L best paths with respect to a metric at every decoding step. However, the correct path might be among the L worst paths due to the imposed penalties. In this case, the correct path is pruned and the decoding process fails. shifted pruning (SP) scheme can recover the correct path by additional decoding attempts when decoding fails, in which the pruning window is shifted by In the successive cancellation (SC) list decoding, the tree pruning operation retains the  Polar codes proposed by Arıkan in  are the When SC or SCL decoding fails, we may be able to correct the error(s) in additional decoding attempts. Bit-flipping  is a popL) by a factor of two until reaching the predefined maximum list size or succeeding in decoding. However, increasing the list size not only contributes to a larger computational time complexity but also requires enough hardware resources to support that. Additionally, as the results showed, the performance gain beyond list size L paths in the sorted list instead of the best ones) at the critical bit coordinates. This work was submitted to ITW2019 on 10 April 2019, presented on August 26 in Visby, Sweden, and published on IEEE Xplore on 10 February 2020. Later, this scheme was improved in | (c.f. ), the poL worst paths in terms of path metric due to high possibility of bit error for all the paths.Clearly, In practice, we prefer addition over division. Hence, we take the logarithm of  and as PT, as shown in line 12. The shifted pruning operation is performed in line 27 over only one bit at each decoding attempt. Note that in this algorithm, Algorithm 1 illustrates a modified list decoder to implement the shifted pruning scheme. In this algorithm, the length of the shift is equal to Algorithm 1: List decoder with shifted pruning.M sub-blocks of size To understand the need for multiple shifting, in particular in the segmented scheme that is discussed later. We present a simple model for error occurrence in the list decoding in this section. Before that, let us look at some numerical results. i, the possibility of the second penalty occurring increases by a factor of i, while in case of the occurrence of the second penalty on bit i, j and j in a segment, respectively. In general, the probability that the j, r, and s, respectively. The sum in ≈pe,0,1,… isPj(ν)≈pe,ns Ew in  is consiNote that the probability of elimination of the correct path differs from the probability of Based on our findings from the study on the distribution of the location of path elimination in the model above and The elimination of the correct path may not be prevented by just a one-time shifting throughout the decoding of a codeword.Let us first look at some statistics, and then we refine our scope of the investigation and propose a scheme. Suppose that there exists an oracle in the list decoder that can avoid the elimination at any bit indices by shifting the pruning window. Now, let us observe the performance improvement due to multiple shifts. Given that the error prevention by more than two shifts occurs rarely at the high SNR regimes, due to the difficulty and need for a computationally expensive search for finding the right combination of positions for more than two shifts, we only focus on double shifting in this section. d is the vicinity parameter) or far from each other in two different segments. Two shifts can occur in the vicinity, i.e., We will cover multiple shifting occurring in different segments in In segmented/partitioned list decoding ,20, we hN is fixed, we drop L as a measure of the complexity of the shifted pruning scheme. Now, let denote the total iterations and the total decoded messages during decoding, and the number of segments by t, c, and s, respectively, the computational complexity of non-segmented and segmented decoding schemes are computed by t refers to the iteration in each segment; hence the total iterations are a sum of the iterations at all segments. Furthermore, For computational complexity comparisons, since the block-length T attempts fail, multiple shifting may recover the correct path. We do not always try pair-shifting, but we perform it when the chance of recovery by pair-shifting is high. We use the following criteria: if among the top 5 out of T sorted positions with respect to the metric, the majority of them belong to a specific segment. This could be a sign that we are facing a low-reliability segment. Otherwise, it is not suggested to try multiple shifting, as it is quite costly in terms of time and computational complexity.As the numerical analysis shows, in the presence of severe noise, when the correct path is recovered by shifting the pruning window, it might be eliminated at the next consecutive low-reliability bit or a bit in the vicinity due to the bit-channels correlation. Here, we propose a scheme to avoid elimination by shifting at two close positions within a segment. Obviously, we first try to recover the correct path by a single shift. As T positions, in descending order. Let us call these bit indices critical bits of the segment. The pairs are selected as shown in d is different with After detecting a low-reliability segment, we sort the bit indices that belong to this segment, appearing among the As the shifted pruning scheme performs in a similar fashion as the conventional list decoding, the same design can be used for implementing this scheme. The only difference is in re-running the decoder and shifting the pruning window at certain bit positions. These candidate bit-positions require a different metric. As the path metric used in this work is based on the approximate LLR-based path metric, the metric can be easily computed throughout the first run and sorted at the end. This obviously requires additional circuitry for a network sorter.In terms of the space complexity of the segmented decoding, note that all the intermediate LLRs in the second segment are computed only based on the channel LLRs, which are the same for all the paths, and the partial sums obtained from the first segment. If we decode the first segment correctly, the partial sums of the correct path used for the second segment can be stored or can be computed simply by This scheme can also be simplified based on the special nodes as was discussed in . As mentT, equal to the size of the critical set, which is large and imposes a huge complexity, while double shifting can approach that performance with less complexity.To evaluate the performance of the proposed metric , polar cximation  while opSimilarly, you can observe the performance comparison for P in In this paper, we propose a new metric for shifted pruning scheme that improves the performance of the shifted pruning scheme significantly in comparison with the available metrics; also, this metric is computationally simple and works with LLR-based SCL decoding. Then, a double-shifting scheme is suggested to prevent a portion of errors that require two shifts by an oracle. This scheme improves the FER performance at a reasonable complexity cost. Finally, we adapt the segmented decoding to the shifted pruning scheme, which results in a significant reduction in the complexity of the decoding process, as the number of node visits decreases by targeting smaller segments for additional iterations.


H and decreased ∆W1. Textural properties revealed that hardness was higher in samples containing chickpea flour and adhesiveness, chewiness, and gumminess were lower in samples with chickpea flour and corn flour. Sensorial properties showed that there were no significant differences between samples except the sample containing 6% chickpea flour. Based on the obtained results, it seems that the sample containing 4% corn flour, 6% chickpea flour, and 0.3% HPMC had the best formulation.The present work was carried out to study the effectiveness of substitution of wheat flour with different levels of chickpea flour, corn flour, and hydroxypropyl methylcellulose (HPMC) for the production of gluten-free sausages. The prepared sausages were subjected to physicochemical, color, texture, thermal, and sensory analyses 24 h after production. Proximate chemical composition indicated that the protein content was raised by the addition of chickpea flour. The result of thermal analysis indicated that the addition of chickpea flour increased To, Tp, Tf, and Δ Today, consumers demand food products, which can improve their health status and life quality as well as prevent diseases . This inPseudoplatystoma corruscans) patties with rice, corn, amaranth, or quinoa. Kerimoğlu and Serdaroğlu [Different meat products contain 15-35% fat, and so, they are categorized as high fat foods. Due to the high consumption of these products, they influence the population diet considerably. In recent years, efforts have been made to reduce the fat content of emulsified meat products in order to improve the healthy state of consumers . Few worrdaroğlu  producedDifferent attributes of a food product including function, composition, and rheology affect the food quality. Improving food quality and formulation needs information regarding the properties of its ingredients. Rheological and textural properties of a meat product are among the main factors influencing consumer's acceptance. Hydrocolloids  are among the main ingredients in meet products that develop rheological and textural properties .Chickpea flour is containing proteins such as albumins and globulins. The technofractional properties of proteins confirm that the products of chickpea processing can be used in the technology of cooking foods with a low content of gluten. Also, it contains phosphorus 290 mg/100 g, potassium, and magnesium 126 mg/100 g. For the chickpea varieties studied, high content of essential fatty acids, such as linolenic 30%, linoleic 60%, and oleic 23-28%, is a characteristic .There are limited works on using chickpea flour and corn flour in the formulation of meat products. The aim of the present work was to evaluate the effects of the addition of chickpea flour and corn flour into gluten-free sausage batter and then investigate the physicochemical, textural, and sensorial attributes of the final sausage products.Chickpea flour, corn flour, and wheat flour were purchased from Kesht-e-Sabz Co. . Hydroxypropyl methylcellulose (HPMC) was prepared by Adonis Gol Darou Co. . Fresh cow meat was purchased from a local butcher shop in Kazeroun (Iran). All other used chemical materials were of analytical grade.The chemical composition of different flour types  including protein, ash, and moisture contents was determined based on the ISO 1442:1997, ISO 936:1998, and ISO 937 standard methods, respectively. A.O.C.S. Official Procedure Am 5-04  was used as the guideline to measure fat content. All the measurements were carried out in triplicate .The connective tissues and excessive fat were removed from the cow meat before use, and then, a meat grinder  was used to grind the meat. The ground meat (1.6 kg) and the other ingredients in the formulation including ice (0.4 kg), back fat (0.4 kg), L-ascorbic acid (0.02%), sodium chloride (1.5%), and sodium nitrite (0.01%) were mixed in a cutter  for 30 min. Different amounts of wheat flour (in the control sample), HPMC, corn flour, and chickpea flour were also added to the five formulations .Three batches of each formulation were produced at different times on the same day. Before producing the sausage samples, the batters were kept in a refrigerator (4°C) for half a day and then stuffed in waterproof plastic casings  using a stuffing machine (VF628 Germany). The samples were heated in a water bath at 85°C for 45 min, and after that, the sausages were cooled with tap water for 30 min. The samples were dried and then kept in a refrigerator before use (24 h) . For eacThe chemical composition  of the produced sausages was determined based on the methods mentioned in To measure the pH value, each type of sausage sample was mixed with deionized water and then homogenized using a mixer. A pH meter  with a glass electrode (HI 1131P) and temperature electrode (HI 7669/2 W) was used to determine the pH of the obtained suspension of sausage at 25°C .L∗, a∗, and b∗ in this system show lightness (+: redness and -: greenness and +: yellowness and -: blueness), respectively. Four random points from the surface of the sausage slices were chosen for color measurements. A Minolta CR-400 colorimeter  with a 10° observation angle, illuminant D65, and spectral reflectance included as calibration modes were used for this purpose [The color properties of the sausage samples were determined based on the CIE system.  