in Q4 group. And we found that UA level was also significantly associated with SMI, irrespective of gender. Collectively, these results suggest that UA seems to be a factor in protecting the muscle mass and the strength of the lower limbs. However, we cannot ignore the fact that this study was only a cross-sectional study. The relationship between uric acid and sarcopenia does not imply causality.Our study was consistent with previous studies –9. One sIn our study, we found a gender difference of the relationship between UA and HGS and this could be related with hormone difference. Several studies reported that estrogen promotes UA secretion and leads to increases in UA levels in postmenopausal women , which mThe exact mechanism that explains the positive associations of UA with muscle mass and strength remains unclear. This might be related with the powerful antioxidant capacity of UA which may protect skeletal muscle function from ROS-induced protein oxidative damage . BesidesOn the contrary, higher uric acid predicts obesity, diabetes and chronic kidney disease (CKD) which were all associated with the development of sarcopenia . So maybSome limitations of our study need to be considered. Firstly, people over 50 years old were enrolled in this study and most of them were in the age range of 50–60 years old. The relationship between UA level and sarcopenia could only reflect mostly middle-aged participants. Secondly, although we included many important confounders in our study. More adjustment should be made for confounders. For example, gout and medicine intake which might affect the UA level should be adjusted. Besides, we used 4-m gait speed evaluation instead of 6-m gait speed and this might exist some bias. Lastly, our study was only an observational cross-sectional study which cannot determine the causal relationship between sarcopenia and serum UA level. Further longitudinal research or clinical trial research was needed.In conclusion, our findings showed that higher UA serum levels was related with better HGS/SMI, not with GS. And sarcopenia was negatively associated with UA with a dosage effect. Besides, there existed some gender difference when these relationships were studied in men and women separately.


The laser beam was focalized through a microscope on the sample, kept at defined temperature using a temperature controller, and the temperature was varied in the range of 283–323 K. The Stokes and anti-Stokes scattered light was analyzed through a triple monochromator and detected by a liquid nitrogen-cooled CCD camera. Raw data have been analyzed with Matlab, and Raman spectrum parameters—such as area, intensity, frequency position and width of the peak—have been calculated using a Lorentz fitting curve. Results obtained, calculating the anti-Stokes/Stokes area ratio, demonstrate that the Raman modes of anatase, in particular the Eg one at 143 cm−1, are excellent candidates for the local temperature detection in the visible range.The determination of local temperature at the nanoscale is a key point to govern physical, chemical and biological processes, strongly influenced by temperature. Since a wide range of applications, from nanomedicine to nano- or micro-electronics, requires a precise determination of the local temperature, significant efforts have to be devoted to nanothermometry. The identification of efficient materials and the implementation of detection techniques are still a hot topic in nanothermometry. Many strategies have been already investigated and applied to real cases, but there is an urgent need to develop new protocols allowing for accurate and sensitive temperature determination. The focus of this work is the investigation of efficient optical thermometers, with potential applications in the biological field. Among the different optical techniques, Raman spectroscopy is currently emerging as a very interesting tool. Its main advantages rely on the possibility of carrying out non-destructive and non-contact measurements with high spatial resolution, reaching even the nanoscale. Temperature variations can be determined by following the changes in intensity, frequency position and width of one or more bands. Concerning the materials, Titanium dioxide has been chosen as Raman active material because of its intense cross-section and its biocompatibility, as already demonstrated in literature. Raman measurements have been performed on commercial anatase powder, with a crystallite dimension of hundreds of nm, using 488.0, 514.5, 568.2 and 647.1 nm excitation lines of the CW Ar The determination of temperature with good accuracy and with nano/micro-spatial resolution (nanothermometry) has been matter of intense research efforts since it opens up new perspectives in different research fields like biomedicine, photonics and nanoelectronics ,2,3,4. FIn optical nanothermometry, luminescence is currently the most widely employed detection technique. However, also Raman spectroscopy is emerging as a valuable tool for temperature measurements. Despite being an intrinsically weaker phenomenon than fluorescence (requiring longer integration times), advantages of Raman include the wide range of temperatures detectable, the ease of sample preparation and the ample availability of materials possessing a Raman spectrum ,11,12,13The Raman effect is the inelastic scattering of light and well-defined characteristics of the Raman spectrum, such as intensity, position in frequency and width of peak signals, are related to temperature. It follows that temperature can be measured from Raman spectra by determining the degree of the shift position of a defined peak at different temperatures, or by evaluating the broadening of its linewidth or by measuring the peak intensity ratio of the anti-Stokes signal to the Stokes signal .−1 [A good Raman thermometer material should possess these properties: (a) a large Raman scattering cross-section ; (b) high-intensity Raman peaks at low Raman shifts , indeedQ, calculated as the ratio of uncertainty  and sensitivity Temperature cannot be measured directly, signals, like frequency position or intensity of the anti-Stokes and Stokes Raman peaks, can be used as indication (Q), which is linked to temperature through a mathematical equation . Great care should also be devoted to the determination of the uncertainty of the obtained values, indicating the dispersion of values within which the true temperature value is expected to lie. The measured temperature will be accurate if it is very close to the true value, and if measured temperatures, acquired by replicate measurements on the same object, are close to each other. In addition, sensitivity and thermal resolution are two fundamental parameters to evaluate thermometry. Sensitivity (S) is defined as the derivative of the indication with respect to the temperature, =|∂Q/∂T| ,16, whilWhen Raman thermometers are considered, it is also important to investigate the effect of the laser power on the local temperature, in order to avoid the heating of the sample due to the laser itself. For this purpose, it is interesting to examine the behavior of the system as a function of the laser intensity . In gen2) has also been tested in few works as a Raman thermometer for titanium dioxide microparticles [Studies on temperature with Raman measurements can be found in literature for silicon ,20, gallarticles ,25 and farticles .Titanium dioxide’s general features of chemical stability and nontoxicity make it a very interesting compound for various different applications, including photocatalysis , opticalTest Sample, obtaining results with high sensitivity and low uncertainty and open the way to the use, in the future, of titanium dioxide-based new biosensors.The aim of this study is to obtain a protocol for temperature determination, with a high spatial resolution, of the order of the micro-nanometer dimension, exploiting Raman spectroscopy on anatase powder. As multiple signals are present in the Raman spectrum of Titanium dioxide, the choice of the actual Raman mode to be used has been performed on the base of its sensitivity to temperature. The ratio between Stokes and anti-Stokes signals of the same Raman mode has been investigated as a function of temperature (T), excitation wavelength  providing the lines at 488.0, 514.5, 530.8, 568.2 and 647.1 nm. The laser beam is coupled to a microscope  and focused on the sample by 100×, 50× or 20× objectives . The Raman scattering is collected into the slit of a three-stages subtractive spectrograph  by means of a set of achromatic lenses. The spectrograph is made up of a double monochromator , working as a tunable filter rejecting elastic scattering, and a spectrograph . The Raman signal is detected by a liquid nitrogen-cooled CCD . When an entrance slit of 50 μm is used, a precision of 0.6 cm−1 in the determination of the peak position is obtained, with this experimental set-up.The system is equipped with a CW Ti:Sapphire Laser, tunable in the range 675–1000 nm  and pumped by a CW Optically Pumped Semiconductor Laser , and an ArA temperature-controlled stage  is used to change and control the temperature of the sample, by means of a liquid nitrogen reservoir and heating resistances, giving a control of 0.1 K on the temperature inside the sample chamber. The sample is inserted into the temperature controller stage, and uniformly heated or cooled to reach the desired temperature, with a rate of 5 K/min and a thermalization time of at least 30 min. Before starting the experiment, a procedure of purging air from the stage chamber with nitrogen is performed; by this way the air in the chamber is eliminated and an inert static nitrogen atmosphere is realized, allowing fast temperature variations. Once the thermalization process is done, consecutive Stokes and anti-Stokes measurements are conducted to measure the local temperature of the sample. The wavelength incident on the sample is properly chosen in order to avoid sample heating (by using photons less energetic than the band gap of sample). Raman spectra have been collected in the visible range, by exciting at 488.0, 514.5, 568.2 and 647.1 nm, at different temperatures, ranging from 283 to 323 K. Measurements are repeated, at each wavelength and temperature, to obtain a consistent set of data (from 5 to 10 measurements at each temperature), by collecting the Raman signal in different positions of the sample.Measurements of the anti-Stokes/Stokes ratio have been collected also at different laser powers, in the range 0.1–20 mW, to individuate the region where the signal is independent from the power, and the local temperature is not influenced by the presence of a laser beam. An input power of few mW has been used for temperature measurements.