purpose .The thermal properties of the prepared sausages were evaluated based on the method of Tongnuanchan et al. . BrieflyThermogravimetric analysis was conducted based on the method of Tongnuanchan et al. . The sauTexture Analyzer CT3  and TPA test were used to investigate different textural properties of sausage samples including hardness, springiness, adhesiveness, chewiness, and gumminess. All measurements were performed on the same day in triplicate . The samTo evaluate the sensorial properties of sausage samples including texture, taste, color, appearance, and total acceptance, a seven-point hedonic test (from score 1: dislike very much to score 7: like very much) was used. Ten trained panelists (5 men and 5 women) were chosen from the staff and students in Islamic Azad University of Kazeroun (Department of Food Science and Technology) in the age range of 18-32 years. Samples (two slices of each sausage sample with 0.3 cm thickness and 4 cm diameter) with a three-digital code were given to the panelists. The panelists evaluated samples in standard booths with fluorescence light. Salty crackers and water at ambient temperature were also provided for palate cleaning .P < 0.05) between different sausage formulations, one-way ANOVA was performed. The Duncan test was used to compare the means.All the mentioned experiments were carried out in triplicate. Results were reported as the mean ± standard deviation. To determine significant differences  of chickpea flour, corn flour, and wheat flour are presented in P < 0.05) by adding chickpea flour. This result is in line with the high protein content (17.91%) of chickpea flour compared to the other flours. Sanwo et al. [The results of chemical composition  and pH of the gluten-free sausages with different formulations are presented in o et al.  reportedo et al.  after tho et al.  indicateo et al. . dos Sano et al.  showed tL∗ value decreased (P < 0.05) by adding chickpea flour. This result is in accordance with the lower L∗ value in chickpea flour compared to the other flour types. Pereira et al. [L∗ value of sausage. In a research, it was reported that by adding 10% rice flour, the L∗ value of duck and pork sausages decreased considerably [L∗ value after the addition of 5% glutinous rice flour to meat patties was also reported by Gao et al. [L∗ value in sausages containing chickpea flour. The results also showed that there were no significant differences between a∗ value and b∗ value of different samples. Pereira et al. [a∗ value of sausage. Sirini et al. [a∗ and b∗ values of dry-cured meat sausages significantly.The color property is one of the main aspects of meat products. In addition to the pigments, the structure and composition of meat products have important roles in their color. The color of fresh meat is an indicator of its quality and can promote consumers to buy it. The results of color properties of the different gluten-free sausages are shown in a et al.  reportediderably . Decreaso et al. . These ra et al.  reportedi et al.  reportedH increased (P < 0.05) by adding chickpea flour. The significant differences between the control and other samples were because of the presence of gluten in the control sample. During thermal processing of starch, its crystalline structure is melted, and the small molecules such as amylose are removed from the granules. In wheat flour, gluten surrounds the starch granules [W1 decreased (P < 0.05) by adding chickpea flour and ∆W2 was higher (P < 0.05) in the samples containing chickpea flour and corn flour compared to the control sample. The addition of chickpea flour also increased ∆W3 (P < 0.05). The lowest amount of free water (∆W1) and the highest amount of bonding water (∆W2 and ∆W3) in the sample with 8% chickpea flour were related to the higher protein content and the presence of HPMC. Similar results were also reported by Verbeken et al. [The results of thermal properties of the different gluten-free sausages, which can be observed in granules . The insgranules . Based ogranules  showed tgranules . They shn et al. . The effn et al. .P < 0.05) by adding chickpea flour. Pereira et al. [Agaricus bisporus flour and Pleurotus ostreatus flour improved the chewiness and gumminess of Frankfurter sausages. Choe et al. [Rheological evaluations can be divided into the sensory method and the instrumental method. In the present study, we used the instrumental method (TPA) in order to investigate the textural properties of the samples. The results of textural properties of different gluten-free sausages are presented in a et al.  reporteda et al. . Similara et al. , 34. Howa et al. . Adhesiva et al.  reportede et al.  reportedEvaluation of the different sensory attributes of the produced sausages including color, taste, texture, appearance, and consumer acceptance was carried out by ten panelists, and the results are shown in Substitution of wheat flour with chickpea flour and corn flour caused higher protein levels and lower lightness in the resultant gluten-free sausages. Increasing the chickpea flour level in the formulation improved the thermal stability and textural properties of the samples. Free water content significantly decreased and hardness increased by increasing chickpea flour content. The results of the sensory evaluation showed that chickpea flour could be used in the formulation of gluten-free sausages up to 6% of the total flour composition with no considerable changes in consumer acceptability. Substituting wheat flour with chickpea flour at levels higher than 8% had a negative effect on the acceptability of the products in terms of color, taste, texture, and overall parameters.


Pruning wounds are the main entry points for fungi causing grapevine trunk diseases (GTDs). Several studies identified factors influencing the temporal dynamics of wound susceptibility, which include the fungal species and inoculum dose, weather conditions, grape variety, pruning date, and so forth. Here, we conducted a quantitative analysis of literature data to synthesise outcomes across studies and to identify the factors that most affect the length of pruning wound susceptibility.We extracted data on the frequency at which the inoculated wounds showed GTD symptoms or an inoculated pathogen was reisolated following artificial inoculation at the time of pruning or in the following days. A negative exponential model was fit to these data to describe changes in wound susceptibility as a function of time since pruning, in which the rate parameter changed depending on specific factors.The results show that wound susceptibility is high at the time of pruning, and they remain susceptible to invasion by GTD fungi for months after pruning. Infection incidence on wounds was higher for fungi associated with Botryosphaeria dieback than those associated with Eutypa dieback or Esca complex, and wound susceptibility decreased faster for Eutypa dieback than for other GTD agents. Grapevine variety and pruning season also affected the wound susceptibility period. Sauvignon Blanc remains susceptible to GTDs longer than other varieties. We also found that the time of pruning can affect infection dynamics, especially for more susceptible varieties. The results increase our understanding of GTD epidemiology and should help growers control infections. The casPhaeomoniella chlamydospora and Phaeoacremonium minimum   , and othn Europe , and othountries ; these fountries , and is Botryosphaeria dothidea, Lasiodiplodia theobromae, Dothoriella viticola, Neofusicoccum parvum, N. australe, N. luteum, N. ribis, Diplodia seriata, and D. mutila  to quantitatively appraise and synthetise outcomes across studies on the dynamics of wound susceptibility to GTD fungi; (ii) to determine whether wound susceptibility depends on the pruning season, grapevine variety, the pathogen that is involved, or by the interaction of these factors; and (iii) to identify knowledge gaps that require further research.22.1®)(Grapevine AND Trunk AND Disease$ AND Wound$ AND (susceptibility OR age). The operator AND indicates that the words must occur simultaneously in the search results, while OR indicates that the selected study must contain any of the terms separated by the operator; the dollar sign ($) represents zero or one character . No restrictions on language or publication date were used. The search detected a total of 79 papers that were exported to EndNote™ online  and that were first screened based on the title and abstract. To be selected, papers had to first satisfy the following criteria: (i) the papers were published in peer-reviewed scientific journals, conference proceedings, scientific reports, or peer-reviewed theses; (ii) they describe experiments in which pruning wounds were artificially inoculated with GTD pathogens at different times after pruning; (iii) they contain quantitative information in the main text, tables, or figures on the percentage of inoculated wounds that were infected ; and (iv) the studies had an experimental design with replicates and were conducted with potted plants or field plants. The papers fulfilling these eligibility criteria were considered to be of potential interest, and the full-text manuscripts were retrieved and reviewed. Additionally, a cross reference-based search of relevant studies was performed to include reports of other studies that might have been eligible for the review and to reduce the risk of missing relevant information due to the choice of search terms. This procedure is shown in A systematic literature search was conducted in the Web of Science (WoS) Databases  using thThe cut-off date for publications to be considered for inclusion in this quantitative analysis was 25 July 2022.2.2Data on the percentage of pruning wounds that showed internal disease symptoms or from which the pathogen was re-isolated following artificial inoculation (hereafter referred to as “disease incidence”) at different times after pruning were extracted from the original papers. All the inoculations were done by depositing a spore suspension  on the pruning wound surface. Additional details can be found in via email, but we received only a few replies, in which the authors declared they are not able to provide us with the original data or missing information, with only one exception.Data in the main text or tables were extracted directly, while data in graphs were extracted with WebPlotDigitalizer version 4.3 , an onli0). For instance, in an experiment in which pruning wounds were inoculated with a pathogen immediately after pruning and then 7, 14, 21, and 28 days later, the times were referred to as t0, t7, t14, t21, and t28.Extracted data were organised in a database containing the selected studies , cases within a study , age of the pruning wounds , and the disease incidence (as defined before). The time at which the pathogens were artificially inoculated on pruning woods was expressed as days after pruning (DAP); the day of pruning was considered as time zero (tPm. minimum or Pa. chlamydospora), Botryosphaeria dieback , and Eutypa dieback . There are eight identities for the inoculated pathogens: Pa. chlamydospora, Pm. minimum, D. seriata, L. theobromae, N. parvum, N. luteum and E. lata. Ten grapevine varieties: Cabernet Sauvignon, Chardonnay, Chenin Blanc, Merlot, Sauvignon Blanc, Thompson Seedless, Grenache, Pinot Noir, Shiraz, or Tempranillo.Relevant information on each case was also included in the database in order to extract subsets of data for considering the following main factors that could potentially affect the relationship between disease incidence and DAP were the pruning period (season), identity of the GTD, identity of the inoculated pathogen, and grapevine variety. The pruning period included three “categories”: early-season pruning , mid-season pruning, , and late-season pruning . Identity of GTD included three categories: the Esca complex  models, and perform a formal meta-analysis. Therefore, we made a quantitative analysis of pooled data coming from the different studies considering the among-study variability only. The weaknesses of this approach are discussed in the next paragraphs.Since it was not possible to extract data on the within-study variance of the different studies, it was impossible to fit a multivariate random effects model 0). To assess the differences between the main factors at t0, we used generalised linear models (GLMs). GLMs provide regression analysis and analysis of variance for one dependent variable by one or more factors, allowing the test of the null hypotheses about the effects of other factors on the means of various groupings of a single dependent variable  was used for post hoc comparisons to compute contrasts between each combination of the factors by using Tukey’s method. The raw data used are available as We first used boxplots  to studyvariable . Because0 was set at 1, and the disease incidences at the following inoculation times were rescaled to 1 and expressed on a 0 to 1 scale. For instance, if the disease incidence was 85% for wounds inoculated at t0, and was 55%, 32%, 10%, and 3% for wounds inoculated at 7, 14, 21, and 28 DAP, then t0 = 1.00, t7 = 0.65 , t14 = 0.38 , t21 = 0.12 , and t28 = 0.04 .To study the temporal dynamics of pruning wound susceptibility, the disease incidence at tAverage (and standard error) of the rescaled disease incidence in the different cases was then calculated for each timing after pruning (DAP) and fit to a negative exponential equation in the following form:tY is the rescaled disease incidence  at time t; t is the time of fungal inoculation after pruning ; parameter a is the rate at which Y decreases as t increases, which was estimated for all data, main factors, and interactions  by using nonlinear regression procedures (nls); standard errors of the parameter a given by the nls function were used to calculate the confidence interval of the predicted Y values.where 2, which represents the proportion of experimental variability explained by the selected model (R2 = 1 represents a perfect fit); and the concordance correlation coefficient (CCC), which is calculated as a measure of model accuracy , i.e., the ratio of the mean square error to the variance in the observed data, subtracted from unity ; W index of agreement, i.e., the ratio between mean square error to total potential error (W = 1 represents a perfect fit); root mean square error (RMSE), i.e., the fit standard error of the regression, which is calculated as the square root of the mean square error ; coefficient of residual mass (CRM), which is a measure the tendency of the model to over- or underestimate the observed data (a negative CRM indicates a tendency of the model toward overestimation); Rent at 1 .a of equation [1] estimated for a category of a main factor  was not significantly different from that of another category of the same factor . The latter null hypothesis was tested as follows. Equation [1] was expressed in its linear form through data transformation through the natural logarithm function (ln), and the intercept of the linearised equation was set at ln(1 + 1) shifting the y-values by ln(2). Significant differences between slopes of the linearised, shifted functions were finally tested using the lsmeans package; slopes were extracted using the function lstrends and then compared through the function pairs, which returns a matrix consisting of t-tests for each comparison. There were 529 comparisons in total, and these are shown in To determine whether the temporal dynamics of pruning wound susceptibility were significantly affected by the categories of the main factors and their two-way interactions (three-way interactions were not considered because the number of studies would have been too low), the following null hypothesis was tested: the slope All statistical analyses were conducted with R studio Version 1.4.1717 .33.1via cross-referencing. After full text analysis, 16 papers were used for quantitative data extraction. The selected papers were published between 1980 and 2022, and were conducted in Australia, New Zealand, California, the USA, South Africa, Italy, Spain, and France; most were performed in the Northern Hemisphere. The key information of these 16 studies is summarised in The literature search provided 79 studies, of which 41 were selected for full text analysis. The others were disregarded because they were not considered relevant to this study as they failed to meet the above-mentioned criteria. Two additional papers were selected Pm. minimum or Pa. chlamydospora, nine for Botryosphaeria dieback , and nine for Eutypa dieback (with E. lata)  Table 1.E. lata) , vines w3.20) was 64.0%; the variability, however, was very high with extremes ranging from 0 to 100%, and with 50% of the data ranging from 40.3 to 80.0% (0 was significantly higher for wounds inoculated at mid-pruning than at late pruning (P< 0.001), with early pruning showing intermediate values (P = 0.032), with intermediate values for Esca complex agents. When the whole dataset was split based on grape variety (P > 0.058).In the whole dataset, the median value of disease incidence  at the time of pruning  when the GTD fungi were inoculated Figure 42 (0.25 to 1); NSE (0.1 to 0.99); W (0.46 to 1); RMSE (0.02 to 0.72); CRM (-0.08 to 0.12); and CCC (0.3 to 1)  and their interactions, for a total of 70 combinations, equation [1] fit the observed data rather well with the following ranges of values: R.3 to 1) 5.a = 0.018), mid (a = 0.020), and late (a = 0.021) pruning were not significantly different from a based on the whole dataset  than for the Esca complex (a = 0.016) or Botryosphaeria dieback (a = 0.015), with P< 0.001 (a=0.024) and early pruning (a = 0.019), which were not significantly different from each other (P = 0.999), than for mid-pruning  (The pruning period (as a main factor) was not significantly related to the dynamics of wound susceptibility, because the slope parameters calculated for early  than with other fungal species . The latter species was also different from the two main species involved in the Esca complex, Pa. chlamydospora and Pm. minimum , and from D. seriata , which is part of the same GTD .Wound susceptibility decreased faster when pruning wounds were inoculated with a ranging from 0.095 to 0.040) than for Cabernet Sauvignon, Chardonnay, Chenin Blanc, Tempranillo, and Thompson seedless ; the pairwise comparisons between varieties in the first and second of these groups were in most cases significant groups (a = 0.010), and was different from the first group of varieties but not from the second. No significant differences were found for the pruning period within each variety (P > 0.593), with the exception of Thompson Seedless, in which the susceptibility of pruning wound declined slower with late pruning (a = 0.019) than with early or mid-pruning  (a = 0.01) were susceptible for a longer time than wounds of Cabernet Sauvignon  or Thomson seedless  pruned in the same season. Differences among varieties were not significant with late-season pruning  than for Botryosphaeria dieback or the Esca complex  (a = 0.057) than for Esca complex  (no data available for Botryosphaeria dieback). For Chardonnay, on the contrary, the decline in wound susceptibility was faster for the Esca complex (a = 0.073) than for Botryosphaeria dieback  (a = 0.024) than with mid-season pruning  , significant differences were found as indicated in When the estimates of 4Since the 1970s, researchers have investigated the susceptibility of pruning wounds to GTD fungi as a function of DAP (number of days after pruning when the wounds were inoculated) and other factors. It is commonly accepted that the age of pruning wounds has a considerable effect on the susceptibility of wounds to GTD fungi, i.e., wound susceptibility decreases as the time increases between pruning and inoculation. The decline in susceptibility over time has been related to changes in environmental conditions affecting biological and physiological wound responses . Many ota estimation was not considered.In this work, we conducted a quantitative analysis of the published data in order to synthesise the available information . The stua in equation [1]. We estimated a for the whole data set and for sub-sets referring to specific influencing factors . In equation [1], a is related to the time required for the disease incidence to halve, so that the higher the estimate of a, the shorter the time required for disease incidence to decline from 1.0 to 0.5. To our knowledge, this is the first time an equation was fit to experimental data describing the temporal changes in wound susceptibility after pruning.Our quantitative analysis showed a significant influence of the considered influencing factors on the infection incidence of GTD fungi inoculated at the time of pruning and on the rate of decline in infection incidence over time. Changes in wound susceptibility to fungal invasion after pruning followed a negative exponential distribution, a probability distribution that describes the time between events in a Poisson process, i.e., a process in which events occur continuously and independently at a constant average rate. In our case, the events consisted of the number of times wounds became infected over the total number of cases that wounds were inoculated at different times after pruning , and the constant average rate was the equation parameter 2 = 0.74; based on this equation, wounds remained susceptible to invasion by GTD fungi for months after pruning, and< 50% of the wounds remained susceptible at 6 weeks after pruning , and their interactions had a significant effect on the estimates of parameter  dataset Figure 4Some data dispersion around the fitted line, however, remained unexplained by our quantitative analysis. Indeed, other factors may influence wound infection by GTD fungi and contribute to the imperfect fit of equation [1] to the observed data, which were not considered in our study because of a lack of information in the literature; they include the age of the wood that was pruned, the size of the cut surface, the extent of wound healing, the trellis system, the rootstock, plant vigor. Because most of the studies included in our analysis were conducted in vineyards, natural colonisation of pruning wounds by GTD fungi may have occurred in the period between two artificial inoculations. It follows that the incidence of GTD in the vineyard and weather conditions that may lead to infection and expression of disease symptoms, may also be a source of variability in the data that we used . Further4.1Neofusicoccum luteum, for which wound susceptibility decreased faster than for other Botryosphaeriaceae and E. lata. A shorter susceptibility of wounds to E. lata infection than to infection by other GTDs was observed by Our analysis showed that, at the time of pruning, disease incidence of wounds was higher for the fungi associated with Botryosphaeria dieback than for those associated with Esca complex or Eutypa dieback, which generally agrees with previous reports . In addiP. chlamydospora in order to extensively colonize the wood. Studies on the susceptibility of pruning wounds to infection by basidiomycetes are then needed.An important limitation of our study is that no studies were found for the basidiomycetes involved in the EC. A possible reason is that these fungi have been isolated from infected vine trunks, but often with an incomplete understanding of their role in the disease aetiology . With th4.2E. lata for a longer time than pruning wounds made in March. Similarly, L. theobromae and N. parvum for a longer time when pruning was done in early winter (with susceptibility lasting up to 84 days) than in early spring (with susceptibility lasting up to 12 days). Furthermore, D. seriata  and after pruning. For the t0 data, however, data variability and unbalanced replications caused some under-dispersion in the GLM model (In our quantitative analysis, differences were found among grape varieties for infection incidence of wounds inoculated at pruning (tLM model . Additioa would be more accurate for Cabernet Sauvignon than for other varieties. Additional work is then needed to add robustness to our interpretations.Our analysis showed that pruning wounds remained susceptible to GTDs for a longer time on Sauvignon Blanc than on other varieties. Sauvignon Blanc was previously reported to be especially susceptible to GTDs . Similar5Following the general objectives of systematic literature search and quantitative analysis of literature data, our work makes a synthesis of published information and projects some relevant aspects that need further research. The number of studies we found and used is limited and most of them did not include complete information on the experimental variability, thus not permitting the calculation of the effect sizes and the execution of a formal meta-analysis. Unfortunately, this limitation has been frequently faced by other authors . Meta-anOur work also suffers from the incomplete knowledge about GTDs we discussed before. Systematic literature review, however, does not have the ambition of elucidating all the aspects related to a topic, but it is useful for identifying knowledge gaps and orientating further research. Complex interactions between cultivar, biogeography, wood healing, the wood fungal pathobiome, pruning time, and environmental conditions, need further investigations.Phaeoacremonium spp., Pa. chlamydospore (D. seriata (Furthermore, our work does not claim that pruning wounds are the sole or most important infection pathway for GTD fungi. Other wood injuries caused by frost, hail, and mechanical harvesting can also act as points of entry. Soil is also considered a possible reservoir of inoculum, and roots an entry point for ydospore , and D.  seriata , but for seriata . Soil is seriata  but as m seriata .The original contributions presented in the study are included in the article/MCR was the principal investigator. MCR and SEL extracted the data. MCR and VR conducted the statistical analysis and drafted the manuscript. VR, IS and SEL edited and revised the manuscript. All authors contributed to the article and approved the submitted version.