+/Kr+ laser).All Raman spectra have been collected using a 20× objective (Olympus) with numerical aperture (N.A.) of 0.4 and a working distance of 12 mm, under these conditions the spot of the laser on the sample is expected to be approximately 1.5 μm , with a final thickness of few hundreds μm.The sample is titanium dioxide, a commercial anatase powder , with a crystallite dimensions of ~200 nm; it possesses a band gap of 3.4 eV . Titaniu−1 and four peaks, at 197, 397, 515 and 640 cm−1, with lower intensity.The Stokes Raman spectrum of anatase powder, recorded at room temperature, using a laser at 488.0 nm with an input power of 1.72 mW, is reported in Anatase crystals are characterized by 15 optical modes at the Γ point of the Brillouin zone, described with the following irreducible representation of the normal vibrational modes ,35:1A1g+Bg1 and Ag1 modes, at 512 and 518 cm−1 respectively, are not distinguishable at the experimental conditions, due to their intrinsic amplitude [Among these, only six modes, mplitude ,36.The experimental frequencies, reported in the central column of g mode at 143 cm−1. It is well defined, very intense even at low laser powers and highly sensitive to temperature, as will be demonstrated later, thanks to its low Raman shift.The Stokes (positive Raman shift) and anti-Stokes (negative Raman shift) Raman spectra are reported in −1 Eg mode, excited at 514.5 nm, at different temperatures, is illustrated in Stokes and anti-Stokes Raman spectra of anatase have been collected in the temperature range of 283–323 K, by exciting at 488.0, 514.5, 568.2 and 647.1 nm, using an input power of 1.47, 1.20, 2.20 and 5.86 mW, respectively. The Raman spectra collected at room temperature, at different excitation wavelengths, are reported in All Raman spectra have been analyzed with Matlab, using a Lorentz fitting, to obtain the Raman spectrum parameters, such as frequency position, width, intensity and area of the peak see .−1 peak, by exciting at 514.5 nm at different temperatures, is reported in An example of the data, obtained from the analysis of the Raman spectra centered on the 143 cm−1 and 0.48 and 0.54, respectively, while the peak width varies in between 10.7 and 11.4 cm−1.Corresponding to the increasing in temperature, both the peak frequency position and the anti-Stokes/Stokes ratio clearly show an increment, from 142.5 to 145 cm−1 Eg mode, at 514.5 nm, are shown in In order to test whether the Raman spectra, and the corresponding parameters, are perturbed by the laser irradiation, it is also necessary to investigate the effect of increasing incident laser power by keeping constant all other variables, like excitation wavelength. The laser power was changed in a range between 0.1 and 18 mW (depending on the laser wavelength); the experimental results , peak area and anti-Stokes/ Stokes ratio), for the 143 cmIt is evident that parameters are not increasing with the laser power only in the small range, 0–2 mW, while at higher input power they increase with the increase of the laser power. In order to assume that the temperature of the sample is not influenced by the presence of the laser irradiating the sample, it is necessary to keep the laser power at values lower than 2 mW.Test Sample, thus verifying if the parameters measured all over the sample are uniform or not, and comparable with the results previously obtained. The calculated anti-Stokes/Stokes ratios are reported in A validation of the method can be done by evaluating anti-Stokes and Stokes intensities in various positions of the Raman spectroscopy allows for temperature measurements with two methods: (a) Since the anti-Stokes and Stokes Raman intensities are proportional to the populations of their respective initial vibrational states, described by the Boltzmann distribution, the T of the sample can be estimated from the ratio of the Stokes and anti-Stokes intensities ,38,39; , which is particular of the instrumentation set-up used.Equation (1) was fitted to the experimental values, collected at different temperatures and defined excitation wavelength, leaving C as a free parameter. −1, in the temperature range 283–323 K, excited at 514.5 nm, and the curve resulting from the fitting with Equation (1) (the dashed lines). The corresponding calibration constants are calculated to be 0.9605, 0.9411, 0.9461 and 0.9491, respectively. −1 Raman mode, measured at different excitation wavelengths as a function of the temperature imposed by the thermostat are reported in The anti-Stokes/Stokes experimental ratios of the 143 cmAs we can see qualitatively in 2 Raman modes as a function of the temperature variation. −1 Eg anti-Stokes/Stokes ratio shows the highest value, in comparison with the other Raman modes, it presents values in the range 0.50–0.55, while the 397 cm−1 mode, the 515 cm−1 and the 640 cm−1 modes present values in the ranges 0.15–0.19, 0.08–0.12 and 0.05–0.07, respectively. The total variations of the ratio, in the whole T range S=The derivative with respect to the temperature, has been evaluated at 514.5 nm, for all the Raman modes of anatase. The results, plotted in −1 Eg mode a thermal resolution in the range 1.2 (@ 514.5 nm) to 3.4 K (@ 647.1 nm) have been calculated. All these experimental data are comparable with those already reported in literature [In particular, it is possible to evaluate the sensitivity, at 300 K close to the room temperature, and to compare the obtained values for different wavelengths and different Raman modes of titanium dioxide. The outcome, shown in terature .−1 Raman mode, the lowest in Raman frequency, the more sensitive to Temperature variation. Moreover, the sensitivity calculated starting from the experimental data overlaps well the theoretical ones.The behavior of the theoretical ratio can be compared to the experimental anti-Stokes/Stokes ratios for the four Raman modes, reported in test sample, of the anti-Stokes/Stokes ratio for the 143 cm−1 mode of anatase at 514.5 nm, at 297 K, is reported; the figure also shows the mean temperature calculated for these measurements and the standard deviation. In this context, the standard deviation of temperatures, rather than the errors derived from each measurement, is reported, as it is preferable when repeated measures are conducted; moreover, the two values have the same order of magnitude (few kelvins). Detailed values are reported in By using the calibration constants, it is possible to determine the local temperature. In These results confirm the validation of the method thus verifying that the temperature measured all over the sample is uniform.g one at 143 cm−1, are excellent candidate for the local temperature detection in the visible range. However, the need remains to investigate the behavior at longer wavelengths, towards the near IR, where the biological window is located.It is possible to conclude that the Raman modes of anatase, in particular the E2 based Raman thermometer are compatible with data reported in literature [The performances obtained with this TiOterature ,44; the Temperature is an important parameter influencing physical, chemical and biological processes: For this reason, the investigation of new materials, with enhanced performances, together with the definition of the more performing experimental protocol is a hot topic in the nanothermometry field. The experimental work reported in this article will contribute to the development of a new Raman based biocompatible nanothermometer, by investigating the optical performances of titanium dioxide, as anatase, with Raman technique.Test Sample. Repeated anti-Stokes and Stokes measurements, have been performed on various positions of the sample at room temperature (~297 K), with an incident laser power of 1.5 mW. An excellent agreement between the temperature derived from the anti-Stokes/Stokes ratios and the expected temperature was found, with a standard deviation of repeated temperature measurements calculated to be in between 1 and 3 K, for the most intense peak, located at 143 cm−1, which has been demonstrated to be the most sensitive to temperature. This titanium dioxide