Kinesins, the microtubule-dependent mechanochemical enzymes, power a variety of intracellular movements. Regulation of Kinesin activity and Kinesin-Cargo interactions determine the direction, timing and flux of various intracellular transports. This review examines how phosphorylation of Kinesin subunits and adaptors influence the traffic driven by Kinesin-1, -2, and -3 family motors. Each family of Kinesins are phosphorylated by a partially overlapping set of serine/threonine kinases, and each event produces a unique outcome. For example, phosphorylation of the motor domain inhibits motility, and that of the stalk and tail domains induces cargo loading and unloading effects according to the residue and context. Also, the association of accessory subunits with cargo and adaptor proteins with the motor, respectively, is disrupted by phosphorylation. In some instances, phosphorylation by the same kinase on different Kinesins elicited opposite outcomes. We discuss how this diverse range of effects could manage the logistics of Kinesin-dependent, long-range intracellular transport. Kinesins represent a superfamily of mechanochemical enzymes that bind to microtubules, hydrolyze ATP and either transport cargoes along the microtubule tracks or participate in controlling microtubule dynamics inside a cell . All KinKinesin motor activity, cargo-binding and the flux of cargo transport driven by the motor are tuned according to the cargo availability and cellular environments. Motor activation and spatiotemporally modified cargo affinity are the two aspects of this regulation. An intramolecular interaction strategy, known as autoinhibition, blocks the ATPase activity when the motor is not associated with a cargo . For somvice versa. The information is relayed by cytoskeleton—associated motor proteins, which move the signaling complexes from their sources to the destinations inside a cell -dependent protein kinase (cAMP-PK/PKA), protein kinase C (PKC) and pp60c-src  could phosphorylate r domain . The phoosophila . The necosophila , which iosophila . Therefoin vivo.Together, these results suggest that phosphorylation of different conserved serine residues in the motor domain could attenuate the Kinesin-microtubule interaction and the ATPase rate through distinct mechanisms . Furtherin vivo  signaling in PC12 cells and chick neurons in vivo . The evein vivo . Althougin vivo . The 14-in vivo  Box 2)..in vivo  elegans . Togethe elegans .2+-dependent dephosphorylation of KHC by protein phosphatase-2Bβ (PP2Bβ) enhanced the glucose-dependent insulin release, whereas the CK2-dependent phosphorylation of KHC inhibited the vesicle transport by Kinesin-1 in these cells  can associate with two different Kinesin-1 adaptors, Alcadeinα/Calsyntenin1 (Alcα/Clstn1) and the JNK interacting protein JIP1. Kinesin-1 association with Alcα/Clstn1 at the post-Golgi vesicles is suggested to regulate the APP transport in the axons . Alcα/ClBesides, KLC phosphorylation and the absence of cargo can stabilize the autoinhibited conformation of Kinesin-1. KLC associates with the KHC stalk through the N-terminal coiled-coil domain and KLC-KHC interaction has been implicated in both activation and inactivation of the Kinesin-1 dependent transport . KLC forin vivo. For example, although the consensus AMPK target sequences at T693 and S520 in KHC and KLC, respectively  and MAPKK (MKK7/Hep) bind to APLIP1/JIP1 and inhibit JIP1-KLC interaction in osophila . It was osophila , which iosophila . This phosophila .Drosophila Unc76/Fez1, which forms a complex with Munc18 and Syntaxin-1 on a distinct subset of presynaptic vesicles , also known as TOAD-64, Ulip2, and DRP-2 . The C-tchondria . CRMP2 ichondria . The CDKchondria . It is uvia Parkin which detaches Kinesin-1 from the mitochondrial surface and promotes a minus end-directed movement on the microtubule.A distinctly different molecular mechanism appeared to regulate the mitochondria association with Kinesin-1. The mitochondrial outer membrane protein Miro binds to KHC through Milton/TRAK . The MirThus, phosphorylation of the adaptor-cargo interacting domains appears to promote cargo association in the case of JIP1/3, Fez1 and Clstn-1, and disrupts the interactions with CRMP2 and Miro . TogetheChlamydomonas at the tip of regenerating flagella released the soluble IFT-B particles from the Kinesin-2 complex and facilitated the motor turnover , also known as Intestinal Cell Kinase (ICK), was shown to associate with the IFT-B complex along with Kif3A  family protein TRIM46 has a unique microtubule crosslinking activity essential for the organization of the axon-initial segment (AIS) , which dosophila . The resosophila . Similarosophila , by the subunits . TherefoFor homodimeric Kif17, CaMKII-dependent phosphorylation of S1029 at the tail domain in dendrites disrupted its interaction with the scaffold protein Mint-1, which associates with vesicles carrying the NMDA receptors subunit GluN2B . WhereasIn summary, the tail phosphorylation of the Kinesin-2 family motors appeared to regulate type-specific cargo release and compartment-selective activation. Kinesin-2 tails are natively unfolded and directly bind to several soluble  and vesiKinesin-3 is a highly diverse family of motors consisting of several conserved sub-families , and disBOX 1Kinesin 1: It is also known as the conventional Kinesin. The motor was the first member of the Kinesin family to be discovered  and accessory protein KAP3/2κ -interacting proteins (JIP1-4): JIPs are scaffolding proteins containing a JNK-binding domain at N-terminus and protein-protein interaction domains, such as Src homology 3 (SH3) domain at their C-terminus. JIP family comprised of four genes encoding for JIP1-4. JIP1, most studied for its role as the scaffolding protein in the MAPK signalling cascade, binds to MLK, MKK7 and JNK : Fez1 is the mammalian orthologue of unc-76 : Clstn1 is a transmembrane protein belonging to the cadherin superfamily. Vertebrates have three Clstn genes (Clstn1-3). Clstn-1 binds to the TPR domain of KLC through W-acidic (WD) peptide motifs present in its cytoplasmic domain (c domain  and recrc domain  and othec domain . Recent c domain .SifA-Kinesin interacting protein (SKIP): SKIP binds to SifA, an effector protein secreted by the bacterium Salmonella typhimurium that helps in the multiplication and virulence of the bacterium in the host cell : CRMP2 associates with microtubule. CRMP2 interacts with heterotetrameric Kinesin-1 through the KLC subunit and acts as an adaptor for the Kinesin1-tubulin complex : PTPN21, also known as PTPD1, is a peripheral membrane protein that links the membrane-associated focal adhesion proteins to cytoskeletal proteins  domains, through which they can bind to several phospholipids and an Arf-GTPase domain. There are five different isoforms in Centaurin family of proteins- α1, α2, β, σ and ϒ. Centaurin α1 (CENTA1) has two PH domains. CENTA1 preferentially binds to phosphatidylinositol-3,4,5 triphosphate (PIP3) and phosphoinositol-3,4-biphosphate P2) and the FHA domain of KIF13B through its PH domains ( domains . CENTA1  domains .Discs large 1 (Dlg1): Dlg1 is a member of the membrane-associated guanylate kinase domain (MAGUK) protein family. It interacts with the MAGUK-binding stalk (MBS) domain of the Kinesin-3 subfamily protein KIF13B/GAKIN. Human disc large (hDlg) is present in two different isoforms-hDlg-I2 and hDlg-I3. hDlg has intramolecular interactions and is present in an inhibited conformation. SH2-I3-GUK segment of hDlg-I3 is required to inhibit intramolecular interactions of GAKIN and activate its ATPase activity . They have a variable N-terminal domain and a conserved C-terminal domain having one phosphotyrosine-binding (PTB) sub-domains and two PDZ subdomains. The Mint-1 protein interacts with the tail domain of homodimeric Kinesin-2 KIF17 through one of its PDZ domains and acts as an adaptor for the transport of NMDA receptor subunit 2B (NR2B) into the dendrites of glutamatergic neurons , while the Drosophila genome only codes for 14-3-3 ε and ζ. The 14-3-3 proteins are present as homo or heterodimers. The N-termini of 14-3-3 monomers form a conserved central groove that binds to phosphorylated motifs on the target proteins. 14-3-3 proteins are expressed in all types of cells. In Drosophila oocytes, 14-3-3 ζ interacts with a Kinesin-3 subfamily member Khc73/Kif13B, and 14-3-3 ε interacts with a dynein adaptor NudE. The 14-3-3 heterodimerization brings about a coordination between the various kinesin and dynein motor proteins required for the organization of mitotic spindles (spindles . Similarspindles .BOX 3Adenosine Monophosphate-activated protein kinase (AMPK): An S/T kinase that consists of three subunits. α subunit contains the catalytic domain, β subunit binds glycogen, and the γ subunit contains four ATP/ADP/AMP binding Bateman domains. It phosphorylates S/T residues. The phosphorylation of the T172 of α subunit by upstream kinases, such as the CaMKKβ, and increasing concentration of AMP activates the enzyme allosterically. AMPK acts as the metabolic sensor and targets a wide variety of pathways (pathways .Casein Kinase 2 (CK2): A heterotetramer of two catalytic and two regulatory subunits that acts on a large variety of substrates containing the consensus “S/T-X-X-E/D/pS/pY” motif except for Casein. The enzyme activity is maintained at a low level in normal cells except during embryogenesis and in cancerous tissue, where the levels are much higher. It controls protein synthesis by upregulating the rRNA levels through direct phosphorylation of RNA and spliceosomal machinery and targets a large variety of cytoskeletal and motor proteins, such as myosin-1, X, XVIIIa, Dynactin, Cytoplasmic Dynein Light Intermediate Chain 1 (LIC1), capping protein, Gelsolin, Septin, etc. It reduces the death/survival ratio, favours angiogenesis, and inactivates tumour suppressors in cancerous tissue. CK2 also acts as a scaffold to stabilize microtubule dynamics in cilia/flagella, spindle body, and axon-initial segment : It is a ubiquitous enzyme of the CaMK superfamily with four  highly homologous and tissue-specific isoforms, each containing regulatory and substrate-binding catalytic sites, and involved in cellular Ca2+ mediates signalling. The holoenzyme consists of 12 subunits of homo and heteromeric isoform composition. The Ca2+-dependent Calmodulin (CaM) binding to each subunit of the holoenzyme activates the enzyme independently, and an intra and intersubunit autophosphorylation of a conserved threonine residue at the regulatory domain modulates the enzyme activity. CaMKII targets an extensive repertoire of proteins carrying the consensus S/T residues. It is extensively studied in the context of learning and memory formation, epilepsy, schizophrenia and other neuropsychiatric disorders (isorders .Cyclin-Dependent Kinase 5 (CDK5): Homologous to the family of proline-directed serine/threonine protein kinases of the Cdc2/CDK1 family involved in cell cycle progression, the enzyme is predominantly expressed in terminally differentiated neurons. Unlike the other CDK homologues, CDK5 binds to a non-Cyclin regulatory subunit called p35/39. Cdk5 and its orthologues—Cdk5r1/2 are also expressed in non-neuronal tissues during developmental stages. Myristoylatation of the p35/39 helps to associate Cdk5 to vesicles and plasma membrane. Calpain-dependent proteolysis of p35 at the plasma membrane hyperactivates the enzyme (e enzyme . The enze enzyme .Cyclic-AMP-dependent Proteins Kinase (PKA): A tetrameric holoenzyme complex comprising a regulatory subunit homodimer and two catalytic subunits (R2:C2) that act as a cAMP-dependent allosteric molecular switch and transmits the second messenger signal downstream of several G-protein coupled receptors (eceptors . All PKAeceptors . It has eceptors . The A-keceptors . cAMP bieceptors .Glycogen Synthase Kinase 3β (GSK3β): A CMGC  family S/T kinase, originally identified in glucose metabolism downstream of insulin signaling, was later shown to target a wide variety of proteins underlying several signaling pathways such as the Wnt and Notch signaling. It binds to targets pre-phosphorylated by the priming kinases such as CKII. The phosphorylation of S9 on GSK-3β by AKT/PKB and other kinases inactivates the enzyme, whereas the Y216 phosphorylation through FYN2 and PYK2 activates it : One of the three-tiered mitogen-activated protein kinase (MAPK) cascade enzymes that acts at the penultimate step in relaying the signal. It belongs to the CMGC family, which is activated by a dual threonine and tyrosine phosphorylation of the TPY motif by the MAP2K family enzymes MKK4 and MKK7 downstream of the MAK3K enzymes DLK1 (mes DLK1 . Proteinmes DLK1 .MAP/microtubule affinity-regulating kinases (MARK1-4): These enzyme isoforms were identified in the context of tau hyper-phosphorylation-dependent development of Alzheimer’s pathology . They belong to the AMPK subfamily of the CaMK group. The enzyme, enriched in the brain, kidney and spleen, phosphorylates serine residues. Phosphorylation of a conserved threonine residue at the catalytic domain activates the enzyme, and that of a conserved serine residue inactivates the enzyme (e enzyme . MARK1/2e enzyme . BesidesProtein Kinase C (PKC): An AGC-family kinase that is generally classified as lipid-dependent serine/threonine kinase. PKC isoforms belong to three subfamilies - conventional , non-conventional , and atypical . The N-terminal regulatory domain contains a pseudosubstrate motif that binds to the C-terminal kinase domain and inhibits the enzyme activity. The N-terminal regulatory subdomains of cPKCs bind to Di-acyl-glycerol (DAG) or phorbol 12-myristate 13-acetate ester (PMA) and anionic phospholipids in the presence of Ca2+. The lipid-binding recruits the enzyme onto the membrane and activates the enzyme through a structural reorganization that expels the autoinhibitory, pseudosubstrate motif from the substrate-binding pocket. The cPKC and nPKC activities are controlled by phosphatidic acid and ceramide binding to the regulatory domain and phosphorylation of a threonine residue in the activation look of the kinase domain (e domain . The nPKe domain .Src-Family Kinase (SFK): These are tyrosine phosphorylating enzymes, homologous to the oncogene cSrc/pp60cSrc, and include nine members  (and Yrk) . SFKs arand Yrk) , and theand Yrk) . Phosphoand Yrk) , whereasand Yrk) .2++, which promotes the dense-core vesicles (DCV) association  in the presence of Caociation . Phosphoociation . Mutatioociation . Here, teraction . Since 1ng event .in vitro, it did not appear to alter the motility of an autoinhibition-deficient KIF1A, suggesting that phosphorylation of additional components may be involved in this process. In the case of mammalian Kif1C, the motor-stalk interaction is disrupted by the binding of PTPN21, a FERM-domain containing protein tyrosine phosphatase, or Hook, a cargo adaptor  and the microtubule-binding CAP-Gly (CG) domain at the C-terminal end . UncharaTherefore, the Kinesin-3 phosphorylation, predominantly in the dimerization and tail domains, appeared to regulate the cargo-motor interaction and motor activation independently . These dXenopus melanocytes  leads to aggregation of mitochondria near the microtubule-organizing centre in the perinuclear region . The treSimilarly, a combination of Kinesin-2, Dynein and Myosin V moves melanosomes inside melanocytes . The MyoTogether, the evidence suggests highly divergent effects of motor subunit phosphorylation. The phosphorylation often has opposing effects on the activation of two different types of motors, such as a Kinesin and Dynein associated with the same cargo, altering the directional flux. As demonstrated with the melanosome and lysosome movements, such changes are coupled to external stimuli affecting cellular physiology.The analysis revealed that a host of cytoplasmic kinases involved in signaling and metabolic regulation  target tThe phosphorylation mainly acts as a switch. For example, phosphorylation of the KHC motor domain and the coiled-coil stalk domain of Kinesin-3 disrupted the microtubule-dependent motility and converted the motor to an inactive state. A similar effect was observed in the case of Kinesin-5, -4 and -13 family members . FurtherAmongst this diversity, one could also find some consensus features of phosphoregulation. For example, phosphorylation of KLCs at the C-terminal and the N-terminal part of KAP disrupted cargo association and transport , 2. AlsoThe other exciting idea that emerged from these studies is that the subcellular localization of the kinases may have a substantial impact on the logistics as it spatially segregates the on/off actions to facilitate the intracellular movements from one point to another. For instance, the localization of PINK1 with Parkin and TRAK on mitochondria is essential for regulating its distribution in the cell. Similarly, Kinesin-2 phosphorylation by CaMKII produced opposite outcomes in axons and cilia . In addiThe phosphorylation by the same kinase often produces opposite effects on the Kinesin and Dynein. For example, ERK1/2-dependent phosphorylation of Dynein-intermediate chain, downstream of TRK signaling, recruits and activates the motor, whereas ERK phosphorylation of KLC dissociates it from Clstn1. In contrast, the GSK3β-dependent phosphorylation disrupted both Kinesin and Dynein association with a cargo. An equally diverse set of kinases are shown to act upon the dynein subunits and regulate the cargo association . Evidenc


Verticillium dahliae Kleb is a typical soilborne pathogen that can cause vascular wilt disease on more than 400 plants. Functional analysis of genes related to the growth and virulence is crucial to revealing the molecular mechanism of the pathogenicity of V. dahliae. Glycosidase hydrolases can hydrolyze the glycosidic bond, and some can cause host plant immune response to V. dahliae. Here, we reported a functional validation of VdGAL4 as an α-galactosidase that belongs to glycoside hydrolase family 27. VdGAL4 could cause plant cell death, and its signal peptide plays an important role in cellular immune response. VdGAL4-triggered cell death depends on BAK1 and SOBIR1 in Nicotiana benthamiana. In V. dahliae, the function of VdGAL4 in mycelial growth, conidia, microsclerotium, and pathogenicity was studied by constructing VdGAL4 deletion and complementation mutants. Results showed that the deletion of VdGAL4 reduced the conidial yield and conidial germination rate of V. dahliae and changed the microscopic morphology of conidia; the mycelia were arranged more disorderly and were unable to produce microsclerotium. The VdGAL4 deletion mutants exhibited reduced utilization of different carbon sources, such as raffinose and sucrose. The VdGAL4 deletion mutants were also more sensitive to abiotic stress agents of SDS, sorbitol, low-temperature stress of 16°C, and high-temperature stress of 45°C. In addition, the VdGAL4 deletion mutants lost the ability to penetrate cellophane and its mycelium were disorderly arranged. Remarkably, VdGAL4 deletion mutants exhibited reduced pathogenicity of V. dahliae. These results showed that VdGAL4 played a critical role in the pathogenicity of V. dahliae by regulating mycelial growth, conidial morphology, and the formation of microsclerotium.IMPORTANCE This study showed that α-galactosidase VdGAL4 of V. dahliae could activate plant immune response and plays an important role in conidial morphology and yield, formation of microsclerotia, and mycelial penetration. VdGAL4 deletion mutants significantly reduced the