mode seems to be an excellent candidate for the local temperature detection in the visible range from 488.0 to 647.1 nm, reaching the highest sensitivity in the red region.The spectroscopic characterization of titanium dioxide has been carried out in the visible range, at 488.0, 514.5, 568.2 and 647.1 nm, and the Raman-active modes have been investigated to find the more performing one, as temperature sensor. Both Stokes and anti-Stokes spectra were collected at different temperature, input power and wavelengths, to investigate the temperature range, the temperature resolution, the eventual self-heating (due to the input laser power) of the sample and to identify the working range of the nanothermometer. A key point for the identification of the local temperature is the calibration of the experimental set-up, which allows defining the best experimental protocol. The calibration procedure has been conducted by controlling the sample temperature with a temperature-controlled stage and exploring the Raman signals in the temperature range of 283–323 K (with 5 K increment), as it is of interest for biological applications. The obtained values of the anti-Stokes/Stokes ratio allow the determination of the calibration constant, specified for all anatase Raman modes at each excitation wavelength. The calibration constant permits to determine the sample local temperature and to identify the power range where the local temperature is not affected by the laser power. Working with an incident laser power higher than 2 mW the sample experiences self-heating, while at lower power samples do not experience any self-heating. The validation of the proposed protocol has been finally achieved with the analysis of the Raman spectra of the


Neocortical pyramidal cells have three key classes of excitatory input: forward inputs from the previous cortical area ; recurrent collateral synapses from nearby pyramidal cells; and backprojection inputs from the following cortical area. The neocortex performs three major types of computation: (1) unsupervised learning of new categories, by allocating neurons to respond to combinations of inputs from the preceding cortical stage, which can be performed using competitive learning; (2) short-term memory, which can be performed by an attractor network using the recurrent collaterals; and (3) recall of what has been learned by top–down backprojections from the following cortical area. There is only one type of excitatory neuron involved, pyramidal cells, with these three types of input. It is proposed, and tested by simulations of a neuronal network model, that pyramidal cells can implement all three types of learning simultaneously, and can subsequently usefully categorise the forward inputs; keep them active in short-term memory; and later recall the representations using the backprojection input. This provides a new approach to understanding how one type of excitatory neuron in the neocortex can implement these three major types of computation, and provides a conceptual advance in understanding how the cerebral neocortex may work.The online version contains supplementary material available at 10.1007/s00429-021-02347-z. How the cerebral cortex performs its functions is a key question in neuroscience. A small region of neocortex can perform several crucial functions, namely learning new categories to represent the inputs; short-term memory; top–down recall of information; and top–down attention. The neuroanatomy of the neocortex suggests that it has one main type of principal excitatory neuron, pyramidal cells (supplemented by granule cells in layer 4). In this paper, the question that is asked is whether the known microconnectivity of the neocortex can perform all of these functions with just one main type of excitatory neuron, pyramidal cells.A number of detailed models and summaries of neocortical microarchitecture have been described , and can build sparse distributed representations with a population of neurons active for any one input . A key part of the theory proposed here of the computational operation of neocortical pyramidal cells is that these three neuronal architectures can operate together, on the same single set of neurons, in this case pyramidal cells. One particular issue that is investigated in the simulations is whether the system illustrated in Fig. Each of these neuronal network architectures, competitive learning, attractor networks, and pattern association learning, are normally investigated separately, and programs to illustrate their operation separately are available Rolls , 2021b   to test whether the recurrent collaterals operated correctly as an attractor network to maintain the firing rates in a short-term memory with stable retrieval, i.e. in a stable fixed point.To test whether the top–down inputs had learned correctly by pattern association learning, each of the 28 backprojection inputs was presented serially in turn with no forward input, to investigate whether the categorised outputs appropriate for each of the 28 forward inputs were correctly recalled by the backprojection inputs.The predictions that were tested in the simulations were as follows. First, in the architecture shown in Fig. The full details of the implementation of the network, and the results that are obtained with it, are evident in the Matlab program CompetitiveAttractorBPNetDemo.m that accompanies this paper.The 28 training patterns are shown in Fig. The competitive network operated as expected even though this was a combined architecture, and learned in this case 4 categories of output rate vectors for the 28 overlapping input patterns. Figure The attractor network functionality operated perfectly even though this was a combined architecture, with the output firing produced by the forward input continuing indefinitely without change when the forward input was removed. This is illustrated in the program CompetitiveAttractorBPNetDemo.m, and is not further illustrated, as the firing remains the same as that illustrated in Fig. Figure Top–down attention is implemented in this system using the backprojection inputs. Top–down attention to one of the stimuli would be produced by a weak backprojection signal, which produced weak activation of some of the neurons through the backprojection synapses trained as described here. If the forward inputs that are applied simultaneously are also weak, then the input stimulus that wins the competition is influenced by the top–down, back-projected, recall signal. The exact regimes in which these interactions between the top–down and bottom up signals produce top–down attention have been analysed elsewhere  scale factor of the recurrent collateral inputs relative to the forward inputs was in the region of 0.1 (with considerable tolerance within a range of 0.02–0.2); and when the (fixed) scale factor of the backprojection inputs relative to the forward inputs was 0.1 (with considerable tolerance within the range 0.1–0.15). These scale factors for the different classes of input to a neuron might be set biologically by processes such as the fact that the backprojection inputs are received on the apical dendrites in layer 1, and will tend to be shunted by any forward inputs lower down the dendrite and closer to the cell body.The fixed parameter learning rate The program runs with a new random seed every time it is run. There is, therefore, some difference from run to run, due to the different random initialisation of the synaptic weights. Usually, four categories are formed from the 28 forward input patterns, but sometimes five, with the standard set of parameters. The actual neurons that respond to each category of forward input stimulus vary from run to run. These results show that the findings described are computationally robust. Moreover, the range of parameters within which similar generic results was found was as described quite wide, providing further evidence on the robustness of the findings and network described here.The important conclusion of this research is that it is possible to combine in the same neurons in a model of neocortical computation: (1) categorisation using competitive learning; with (2) attractor network dynamics to maintain the firing when the forward input is removed; and with (3) pattern association learning of backprojection inputs to implement memory recall .The research also showed that the system operates well when all the synaptic weights are initialised to random values. Such random synaptic weights or diluted connectivity are important for competitive learning, by breaking the symmetry between neurons Rolls . But norThe research also shows that the synaptic weight normalisation that is typically used in competitive networks Rolls , 2021b dThere is considerable research that each of the computational processes described here is important for understanding the operation of the cerebral cortex.For example, competitive learning is the key computation that when combined with slow learning over transforms of objects enables transform-invariant visual object recognition to occur in a model of the ventral visual system, VisNet Rolls ; c.Pattern association learning between backprojections from the hippocampal system to the neocortex Rolls  providesTop–down influences of the type implemented by backprojections are fundamental for biologically plausible models of top–down attention Below is the link to the electronic supplementary material.