pathogenicity of V. dahliae. These findings deepened the understanding of pathogenic virulence factors and how the mechanism of pathogenic fungi infected the host, which may help to seek new strategies for effective control of plant diseases caused by pathogenic fungi. Verticillium dahliae Kleb is a soilborne plant-pathogenic fungus that can cause vascular wilt disease in more than 400 plants, including important economic crops and ornamental plants worldwide , which is a basic defense that begins when plant cells recognize conserved pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs)  . PRRs ars (PRRs) , 10. PTIs (PRRs) . Pathoges (PRRs) , 13. Howesponses . SimilarV. dahliae related to virulence have been characterized. V. dahliaeVdSsk1 was homologous to the Saccharomyces cerevisiae and was a response regulator of the two-component system; deletion of VdSsk1 severely reduced the fungal virulence on tobacco seedlings, and VdSsk1 was required for full virulence  VdNop12 was more sensitive to low-temperature stress at 15°C, and VdNop12 was essential for hyphal morphology, cold adaptation, and pathogenicity of V. dahliae  and Aspergillus fumigatus (Aayg1), was involved in production of microsclerotia and melanin biosynthesis and may catalyze two different precursors, one related to melanin synthesis (such as YWA1 and At4HN), and the other was a key factor in the network pathway of microsclerotium formation   protein, XEG1, of fectors) , 23. FoEresponse . V. dahlesponses . GH11 presponses . Althoug dahliae .V. dahliae was identified as a pathogenicity-related protein by RNA-Seq analysis; its expression level in a high-virulent isolate was 22-fold higher than in a low-virulent isolate in quantitative reverse transcription PCR (RT-qPCR) analysis . VdGAL4 was an alpha-galactosidase that belongs to GH27. VdGAL4 could cause plant cell death, which depended on the secretary function of signal peptide. VdGA4-triggered cell death depended on BAK1 and SOBIR1 in N. benthamiana. The deletion of VdGAL4 led to changes in the morphology of conidia and mycelia of V. dahliae, inhibited the production of microsclerotia, and enhanced the resistance to abiotic stress. In addition, we obtained data that suggested that the VdGAL4 deletion mutant also had reduced virulence of V. dahliae. Collectively, VdGAL4 played a critical role in the fungal development and pathogenicity of V. dahliae as a virulence factor.Recently, VdGAL4 (VDAG_04757) in VDAG_04757) was identified in V. dahliae strain VdLs.17 (https://www.ncbi.nlm.nih.gov/genome/832), and it had a conserved glycosyl hydrolase family 27 (GH27) domain and was named VdGAL4. The full-length cDNA of VdGAL4 was cloned from the strong pathogenic defoliating strain Vd080, which contained 1,215 bp and encoded a protein of 404 amino acids.An alpha-galactosidase gene , but only VdGAL4 had a transmembrane domain  with GH27 domain-containing proteins were identified in Agrobacterium tumefaciens-mediated transformation was used to transiently express each of the five proteins of GH27 in V. dahliae on N. benthamiana leaves. The sequences carrying VdGAL1, VdGAL2, VdGAL3, VdGAL4, and VdGAL5 were cloned individually into the PVX vector pGR107. Interestingly, transient expression of these five genes showed that only VdGAL4 triggered typical cell death in N. benthamiana leaves 7 days after infiltration (N. benthamiana leaves confirmed effective protein production by VdGAL1 to VdGAL5 (N. benthamiana and may be involved in immunity manipulation in host-V. dahliae interactions.ltration . Westerno VdGAL5 . The resSP. The recombined plasmid was transformed into the yeast YTK12. YTK12::pSUC2-VdGAL4SP grew well in the YPRAA medium (2% raffinose) with raffinose as the sole carbon source, and the insoluble 2,3,5-triphenyltetrazolium chloride (TTC) could be reduced to insoluble red 1,3,5-triphenylformazan (TPF)  of VdGAL4 was composed of 31 amino acids at the N terminus. The SPs of VdGAL4 were fused to the vector pSUC2 and generated pSUC2-VdGAL4an (TPF) . These rV. dahliae as a virulence factor. We found that VdGAL4ΔSP could not induce cell death in N. benthamiana, which indicated that SP was required for VdGAL4 to induce cell death and VdGAL4 must target the extracellular space to induce cell death  (PBI121-VdGAL4-RFP) and transiently expressed in N. benthamiana leaves. The result showed that VdGAL4 was located on the cell membrane and nucleus of N. benthamiana .To further detect the subcellular location of N. benthamiana leaves and confirmed the silencing of NbBAK1 and NbSOBIR1 3 weeks after virus inoculation, and the expression of two genes was significantly decreased  to silence NbBAK1 and NbSOBIR1 in ecreased . In addithamiana . ImmunobTRV2-GFP . Thus, tVdGAL4, deletion mutants were constructed by replacing the coding sequence of VdGAL4 in the wild-type (WT) strain Vd080 with a hygromycin resistance cassette via homologous recombination. After PCR verification, two independent deletion transformants were obtained, and Southern hybridization was used to verify that Hyg was a single copy in the two deletion mutants . Then, the genome sequence and promoter region of VdGAL4 were reintroduced into the deletion mutants, and the corresponding complementary mutant strains were obtained by PCR verification .In order to study the function of ΔVdGAL4 strains (ΔVdGAL4-1 and ΔVdGAL4-2 deletion mutant strains) were only 57.04 ± 21.46 U/g and 87.60 ± 18.67 U/g, respectively, and were only 4.85% to 7.40% of that of the WT strain  and 10.94 ± 0.20 mm (ΔVdGAL4-1), which were significantly lower than WT and C-ΔVdGAL4 strains; the same was true in the water agar medium amended with melibiose with the corresponding values of 10.47 ± 0.34 mm and 10.62 ± 0.40 mm  were similar to that of the WT and C-ΔVdGAL4 strains (C-ΔVdGAL4-1 and C-ΔVdGAL4-2), and there was no significant difference in radial growth diameter (After being cultured on potato-dextrose agar (PDA) medium at 25°C for 14 days, the phenotypes of the diameter  and C.C-ΔVdGAL4 strains were plump and smooth, while the conidia of the ΔVdGAL4 strain showed shrinkage with dents and irregular shapes on the surface . It is further explained that there is an important role of VdGAL4 in the conidial production of V. dahliae.The conidia of the WT and  surface . By comp strains . Under nC-ΔVdGAL4 strains was relatively regular, while the hyphal growth of ΔVdGAL4 strains had more branches  medium for 40 days, cultures of WT and  strains , and theVayg1, VT4HR, VaflM, VdSCD, VDH1, and VdLAC, were significantly lower than the WT and C-ΔVdGAL4 strains   . After i strains . These rΔVdGAL4, and C-ΔVdGAL4 strains in PDA for 14 days , mock-inoculated cotton seedlings were growing well without any disease symptoms, while cotton seedlings inoculated with the WT and browning  to C. Th strains . The dis reduced  and D. F strains . Quantif strains . TherefoF. oxysporum, Trichoderma reesei, Pichia pastoris, and Aspergillus niger  are enzymes that hydrolyze glycosidic bonds and play an important role in the hydrolysis and synthesis of sugars and glycoconjugates in organisms. Previous studies have shown that the different GH family members  in different plant pathogens can cause plant cell necrosis through different ways, stimulate plant defense response, and have different effects on pathogenicity  is widely present in plants, animals, and microorganisms, including us niger . In thisgenicity , 28. In V. dahliae that have been identified, such as the hydrophobin VdHP1, that can cause plant cell death and act as an effector to induce plant immunity (F. oxysporum and PsXEG1 of Phytophthora sojae acted as a pathogen-related molecular pattern (PAMP) on the extracellular matrix of plants to induce cell death  in N. benthamiana, and VdGAL4 SP can secrete invertase in yeast and transfer VdGAL4 into the extracellular space -melanin during microsclerotium formation and maturation, which helps them resist various environmental stresses and is important for the long-term survival of the pathogen , potato-dextrose broth (PDB), or Czapek-Dox medium unless otherwise specified. The upland cotton (Gossypium hirsutum) cultivar Jimian11, susceptive to V. dahliae, was grown in a greenhouse at 25°C (8 h/16 h dark/light cycle). The culture condition of N. benthamiana is the same as the Jimian11.The highly virulent, defoliating VdGAL4 gene was amplified from cDNA or gDNA samples of Vd080 based on the VdGAL4 sequence in the V. dahliae genome database. The specific primers were designed using Primer5.0 and multiple-sequence alignment using DNAMAN. The signal peptide analysis was implemented using SignalP5.0 . The transmembrane domain of VdGAL4 protein was predicted with TMHMM-2.0 . SMART was used to predict the conserved domain of VdGAL4 protein (http://smart.embl.de/smart/set_mode.cgi?NORMAL=1). The primers used are listed in Table S1 in the supplemental material.The SP and pSUC2::Mg87SP, which were used as positive and negative controls, respectively, and were then incubated in YPRAA medium (2% raffinose) to observe their growth. In addition, the sucrose convertase activity of SP was detected by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to an insoluble, red-colored 1,3,5-triphenylformazan (TPF). These transformed yeast strains were cultured in CMD-W medium  and then collected and stained with 2% TTC . Finally, invertase activity was determined by observing and comparing TTC color changes. The primers used are listed in Table S1 in the supplemental material.Functional verification of predicted signal peptides of VdGAL4 was prepared as described previously , 64. TheVdGAL1, VdGAL2, VdGAL3, VdGAL4, VdGAL5, and the VdGAL4 without signal peptide (ΔSPVdGAL4). All of these sequences were cloned severally into the PVX vector pGR107 and transformed into the Agrobacterium tumefaciens strain GV3101. Transient gene expression assays were performed on N. benthamiana plants; Bcl-2-associated X protein (BAX) and green fluorescent protein (GFP) were used as positive and negative controls, respectively  of 0.8 with infection solution, and then injected into 4-week-old N. benthamiana leaves. Symptom development was monitored from 3 to 7 days postinfiltration. Each assay was performed on 3 leaves from 3 individual plants and repeated 3 times. The primers used are listed in Table S1.The tested genes were amplified from cDNA of Vd080 using the indicated primers, including ectively . All AgrN. benthamiana leaves, the total proteins were extracted from the leaves of N. benthamiana plants after Agrobacterium infiltration for 48 h. The infiltrated leaves were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer  at 4°C for 30 min. The lysed samples were centrifuged at 4°C for 10 min, and the supernatants were extracted as the crude protein samples. The protein samples were mixed with protein loading buffer and inactivated at high temperatures for 10 min. The proteins were analyzed by SDS-PAGE and electroporated onto polyvinylidene fluoride (PVDF) membranes .To detect protein expression in A. tumefaciens GV3101. The A. tumefaciens strains pTRV1 and pTRV2 were mixed at a ratio of 1: 1, and the final OD600 was adjusted to 0.8, which was injected into 3-week-old N. benthamiana leaves  and transformed into the A. tumefaciens strain GV3101. Subsequently, the suspension (OD = 1.0) was injected into N. benthamiana leaves using a previously reported method  method. The VdGAL4 deletion mutant was constructed using B303 vector. The 1.2-kb upstream fragment and 1.2-kb downstream fragment of VdGAL4 were separately amplified via PCR with the primer pairs B303-VdGAL4-UP-F/R and B303-VdGAL4-DOWN-F/R (Table S1). The hygromycin-resistant fragment was amplified from the B303 vector with B303-VdGAL4-Hyg-F/R (Table S1). Then, those fragments were connected and introduced into B303 vector by the method of homologous recombination. The positive recombinant vector (B303-VdGAL4-UP/Hyg/DOWN) was transferred into A. tumefaciens strain AGL-1 for fungal transformation (Gene deletion and complementation transformants were generated by the ormation . The posΔVdGAL4-1 and ΔVdGAL4-2 strains) was digested by HindIII. A 698-bp Hyg fragment (selective marker gene) was used as probe.The obtained deletion mutants were further verified by Southern blotting. Southern blot analysis was performed using digoxigenin (DIG) high prime DNA labeling and detection starter kit I . Genomic DNA of the WT strain and deletion mutants (VdGAL4 complementation mutant was constructed using pSULPH-mut-RG#PB vector. The 1.2-kb upstream fragment and VdGAL4 genomic DNA were amplified with the primer pairs pSULPH-VdGAL4-F/R (Table S1). The positive recombinant vector (pSULPH-VdGAL4) was transferred into AGL-1. Subsequently, the obtained deletion mutants were used for fungal transformation of complementation  activity assay kit . α-Galactosidase can decompose p-nitrophenyl-α-d-galactopyranoside into p-nitrophenol, so the production of 1 nmol p-nitrophenol per gram mycelia per hour is defined as an enzyme activity unit. Experiments were triple replicated.For analysis of α-galactosidase activity of VdGAL4, WT, ΔVdGAL4, and C-VdGAL4 strains were cultured on water agar medium amended with isomaltose or melibiose. After 16 h at 25°C, the dishes were stained with iodine (0.2%)-KI (2%) (volume of 1:1) solution. Experiments were triple replicated.To analyze the hydrolytic activity of VdGAL4, the conidial suspensions of WT, 6 conidia/mL of the WT, ΔVdGAL4, and C-VdGAL4 strains was placed in the center of PDA plates and incubated at 25°C for 14 days. Experiments were triple replicated.For the radial growth rate assay, 5 μL of conidial suspension with a concentration of 5 × 10ΔVdGAL4, and C-VdGAL4 strains, agar plugs were collected using an 8-mm-diameter borer from the edge of mycelium on PDA plates that were cultured for 7 days. Then, the plug was shaken in 1 mL of sterile water, and the number of conidia was quantified under microscope (3 conidia/mL) was added as a hanging drop to the glass slide and cultured at 25°C for 5 h. The germination rate of conidia was measured with the optical microscope  of the respective strains were evenly spread on a plate of BMM   . The plaΔVdGAL4, and C-VdGAL4 strains were cultured in Czapek-Dox medium (without sucrose) amended with different carbon sources   . Colony The sensitivity of mutants to abiotic stress agents was detected using PDA containing 0.002% SDS, 0.02% Congo red, 1 M sorbitol, 1 M KCl, and 1 M NaCl . The rel3 conidia/mL) were heated at 45°C for 1 h and then added as a hanging drop to the glass slide and cultured at 25°C for 5 h  onto the center of sterilized cellophane membrane covering PDA medium at 25°C for 3 days  of WT, deletion, and complementation strains were inoculated using a root dip inoculation method  fungal DNA kit (Omega Bio-tek) and quantified by NanoDrop 2000, and for each sample, 100 ng of DNA was employed for quantitative real-time PCR (qPCR) reaction. The qPCR was performed using the ChamQ universal SYBR qPCR master mix (Vazyme) with the primers listed in Table S1. V. dahliae β-tubulin (GenBank accession no. DQ266153) was used to quantify fungal colonization. The cotton UBQ7 gene was used as endogenous plant control. Three independent biological and technical repeats were performed.Cotton tissue samples from the stem of inoculated plants were collected at 26 dpi for VdNLP1 (VDAG_04701.1), VdPLP (VDAG_00942), and Vdpf (VDAG_08521.1), were used for RT-qPCR analysis. For further analyses the effect of VdGAL4 deficiency on V. dahliae infection structure, such as the infection peg-related genes VdNoxB (VDAG_09930) and VdPls1 (VDAG_01769); regulating infection peg development of transcription activator VdCrz1 (VDAG_03208); assembling mycelium neck ring-related secretary protein VdSep5 (VDAG_04382); regulating appressorium formation-related gene VdCSIN1 (VDAG_05652); and plant root adhesion, penetration, and colonization of V. dahliae-related transcription factors Som1 (VDAG_JR2_Chr1g09120a) and Vta3 (VDAG_Chr1g07600a). To further investigate melanin, microsclerotium formation, and cell wall biogenesis, six genes, including Vayg1 (VDAG_04954), VT4HR (VDAG_03665), VaflM (VDAG_00183), VdSCD (VDAG_03393), VDH1 (VDAG_02273), and VdLAC (VDAG_00189), were used for RT-qPCR analysis  method. The mean and standard error of gene expressions were estimated from three biological replicates.All of the strains, including WT, deletion, and complementation mutant strains, were cultured in PDA at 25°C for 7 days, and the mycelia were collected after filtering with a 4-layer sponge. Total RNA was extracted with fungal total RNA isolation kit (Sangon Biotech), and cDNA was synthesized with the HiScript II Q RT supermix for qPCR (+genomic DNA [gDNA] wiper) (Vazyme) following the manufacturer’s procedure. The RT-qPCR of the above-described genes (Table S1) was performed with the β-tubulin gene (Table S1) of P values of 0.05 or 0.01.Statistical analyses were performed using SPSS statistical software package (v22.0). One-way analysis of variance (ANOVA) was applied and followed by the Student-Newman-Keuls (SNK) test to determine significant differences between treatments at


The rise in the flow of narratives is directly associated with technological advances in communication. In pandemic times, the narratives have produced profound consequences in many dimensions of human life, such as individuals' belief systems. Narratives operate as a background of the self's functioning and present substantial importance to personality development. On the other side, narratives can influence the functioning of the self's disorders. It occurs when narratives do not respect a hierarchical belief system in the individuals. The disrespect to the central beliefs that operate in the individuals' personalities often fosters a psychosomatic process that distances them from authentic contact with themselves. Consequently, it enhances mental suffering and psychic illness. The current paper aimed to discuss some intersections between narratives and states of psychic suffering. The Covid-19 pandemic has specific characteristics which differ from other global catastrophes, such as the speed of information transmission , 3, sociAlthough, narratives have other vital functions than distorting reality and fostering fear in the population with false or partial information. Narratives are aligned with human development in different ways. Even fictional (imaginary world) or nonfictional , the narratives comprehend an ontological semantic core . Thus, tDespite the advances in COVID-19’s vaccines program worldwide, Kalin  has warnThe study of Gubrium and Gubrium  brought The connectivity, speed of information, and broad access for content production are specific and significant characteristics to consider in the Covid-19 pandemic crisis. The possibility to confirm an affirmation or negation is not always constructive and beneficial. The attribution of meaning in the belief system of individuals does not work in the same way and with the same speed as the information is produced. Besides, dealing with many possibilities of meanings or truth beyond the narratives is often complicated and exhausting. Psychological preparation is required because the flow of meanings can contribute to the best attitudes but also influence the worst.Among the most elementary foundations of the human psyche, belief systems present essential contributions to understanding individuals’ mental functioning and personality development. From the trajectory of people’s lives, it is possible to notice that they develop and qualify their beliefs from various