Kidney renal papillary cell carcinoma (KIRP) is a type of low-grade malignant renal cell carcinoma. Huge challenges remain in the treatment of KIRP. Cell division cycle associated 3 (CDCA3) participates in human physiological and pathological processes. However, its role in KIRP has not been established. Here, we evaluated the prognostic value of CDCA3 in KIRP using a comprehensive bioinformatics approach. Data for CDCA3 expression in KIRP were obtained from online database. Different expression genes between high and low CDCA3 expression groups were identified and evaluated by performing Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. A gene set enrichment analysis was performed to elucidate the function and pathway differences between the different. Differences in immune cell infiltration between low and high CDCA3 expression groups were analyzed by a single-sample GSEA method for immune cells. A protein-protein interaction network was generated and hub genes were identified. UALCAN was used to analyze associations between the mRNA expression levels of CDCA3 in KIRP tissues with clinicopathologic parameters. The diagnostic efficacy of CDCA3 for KIRP was analyzed by ROC analysis. Logistic regression was used to analyze relationships between the clinicopathological characteristics and CDCA3 expression. Our results indicated that high CDCA3 mRNA expression is significantly associated with some clinicopathologic parameters in KIRP patients High CDCA3 mRNA expression associated with a shorter overall survival, progression-free interval, and disease-special survival. Taken together, CDCA3 is a potential target for the development of anti-KIRP therapeutics and is an efficient prognostic marker. Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, and its morbidity and mortality are on the rise worldwide . Kidney Currently, imaging examinations such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) are used to diagnose KIRP, however, these approaches do not show sufficient specificity . TherefoAlthough KIRP has a low rates of metastasis and recurrence , 11, proGene encoding CDCA3 is located on chromosome 12p12 and the protein is composed of 268 amino acids with a molecular weight of 29 kDa. CDCA3 contributes to human physiological and pathological processes by regulating various downstream cytokines. Studies have shown that CDCA3 plays an important role in the development of various tumors –19. HoweIn this study, we addressed this issue by identifying the transcriptional expression patterns of CDCA3 based on The Cancer Genome Atlas (TCGA) database and the Genotype-Tissue Expression (GTEx) database. We further evaluated Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of CDCA3 related to CDCA3 and associated differential expression genes (DEGs) in KIRP. Furthermore, we performed a gene set enrichment analysis (GSEA), immune infiltration analysis, protein-protein interaction (PPI) network analysis, clinicopathologic analysis, and analyzed the prognostic value of CDCA3 in KIRP. Our study clarify the biological functionality and prognostic value of CDCA3, which is expected to be beneficial for the diagnosis and treatment of KIRP.P < 0.001 were considered statically significant.The TCGA database was used to investigate CDCA3 expression in patients with KIRP and analyze the association between expression levels and the prognosis. In total, 320 samples were selected as the TCGA cohort including 288 KIRP samples and 32 normal samples. Level 3 high-throughput RNA-sequencing data and corresponding clinical information data were downloaded from the KIRP project of the TCGA GDC data portal. RNAseq data in FPKM (fragments per kilobase per million) format were converted into TPM (transcripts per million reads) format for comparisons of CDCA3 expression levels between samples. The Wilcoxon rank-sum test was used to compare the gene expression levels of CDCA3 in 32 normal samples and 288 KIRP samples and between 31 KIRP samples and the paired adjacent normal tissues were compared. Results with https://xenabrowser.net/datapages/), and these data were processed in a unified way through the Toil process [P < 0.001 were considered statically significant.RNAseq data were downloaded in TPM format from UCSC XENA  in KIRP from TCGA database, all KIRP samples were divided into two groups: CDCA3-high expression group and CDCA3-low expression group. The DESeq2 package  was usedhttp://metascape.org) was used to analyze the functional and pathway enrichment of DEGs and generate PPI networks associated with CDCA3 alterations in KIRP. GO and KEGG pathways enrichment was analyzed using Metascape [P < 0.01, a minimum count of 3, and the enrichment factor > 1.5 were thresholds for statistical significance.Metascape  was used to analyze the functional interactions between proteins [The STRING database  , and the ordinate was the true positive rate (TPR).P < 0.05 were considered statically significant.The prognostic value of the CDCA3 mRNA expression level in KIRP was analyzed using the survminer package of R. Based on the median values of CDCA3 expression (TPM), patients with KIRP were divided into CDCA3-low expression group and CDCA3-high expression group. Results with As all data used in this study were obtained from the TCGA database. Hence, ethics approval and informed consent were not required. Our study was performed in accordance with the publication guidelines of TCGA.All statistical analyses and the generation of plots were performed using R (v.3.5.1). The Wilcoxon rank-sum test and Wilcoxon signed-rank test were used to compare the expression of CDCA3 in unpaired samples and paired samples, respectively. The Kruskal-Wallis test, Wilcoxon signed-rank test, and logistic regression were used to evaluate the relationships between clinical-pathologic features and CDCA3 expression. Cox regression analyses and the Kaplan-Meier method were used to evaluate prognostic factors. A multivariate Cox analysis was used to evaluate the impact of CDCA3 expression on survival along with other clinical traits.We analyzed CDCA3 expression in normal samples from the GTEx database and the TCGA and 33 tumor samples in TCGA. CDCA3 expression was significantly up-regulated in bladder urothelial carcinoma, cervical squamous cell carcinoma and adenocarcinoma, KIRP, KIRC, and other cancer types . An analTo detect the differences in the CDCA3 mRNA expression level between tumor and non-cancerous tissues, RNAseq data for 288 KIRP samples and 32 normal samples were analyzed. As was shown in These results indicated that the expression of CDCA3 is up-regulated in various types of tumor tissues, including KIRP, in which it is significantly overexpressed compared with levels in normal kidney tissues or paired paracancerous normal samples.padj < 0.05 between groups. A volcano graph was generated to visualize the results of the DEGs analysis. Among the DEGs, 565 had logFC > 1.5 and padj < 0.05, and 174 had logFC < -1.5 and padj < 0.05 , GO: 0048285 (organelle fission), GO: 0000280 (nuclear division), GO: 0003002 , GO: 0140014 (mitotic nuclear division), GO0001708 (cell fate specification), GO: 0048663 (neuron fate commitment) and GO: 0048665 (neuron fate specification) were significantly associated with alterations in CDCA3 expression . AdditioIn a KEGG analysis, these pathways including hsa04080 (Neuroactive ligand-receptor interaction), hsa04727 (GABAergic synapse), hsa05032 (Morphine addiction), hsa04960 (Aldosterone-regulated sodium reabsorption) and hsa04950 (Maturity onset diabetes of the young) pathways associated with CDCA3 function in KIRP .p.adj = 0.013, FDR = 0.005), HALLMARK_MITOTIC_SPINDLE , HALLMARK_KRAS_SIGNALING_DN , and HALLMARK_G2M_CHECKPOINT . The data sets were significantly enriched in CDCA3-high expression group.GSEA was used to identify signaling pathways involved in the difference between CDCA3-low expression group and CDCA3-high expression group in KIRP. + T cells, T cells, Tcm, pDCs, T helper cells and Th2 cells  and the TCGA combined with the GTEX database (AUC = 0.823) , 8B.p < 0.001), clinical N stage (p = 0.003), clinical M stage (p = 0.041), and Clinical stage (p = 0.027)  .p < 0.001) (p < 0.001) and DSS analysis (HR = 15.90(3.77-67.05), p < 0.001) analyses , progression-free interval (PFI), and disease-specific survival (DSS) in CDCA3 expression subgroups in KIRP. High CDCA3 expression in KIRP associated with a worse OS (HR = 3.75(1.93-7.31), < 0.001) . Similaranalyses , 9C.p = 0.012), clinical M stage (p = 0.008), and CDCA3 expression (p = 0.017) were independent factors associated with a poor OS , clinical N stage (p < 0.001), clinical M stage (p < 0.001), clinical stage (p <0.001), gender (p = 0.026), hemoglobin (p = 0.039), and CDCA3 expression (p < 0.001) were associated with a worse PFI. A multivariate Cox regression further showed that the clinical N stage (p = 0.006) and CDCA3 expression (p = 0.017) were independent prognostic factors based on PFI (p = 0.012), clinical M stage (p = 0.008), and CDCA3 expression (p = 0.017) were independent factors associated with a poorer DSS , and the M0 subgroup of the clinical M stage (HR = 3.307(1.446-7.563), p = 0.005), clinical stage II, stage III, and stage IV subgroups of clinical stage (HR = 10.106(2.326-43.908), p = 0.002), male subgroup (HR = 3.189(1.494-6.807), p = 0.003), female subgroup (HR = 5.959(1.315-26.995), p = 0.021), white subgroup of race (HR = 3.684(1.759-7.717), p < 0.001), age less than 60 years old subgroup (HR = 14.831 (1.979-111.161), p = 0.009), age over 60 years old subgroup (HR = 3.176 (1.413-7.138), p = 0.005), non-smoking subgroup (HR = 4.250 (1.426-12.664), p = 0.009), smoking subgroup (HR = 3.173 (1.155-8.715), p = 0.025), normal serum calcium subgroup (HR = 6.427(1.775-23.269), p = 0.005), elevated and low hemoglobin subgroup (HR = 4.488(1.735-11.607), p = 0.002), left laterality subgroup (HR = 4.132(1.813-9.421), p < 0.001), and wild type subgroup (HR = 3.536(1.746-7.160), p < 0.001). The analyses of the prognostic value of CDCA3 expression in each KIRP subgroup based on PFI and DSS also yielded similar results.Finally, we analyzed the prognostic value of CDCA3 expression based on OS, PFI, and DSS in each clinicopathological subgroups of KIRP. As shown in KIRP accounts for approximately 10-20% of RCC cases. KIRP tends to occur in individuals over 50 years of age and affects more men than women, with a genetic predisposition. KIRP is typically discovered incidentally during physical examination. Some patients have typical clinical manifestations of RCC, such as hematuria, lumbago, and abdominal masses. The pathological features of KIRP are solid tumors in the renal cortex with clear boundaries. . The proSeveral cytokines, hormones, and proteins are involved in the development and progression of RCC and KIRP. Galectin-3 is widely expressed in RCC, and promotes the invasiveness, and suggestiveness via CXCR2, thereby affecting the occurrence and development of RCC . Activatin vivo and in vitro decreases the proliferation of colorectal cancer cells [in vivo and in vitro promotes the growth and colony formation ability of gastric cancer cells, while inhibiting CDCA3 expression mitigates these effects [CDCA3, as a part of the skp1-cullin-f-box ubiquitin ligase complex, regulates the cell cycle by acting as an endogenous cell cycle inhibitor. CDCA3 participates in human physiological and pathological processes via regulating various downstream cytokines, hormones, and proteins. As shown in er cells . In parter cells . CDCA3 e effects . Further effects . In leuk effects . CDCA3 i effects  and its  effects .The prognosis value of CDCA3 in KIRP remains unclear and was the focus of this study. We observed that CDCA3 in KIRP tissues was significantly up-regulated compared to level in normal or paired paracancerous normal tissues , 1D. OurStudies have shown that the expression levels of CDCA3 and CENPF are correlated in esophageal carcinoma . Levels in silico analyses were performed, further in vivo and in vitro studies are required to verify our results. Second, we found that CDCA3 was related to KIRP and could be used as a potential predictor of the prognosis. However, the underlying mechanisms by which CDCA3 regulates the occurrence and development of KIRP remains unclear. Further research studies to reveal the detailed mechanism underlying the relationship between CDCA3 and KIRP.Despite presenting some credible data and experimental evidence, this study has some limitations. First, all the data were obtained from online databases and only In conclusion, our results showed that CDCA3 is overexpressed in KIRP. The infiltration of various immune cells was notably related to CDCA3 mRNA expressions. Moreover, CDCA3 was significantly associated with the clinical T stage, clinical N stage, clinical M stage, clinical stage, age and hemoglobin in KIRP. Furthermore, high expression level of CDCA3 were significantly related to a shorter OS, PFI, and DSS in KIRP. Accordingly, CDCA3 is a potential target for the development of anti-KIRP therapeutics and an efficient prognostic marker for KIRP.


Together, these pathways form an extensive communication network that regulates basic cell activities and coordinates the function of multiple cells or tissues. The process of cell signaling imposes many demands on the proteins that comprise these pathways, including the abilities to form active and inactive states, and to engage in multiple protein interactions. Furthermore, successful signaling often requires amplifying the signal, regulating or tuning the response to the signal, combining information sourced from multiple pathways, all while ensuring fidelity of the process. This sensitivity, adaptability, and tunability are possible, in part, due to the inclusion of intrinsically disordered regions in many proteins involved in cell signaling. The goal of this collection is to highlight the many roles of intrinsic disorder in cell signaling. Following an overview of resources that can be used to study intrinsically disordered proteins, this review highlights the critical role of intrinsically disordered proteins for signaling in widely diverse organisms , in every category of cell signaling pathway  and at each stage  in the cell signaling process. Thus, a cell signaling pathway cannot be fully described without understanding how intrinsically disordered protein regions contribute to its function. The ubiquitous presence of intrinsic disorder in different stages of diverse cell signaling pathways suggest that more mechanisms by which disorder modulates intra- and inter-cell signals remain to be discovered.Signaling pathways allow cells to detect and respond to a wide variety of chemical (e.g. Ca A wide variety of signaling pathways form a vast communication network that regulates basic cell activities and coordinates biological functions within an organism. Some signaling pathways even allow chemical communication between different organisms or species. Cells communicate with each other to coordinate a variety of functions between cells, tissues, and organs, and all cells must respond to environmental cues. Many mechanisms have evolved to transmit these signals. Extra-cellular signaling involves releasing or displaying any of a variety of chemicals, which are specifically recognized by a receiving cell that then activates an intracellular signal transduction pathway to respond to the signaling event. These events typically rely upon a series of protein–ligand and protein–protein interactions. Such signals must effectively propagate across long distances and even across barriers, such as the cell membrane. Each step in the signaling pathway must be highly specific, both to preserve the integrity of the signaling pathway’s ability to generate a reliable response, and to ensure that the pathway is not erroneously activated. Because a cell’s response to a signal must be transitory, each of these molecular interactions must be reversible. Ligand recognition must both augment and stabilize the response to the signal. These requirements of cell signaling impose unique, and often conflicting requirements on the proteins that constitute the signaling pathways, such as signal integrity versus crosstalk. These apparent conflicts extend to the individual interactions that propagate the signal, an apparent conflict between specificity (large interaction interface) versus reversibility (low free energy of interaction).These conflicting needs have been resolved to a large extent by including intrinsically disordered proteins in cell signaling pathways through biological evolution. Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of proteins fail to fold into stable, defined structures as free monomers. Numerous functional advantages of intrinsic disorder are outlined in dedicated studies -like organization of the cellular signaling, which represents a complex network of networks, with even a single node in a protein–protein interaction network representing a multileveled network itself . Here, aUnder physiological conditions, ordered proteins are known to possess unique three-dimensional 3D) structures, in which each atom of the polypeptide chain has a unique position in space. These structures are determined by a vast number of intrachain non-covalent side chain-side chain, side chain-backbone, and backbone-backbone interactions. Such structure-determining conformational interactions include hydrophobic interactions, hydrogen bonding, Van der Waals forces, and ionic/electrostatic interactions. These interactions have different physico-chemical natures and their strengths depend on the amino acids involved and on the peculiarities of the interactions of a polypeptide chain with solvent. Kinetically, the self-organization of a protein molecule from its unfolded state to a unique 3D structure represents a complex sequential process accompanied by the formation of several partially folded intermediates –62. FurtD structuThe situation is further complicated by the fact that not only the entire protein, but its different parts can be disordered to different degrees. As a result, IDPs are not homogeneous, but represent a very complex mixture of a broad variety of potentially foldable, partially foldable, differently foldable, or not foldable segments –72. MoreAgain, this structural heterogeneity is determined by specific features of the amino acid sequences of IDPs/IDRs. In fact, in comparison with ordered proteins and domains, most IDPs/IDRs are systematically depleted in order-promoting residues , while being simultaneously enriched in disorder-promoting residues  , 76–82 aApplication of these algorithms to various proteomes indicate that IDPs and IDRs are abundantly present in any given proteome, although eukaryotes have much more disorder than prokaryotes , 103–107Contrarily to mostly catalytic, transport, and protein interaction domain functions of ordered proteins and domains , 109, IDMPM [Often, disorder-to-order-transition-based recognition is mediated by the specific functional elements known as molecular recognition features (MoRFs) –122, whiMPM , and MoRMPM .An alternative approach has been to identify DBRs from their sequence patterns or motifs. Examples of this approach include eukaryotic linear motifs (ELMs) , 140, liProtein–protein interactions have been further studied by high throughput methods such as the yeast 2 hybrid assay . Such hiHub proteins have been shown to employ IDRs for multiple partner binding using two completely different mechanisms : namely,There are also computational tools for predicting disorder-based sites responsible for interaction with RNA and DNA , which links cell wall synthesis and cell wall integrity with morphogenesis and cell cycle progression . Both thIDPs/IDRs are found in pathways initiated by a variety of molecular signals, ranging in size from single-atom ions, small molecules such as steroid hormones, and biomacromolecules like nucleic acids and proteins , 167. ThIons Multiple proteins in the calcium signaling pathway are intrinsically disordered. First, calcium channels permit the passive transport of Ca2+ into a cell, either by voltage-gated and/or ligand-gated mechanisms. In the spine, the N-methyl-D-aspartate (NMDA) receptor is a tetrameric Ca2+ ion channel which induces different cellular responses—long-term potentiation or long-term depression—based on the intracellular concentration of Ca2+ and frequency of stimulation by which it is activated [2+ from blocking NMDA receptor activity [2+ binds calmodulin, and either increases synapse response (long-term potentiation) or decreases synapse response (long-term depression). Protein complexes formed by the intrinsically disordered intracellular tail of the NMDA receptor modulates the cellular response to NMDA activity. High concentrations of Ca2+ activate calmodulin-dependent kinases, such as calmodulin-dependent kinase II, and thus long-term potentiation, whereas low concentrations of calcium activate the only phosphatase, calcineurin, and thus stimulate long-term depression [ctivated . NMDA reactivity , and binpression –173.2+ trafficking through the channel [The intrinsically disordered long C-terminal tail of the NMDA receptor also regulates calcium signaling by altering the properties of the channel and Ca channel . This ta channel  and calp channel , while P channel , 171. Th channel . This pr channel .2+ sensors and responders [2+ but the absence of calmodulin, the regulatory domain is unfolded and solvent-exposed. Calmodulin binding to the regulatory region induces folding to a helix, and release of an auto-inhibitory domain from the active site of calcineurin. Such coupling of protein (or ligand) binding with protein folding is another common feature of intrinsically disordered proteins [Calcineurin provides a second example of the role of intrinsic disorder in ion signaling, which is described detail by Trevor Creamer in this collection . Togethesponders –180. Botsponders . In the proteins , 175.Hormones Coupling ligand binding to the folding of an intrinsically disordered region expands the range of binding free energies at which allosteric regulation of protein function can occur [an occur . One proan occur , 177. Than occur . AF1 is an occur ). Interean occur .Fig. 2InThe presence of intrinsically disordered regions in a protein allows the allosteric response to be tuned . Prior tThe disorder-to-order transition in the N-terminal domain is regulated by ligand binding and many other factors as well, including interactions with many other proteins, DNA binding, and phosphorylation of the AF1 domain , 183. ThIntrinsic disorder in the GR receptor not only enables multiple allosteric regulatory interactions to impact function, but also allows deployment of different surfaces of the protein to enable binding to many different sets of macromolecules, and regulation of these interactions via mRNA splicing and phosphorylation . In addiLipids In addition to forming membranes and storing energy, lipids can also activate or regulate cell signaling. Lipid-activated cell signaling also relies on IDPs/IDRs. For example, the Phosphatase and Tensin homolog (PTEN) protein inhibits signaling via the PI3K/AKT/mTOR proliferative pathway, in which phospholipids act as a second messenger. PI3 Kinase bound to an activated receptor phosphorylates PIP2 to create PIP3, which in turn activates cell proliferation and survival via AKT and promotes cytoskeletal changes via Rac/Rho/cdc42. PTEN dephosphorylates PIP3 to inhibit signaling and thus proliferation. Consequently, PTEN is not only a powerful tumor suppressor, but also a prognostic marker that predicts response in many human cancers [ cancers . Inactiv cancers . Recruit cancers ). Intere cancers .2 binding site, and a structured catalytic domain followed by an intrinsically disordered auto-inhibitory C-terminal tail which culminates in a PDZ binding domain [The PTEN protein contains an N-terminal PIPg domain , 189. Thg domain , 190. Phg domain . The disg domain . Interacg domain , 192.Multiple alternate translation and splicing start sites in the PTEN gene creates many versions of the protein, most notably PTEN-L, which contains an extra 173 amino acids on the N-terminus , 193. ThProteins Signaling pathways activated by proteins often are regulated by IDPs/IDRs in multiple steps of the pathway. In canonical cell signaling, an extracellular ligand is recognized by a membrane protein which transmits the signal, typically through phosphorylation through a series of cytoplasmic/nuclear proteins, culminating in the regulation of a transcription factor which alters transcription of specific genes. The use of proteins as the signal provides extra regulatory opportunities through modulating signal production (transcription and translation), signaling transport and availability via binding to extracellular matrix proteins, and signal activity via PTMs.There are many categories of proteins that serve as cell signals. For example, cytokines are small secreted immunomodulatory protein signals. Osteopontin is a multifunctional cytokine with key roles in inflammation, cell viability, and tissue repair, which also functions as a bone matrix protein that mediates osteoclast adhesion , 197. Thvegf mRNA is alternatively spliced to produce a family of protein isoforms with varying affinities for different VEGF receptors [vegf-a alter the lengths of these tails (e.g. VEGF165 versus VEGF143). The VEGF “B” splice variants are generated by use of an alternate 3’ acceptor site for exon 8, lengthening the intrinsically disordered C-terminal tail by an additional 6 amino acids  [165 is a potent stimulator of angiogenesis, VEGF165b binds VEGF receptors but fails to activate them in a robust, sustained manner. By ineffectively occupying a binding site on the receptor, the VEGFXXXb isoforms inhibit the function of the VEGFXXX variants [While cytokines regulate the immune system, growth factors are protein signals that primarily target other types of cells. Vascular Endothelial Growth Factor (VEGF) regulates angiogenesis, and thus plays significant roles in animal development, wound healing, and carcinogenesis. The eceptors . Active EGF165b) , 200. Thvariants , 200. Invariants .Multiple chemical stimuli Some signaling pathways are capable of responding to a variety of chemical stimuli. For example, the GPCR-G protein signaling system is a complex machine responsible for the recognition of a wide variety of extracellular signals and controls various cellular responses to these signals by triggering the numerous intracellular signaling cascades. The complexity of this machinery is determined by the multitude of the members of the GPCR family  thant GPCRs , 204. Atnt GPCRs –210. In nt GPCRs . Furthernt GPCRs . All thint GPCRs . In factnt GPCRs . Therefont GPCRs . Finallynt GPCRs .Environmental conditions as signals Unlike many structured protein domains, the function of IDPs/IDRs can persist in extreme environmental conditions. This trait allows IDPs to reliably sense extreme conditions and instigate responsive signaling pathways [pathways . The proLight Plants must sense and adapt to light in order to optimize energy production, to limit photodamage, and to set/maintain their circadian clock. The UVR8 photoreceptor in plants is crucial for generating photomorphogenic and protective responses to UV light [UV light . The UVRUV light . UVR8 isUV light . ConversEctothiorhodospira halophila. This water-soluble ~ 14 kDa protein contains a thioester-linked p-coumaric acid cofactor and acts as the photosensor [trans/cis isomerization of a double-bond in the chromophore triggers a cycle of structural events yielding a long-lived, blue-shifted intermediate (known as pB) with a life-time on the order of 1 s [To avoid harmful light exposure, blue light is used by free-swimming bacteria to modulate both the length and directionality of their run . To thistosensor –218. Upor of 1 s , 219. Hir of 1 s . Althougr of 1 s .Redox potential The conditionally disordered chloroplast protein of 12 kDa (CP12), found in the chloroplasts of photosynthetic organisms such as plants, cyanobacteria, algae, and cyanophages. CP12 regulates the Calvin-Benson-Bassham cycle, which is a series of redox reactions that converts carbon dioxide into glucose [ glucose . The extMechanical forces Many cellular processes that are regulated by chemical stimuli, such as proliferation, differentiation, motility, and survival, are also influenced by the mechanical properties of the substrate supporting the cells [he cells . Mechanohe cells . Mechanihe cells . The ABEhe cells . This sthe cells .In a second example, the mouse protein CasSD includes an intracellular, proline-rich disordered domain. In the absence of mechanical stretching forces, this region formed polyproline II helices hypothesized to bind LIM domain proteins, thus protecting CasSD from phosphorylation. Application of mechanical stretch has been proposed to unfold the PPII conformation, precluding LIM protein binding, thus enabling CasSD phosphorylation and signal propagation . MechanipH The low pH of the mammalian stomach is one of the natural barriers to protect the organism from microbial infection. Enteric bacteria, such as E. coli, have adapted to sense, respond to, and survive in acidic environments. Sudden immersion in acid requires an immediate adaptive response. Thus, the extended signaling pathways used in non-life-threatening situations in other organisms are reduced to single protein sense-and-respond units to detect acid in bacteria. For instance, the CadC protein is a pH-responsive one-component signaling system composed of an N-terminal winged helix-turn-helix DNA binding domain, followed by a 50 amino acid intrinsically disordered region linking to a single transmembrane helix and ending in a C-terminal pH sensing domain which extends into the periplasm [E. coli periplasmic protein HdeA behaves as an acid-inducible chaperone [eriplasm . Intriguhaperone . At neutHydration Dehydrins are a family of intrinsically disordered proteins that act as effectors whose expression is induced by signaling pathways that sense abiotic stresses, such as cold or draught. Dehydrins protect plants from dehydration and from cold [rom cold . Althougrom cold . Hydrophrom cold . Dehydrirom cold . In vitrrom cold , 229, whrom cold .Heat The dehydrin protein family discussed above may also protect from heat. The Arabidopsis thaliana dehydrin Early Response to Dehydration 14 (ERD14) can increase the viability of E. coli subjected to 15 min at 50 °C [at 50 °C . This prBased on the source of the signal and the relative location of the responding cell, cell signaling is divided into autocrine, juxtacrine, intracrine, paracrine, and endocrine pathways. Despite the large variety of signal transmission mechanisms used, IDRs/IDPs act as crucial components in each of these different categories.Intracrine signaling self-regulates by producing hormones that bind intracellular receptors. Thus the cell stimulates itself because the signal, and hence the signaling cascade, never crosses the cell membrane. The nature of intracrines can vary: structurally diverse hormones , growth factors, DNA-binding proteins, and enzymes all can have intracrine activity . Further2+, which is present outside neurons, creates distinct conformations and dramatically alters prodomain oligomerization.Autocrine signaling occurs when a cell is stimulated by a signal that was produced and secreted by that same cell. Autocrine brain-derived neurotrophic factor (BDNF) has been implicated in the structural and functional plasticity of dendritic spines . BDNF alDrosophila Suppressor of Hairless, and C. elegans Lag-1), the coactivator Mastermind, and Notch target DNA [Juxtacrine signaling requires contact between cells, allowing a ligand on one cell surface to bind a receptor on an adjacent cell. In metazoans, Notch signaling plays key roles in early stages of embryonic development, as well as development of the cardiovascular system, the central nervous system, and the respiratory system, among others, and has well-defined roles in cancer progression , 250. Norget DNA . Within rget DNA .A specialized type of juxtacrine signaling is enabled by gap junctions. Gap junctions allow ions and small metabolites to exchange between adjacent cells, and are formed by two connexin proteins, one provided by each cell . The intParacrine signaling involves the release of diffusible chemical signals to communicate between nearby cells in which cell–cell contact is not required. One prominent example is neurotransmission. Glutamate is the primary neurotransmitter for excitatory stimulation. Signaling via glutamate is a critical component of long-term potentiation and long-term depression, which alter the strength of downstream signaling in response to glutamine binding to receptor. These adaptations are crucial events in learning and the formation of memory . GlutamaDrosophila body-plan-controlling developmental protein (Winged) were identified as homologues, leading to the portmanteau Wnt as the family name for these proteins [In endocrine signaling, endocrine cells produce signals that target distant cells in the body. Most of the intrinsically disordered proteins with well-studied roles in signaling operate in endocrine signaling pathways. The Wnt signaling pathway exemplifies how intrinsic disorder can play multiple roles in a single pathway Fig. . A mouseproteins . The Wntproteins . While sproteins .Prior to Wnt signaling, the transcription factor β-catenin is maintained at low levels by the β-catenin destruction complex, which is an assembly of the 852 residue mostly disordered scaffold protein axin, the 2,843 residue massively disordered protein adenomatous polyposis coli (APC), and the three mostly structured proteins β-catenin, casein kinase Ia (CKI-a), and glycogen synthetase kinase 3b (GSK3b). The two kinases phosphorylate residues within a long IDR at β-catenin’s amino terminus , 260. InThe two kinases and β-catenin bind to Axin’s long intrinsically disordered region, thereby connecting structured proteins with flexible linkers. Random movements of the bound proteins enabled by the flexible linkers bring about frequent kinase-substrate collisions, which, in turn, lead to efficient phosphorylation. Thus, the destruction complex works by random movements of a ‘‘stochastic machine,’’ not by cooperative conformational changes .Laboratory experiments show that a 55 residue segment of axin containing binding sites for both β-catenin and GSK3b has the biophysical properties of an unstructured protein , which iUpon encountering the target cell, Wnt binds to two co-receptors, the seven transmembrane helix protein Frizzled (Fzd) and the single pass lipo-related-receptor protein 5/6 (LRP5/6). This binding promotes recruitment of the scaffolding protein disheveled (Dvl), which results in the phosphorylation of the cytoplasmic domain of LRP5/6, a domain whose disorder , 265 refDue to its prominent physiological function, the Wnt/β-catenin signaling must be strictly controlled because its dysregulation, which is caused by different stimuli and also by many different mutations that lead to alterations in cell proliferation, apoptosis, inflammation-associated cancer or alterations in stem cell proliferation or self-renewal, for both embryonic and various types of adult stem cells .The sections above highlight the different structures of cell signaling pathways. Intrinsic disorder may be present, and provide regulatory opportunities, for any of the following steps: ligand production, ligand activity, ligand bioavailability, receptor structure, intracellular transmission, termination/intracellular trafficking, and effector proteins Fig. . Indeed,Ligand production The production of many signaling molecules is highly regulated at the level of gene transcription. Furthermore, the transcription factors involved are often regulated by other signaling pathways , it is Integrins not only mediate two-way communication between the cell interior and the extracellular matrix, but they also regulate ion channels, growth factor receptors, and the activity of cytoplasmic kinases . These rSimilar regulatory mechanisms have been established for G-Protein Coupled Receptors Fig. , which wLarge multi-site docking proteins (LMDs) leverage the protein binding capacity of intrinsically disordered tails. Many cell signaling pathways require large multi-site docking proteins to transduce signal from the activated receptor to downstream intracellular effectors [ffectors . Signaliffectors . Scaffolffectors . Thus, Lffectors , 308. Foffectors , 309. Foffectors . Gab2 isffectors  and tranffectors , 312. Upffectors .Although most of the 74 kDa Gab2 protein is intrinsically disordered, it does contain a folded PH domain at its N-terminus, which anchors Gab2 to the membrane via interactions with the lipid PIP3 . Gab2 fuProteins that bind to Gab2 often contain an SH2 protein interaction domain , 313. SHIntracellular transmission of signals relies on a series of protein interactions. Many kinases include IDRs, which facilitate the intermolecular interactions critical for the function and specificity of the signaling cascade . For intThe function of K-Ras, a member of the Ras family with a lysine-rich tail, can be affected by alterative mRNA splicing which alters the amino acid sequence of the tail . K-Ras4B is an oncogenic isoform of Ras, in which GTP hydrolysis, aided by the GAP protein, is sterically obstructed . ConsequTranscription factors Greater than 90% of transcription factors either contain IDRs or are entirely intrinsically disordered, thus it is not surprising that transcription factors regulated by cell signaling are also likely to include intrinsically disorder [disorder , 323. Madisorder ). Converdisorder . In respdisorder . A finaldisorder . Smad dyDrosophila). Groucho is composed of structured domains near both termini, and a central disordered domain that prevents promiscuous binding and unrestrained repression of transcription [Most gene-specific transcription factors regulate transcription by recruiting components of general transcription activation or repression complexes. These components also include IDPs/IDRs. To continue the example of LEF-1, in the absence of Wnt signaling LEF-1 binds the corepressor TLE , in every category of cell signaling pathways  and at each stage  in the cell signaling process. Clearly, any particular cell signaling pathway cannot be fully described without understanding the mechanisms by which intrinsically disordered protein regions contribute to that pathway. Understanding these mechanisms requires not only understanding the statistics of the conformational ensembles generated by intrinsically disordered protein regions , but als


ORAI1 (rs3741596) and BLK (rs2254546). In summary, the refractory nature of the present case could be explained by the presence of combined SNVs in susceptibility genes associated with upregulation of the calcineurin/NFAT signaling pathway. It may provide insights for stratifying KD patients based on the SNVs in their susceptibility genes.Kawasaki disease (KD) is a systemic vasculitis with an unknown etiology affecting young children. Although intravenous immunoglobulin (IVIG) plus acetylsalicylic acid is effective in most cases, approximately 10–20% of patients do not respond to this therapy. An 8-month-old boy was admitted to a local hospital with the presumptive diagnosis of KD. He received IVIG twice and four series of methylprednisolone pulse therapy from the third to the tenth day of illness. Despite these treatments, his fever persisted with the development of moderate dilatations of the coronary arteries. A diagnosis of refractory KD was made, and infliximab with oral prednisolone was administered without success. Defervescence was finally achieved by cyclosporine A, an inhibitor of the signaling pathway of the calcineurin/nuclear factor of activated T cells (NFAT). Whole-genome sequencing of his deoxyribonucleic acid samples disclosed two single nucleotide variants (SNVs) in disease-susceptibility genes in Japanese KD patients,  Kawasaki disease (KD) is a systemic vasculitis affecting young children between 6 months and 4 years old. Although the therapeutic efficacy of intravenous immunoglobulin (IVIG) plus acetylsalicylic acid has been established for KD, approximately 10–20% of patients do not respond to IVIG [Recent studies have shown that some refractory KD cases had genetic backgrounds characterized by single nucleotide variants (SNVs) in disease-susceptibility genes ,3,4.Here, an infant with refractory KD who did not respond to repeated IVIG, corticosteroids, and IFX, but responded to cyclosporine A (CsA), an inhibitor of the calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway, is presented. Whole-genome sequencing (WGS) revealed that he had combined SNVs in susceptibility genes reported in Japanese KD patients. We also discuss an association of the refractory nature of KD with SNVs.The patient’s clinical course during admission is shown in From these findings, a diagnosis of refractory KD complicated by moderate coronary arterial lesions (CALs) was made, and IFX 5 mg/kg) with oral prednisolone (1 mg/kg/day) was administered intravenously on the 12th day of illness without success. Then, according to previous observations of efficacy for refractory KD [ mg/kg wiAt the outpatient clinic, at 1 year of age, echocardiographic examination demonstrated moderate CALs, necessitating the administration of warfarin, dipyridamole, and aspirin.The patient’s parents provided informed consent and agreed with the submission of the case report to the journal.To determine the mechanism by which CsA was effective in this patient, WGS was performed to search for SNVs in the susceptibility genes in Japanese KD patients using NovaSeq6000 and the TruSeqDNA PCR-free library . The primary data from WGS were automatically processed for assembly and mapping. The obtained whole-genome data were subjected to structural annotation.ORAI1 , FCGR2A , BLK (B lymphoid tyrosine kinase), CD40 (CD40), ITPKC , CASP3 (caspase-3), and HLA (human leukocyte antigen) (gene products were shown in parentheses). The present case had a total of 17,707 SNVs in these susceptibility genes. Among them, 17,692 SNVs were excluded from further analysis as they were not listed in ORAI1, 4 in CD40, and 7 in BLK, were targeted for the next stage. After leaving out 12 SNVs of synonymous amino acid substitution, 3 SNVs, ORAI1 (rs3741596), CD40 (rs4813003), and BLK (rs2254546), remained as candidate SNVs associated with KD in the case. Among them, the SNV of CD40 was determined to be neutral polymorphism based on the previous reports demonstrating that CD40 (rs4813003) neither promoted nor inhibited the calcineurin/NFAT signaling pathway, preserving the function of CD40L-mediated signaling between B and T cells [First, the susceptibility genes of Japanese KD were listed by a literature search as the following ,3,4,6: O CD40 rs413003, anORAI1 (rs3741596) and BLK (rs2254546) were finally identified as significant SNVs in the case.Thus, Despite extensive research, the underlying mechanisms of KD remain unknown . The curThe therapeutic efficacy of IVIG has been established for KD, as IVIG reduces inflammation  and prevents the development of CAL. IVIG appears to have a generalized anti-inflammatory effect, and possible mechanisms include the enhancement of regulatory T cell activity, the neutralization of bacterial super-antigens or other unknown pathogenic agents, the regulation of cytokine production, the suppression of antibody synthesis and inflammatory markers, the provision of anti-idiotypic antibodies, the Fc-gamma receptor and IL-1β, and balancing the T helper (Th) Th1/Th2 immune responses . HoweverITPKC, ORAI1, STIM1), dysregulated leukocyte signaling , decreased apoptosis (CASP3), and altered transforming growth factor-beta signaling  [ITPKC (rs28493229), ORAI1 , FCGR2A (rs1801274), CD40 (rs4813003), BLK (rs2254546), CASP3 (rs113420705), and HLA (rs2857151) [ITPKC and CASP3 are reported to be associated with resistance to IVIG [ORAI1 in a refractory patient caused a gain of function, leading to constitutive Ca2+ entry into immune cells, which upregulated the calcineurin/NFAT signaling pathway [ORAI1 (rs3741596) detected in the present case might be associated with its refractory nature. Though the pathogenesis is poorly understood, recent advances in genome-wide association studies (GWAS) and linkage analyses of KD have identified the SNVs in several disease-susceptibility genes. Candidate genes can be classified into four groups: enhanced T cell activation  found in the case may also be associated with the activation of the calcineurin/NFAT signaling pathway via cross-interaction between T cells and B cells [BLK, including rs2254546, demonstrated a decreased expression of BLK on B cells with reduced B cell receptor signaling activity, leading to the activation of T cells via the CD86 molecule [BLK detected in the present case (rs2254546) could amplify the upregulation of the calcineurin/NFAT signaling pathway.Meanwhile, another SNV in  B cells ,20. In tmolecule . This reRecent observation confirmed the involvement of this pathway in KD, demonstrating that the serum levels of calcineurin and NFAT increased significantly in the acute stage and decreased progressively in the afebrile and subacute stage, reducing the CRP, white blood cell, and neutrophil counts . FurtherFrom these findings, CsA, a calcineurin inhibitor, is expected to suppress the upregulation of the calcineurin/NFAT signaling pathway in refractory Japanese KD patients. The recent clinical trial from Japan revealed the efficacy of CsA in KD; an open-label, randomized, multicenter study of 173 patients with KD showed that IVIG plus CsA resulted in earlier fever resolution, lower CRP, and a lower CAL formation rate than IVIG alone. There was no difference in adverse events between the two groups, confirming that CsA is helpful in clinical practice for calming inflammation and preventing CAL formation in KD .2+ concentration due to functional changes in ORAI1 due to SNV in ORAI1 (rs3741596); another SNV in BLK (rs2254546) might indirectly aggravate the calcineurin/NFAT signaling pathway, with an increase in T cell–B cell collaboration; as a result, upregulated calcineurin/NFAT signaling pathway caused an enhanced proinflammatory cytokine production leading to multidrug resistance including IVIG, corticosteroids, and IFX; CsA effectively suppressed the upregulated calcineurin/NFAT signaling pathway resulting in remission, which could not be obtained by other therapeutic agents including repeated IVIG or IFX.Based on these findings, the following mechanisms might be involved in the efficacy of CsA in the present case: the calcineurin/NFAT signaling pathway was constitutively upregulated because of an elevated cytosolic CaOur present case study has two limitations. First, our concept concerning the relationship between multidrug resistance and the susceptibility genes in KD is based on only a single case. To verify this, further studies with more cases are necessary. Second, the genetic examination was performed using the targeted analysis for the annotation. Thus, several SNVs of other candidate genes have not been considered for their association with KD. The refractory nature of KD patients who did not respond to conventional therapies could be explained by the presence of combined SNVs in susceptibility genes associated with the upregulation of the calcineurin/NFAT signaling pathway. Our findings may provide insights for stratifying KD patients based on the SNVs in their susceptibility genes.