aspects of their experiences. Beliefs do not have the same meaning and value  for everA priori, the hierarchical belief system cannot be understood as a positive or negative attitude because it describes a structural functioning. Positive or negative attitudes are consequences of a complex functioning in which beliefs comprehend a structural part of attitude's phenomenon. The differentiation of meanings and values in the belief’ system is not an exceptional condition for some individuals. Indeed, it is a fundamental and elementary principle for developing the individuals' singularities from a specific to a broad perspective.Merit is one of the central beliefs of a hierarchical system, and its development influences crucial domains of personality and mental health, such as self-esteem, identity, and empathy. An individual can corrupt or mischaracterize his personal merit beliefs by influencing narratives. In a hypothetical case, an individual who transfers all his responsibilities to other agents, institutions, or conditions removes valuable resources to improve some self's characteristics. The narratives that totalized the social inequalities in the collective domains offer an 'ideal' rhetorical condition for someone hiding their intimacy in the social dimension. Besides, the generalization of social inequalities as an end in themselves inserts individualities as a simplistic attribute in the real modifications of life conditions.In addition, the distinction of meaning and values in the belief' system permits individuals to recognize the elements of reality more properly and less propitious to alienated positions. The production of opposite or contradicting narratives potentiates uncertainties, leaving individuals more conducive to driving that somehow calms their fears. It is a propitious scenario for the implementation of authoritarianism. In the current Covid-19 pandemic, when Big Techs still have hard influences on the production and development of narratives, alienated' beliefs and positions reveal a dangerous condition to mental control and mass psychosis . The conNarratives can corrupt the beliefs system of individuals and further enhance inequalities when the conditions of real impediments are not differentiated from supposed or ideological impediments. Thus, it is crucial to understand how the narratives impact individuals' belief systems and vice versa. On the one side, narratives can contribute to the healthy development of individuals' personalities. However, disrespecting the central beliefs that operate in their personalities fosters a psychosomatic process that distances them from authentic contact with themselves. Consequently, it enhances mental suffering and psychic illness.It is understood that the clarity and appropriation of the influences of narratives in mental functioning can contribute to better comprehension and diagnostics on many fronts associated with mental suffering and social intolerance. The denial of a hierarchical belief system of individuals exposes them to conditions of psychic suffering. Furthermore, it fosters more inequalities and social tensions because it distorts the individuals' singularities so that there is no possibility of healthy social participation. Besides, narratives can foster the sensation of uncertainty and influence people to become more conducive to accepting everything, even destructive rules.Narratives significantly influence the self’s development and the relationships between individuals worldwide. They can even influence the functioning of the self positively and healthily. However, understanding how they affect the individual and the collective in which they participate is crucial, especially as mental suffering in the pandemic has increased exponentially.


Drosophila, which is emerging as a powerful model in the field, due to the extensive, well-characterized and stereotypic remodeling events occurring throughout its nervous system during metamorphosis, combined with the wide and constantly growing toolkit to identify CAM binding and resulting cellular interactions in vivo. We believe that its many advantages pose Drosophila as a leading candidate for future breakthroughs in the field of neuronal remodeling in general, and spatiotemporal control by CAMs specifically.Developmental neuronal remodeling is required for shaping the precise connectivity of the mature nervous system. Remodeling involves pruning of exuberant neural connections, often followed by regrowth of adult-specific ones, as a strategy to refine neural circuits. Errors in remodeling are associated with neurodevelopmental disorders such as schizophrenia and autism. Despite its fundamental nature, our understanding of the mechanisms governing neuronal remodeling is far from complete. Specifically, how precise spatiotemporal control of remodeling and rewiring is achieved is largely unknown. In recent years, cell adhesion molecules (CAMs), and other cell surface and secreted proteins of various families, have been implicated in processes of neurite pruning and wiring specificity during circuit reassembly. Here, we review some of the known as well as speculated roles of CAMs in these processes, highlighting recent advances in uncovering spatiotemporal aspects of regulation. Our focus is on the fruit fly  Following their initial establishment, developing neural circuits are further refined by a combination of degenerative and regenerative events. Collectively known as developmental neuronal remodeling, such processes are essential for shaping the connectivity of functional circuits, and represent a conserved strategy occurring throughout the animal kingdom and across the peripheral and central nervous systems. Remodeling varies in scale, from retraction of single synapses, up to degeneration of long stretches of axons or dendrites, often with remarkable spatiotemporal precision. Regressive steps are generally followed by progressive ones including stabilization and even reformation of new, adult-specific connections . DefectsIn recent years, it is becoming increasingly evident that neuronal remodeling is not solely governed by intrinsic genetic programs and cell-autonomous mechanisms . Of course, focusing on Drosophila does not underestimate the significant contributions of research in mammalian models, mostly to understanding the roles of CAMs in synapse retraction/stabilization  is a well-characterized circuit in the fly brain that is comprised of three types of intrinsic neurons, known as Kenyon cells (KCs), which are sequentially born from the same neuroblasts. The first-born KCs – called γ-KCs – undergo stereotypic remodeling during metamorphosis, in which they prune their dendrites completely, and their bifurcated axons up to their branchpoint. Later during the pupal stage, γ-KCs regrow their dendrites, and their axons to form adult-specific projections  expressing the same odorant receptor converge onto one of ∼50 discrete glomeruli in the antennal lobe – a structure analogous to the mammalian olfactory bulb – where they synapse with a single class of projection neurons in each glomerulus . It was previously shown that embryonic-born PNs participate in both the larval and adult olfactory circuits, in which they innervate different glomeruli. Developmental studies indicate that during metamorphosis, PNs undergo local pruning of their dendritic and axonal terminal branches, followed by re-extension of adult projections. In contrast, larval-born PNs, which constitute the majority of PN types, only participate in the adult circuitry and do not remodel wiring are just beginning to be unraveled.via cellular processes that affect delivery to the membrane (such as trafficking and exocytosis), stabilization within the membrane (such as interactions with the cytoskeleton) and, finally, removal from the membrane  neurons, which extend highly branched dendrites along the body wall. Proper dendritic coverage during the initial elaboration of da dendrites requires self-avoidance and tiling mechanisms which are both mediated by CAMs . This indicates that the temporal specificity of dendrite pruning is dictated, at least in part, by precise timing of Nrg internalization. Interestingly, while Nrg is expressed, and internalized to endosomes, in both axons and dendrites, its loss selectively affects dendrites, and does not “force” ectopic pruning of axons. Therefore, the mechanism underlying compartment-specific pruning, and whether and how it relates to Nrg, remains unclear. Perhaps Nrg downregulation renders da neurons more susceptible to pruning by reducing their adhesion – most likely to the epidermis – and another, Nrg-independent mechanism protects axons from a similar fate. Since its identification, many additional regulators of Nrg endocytosis-mediated pruning have been uncovered, including members of the secretory pathway, protein trafficking, and endo-lysosomal degradation (Drosophila Giant Fiber (GF) circuitry, which exhibits pruning of extraneous axonal branches during the pupal stage , as their interactions induce intracellular signals that eventually lead to spine collapse -mediated reduction in the membranal levels of the IgSF CAM Fascilin II (FasII), the ortholog of the mammalian neural cell adhesion molecule (NCAM). While trafficking was ruled out as the major regulator of FasII membranal levels, whether FasII downregulation also occurs via endocytosis, or by an alternative destabilizing mechanism, is unknown. Mutating JNK, or overexpressing FasII, is sufficient to inhibit pruning of γ axons, but not dendrites. Moreover, overexpressing other CAMs has a similar effect, suggesting that increased axo-axonal adhesion, in general, prevents normal progression of pruning. Manipulations of JNK or FasII are the first case of selective regulation of axon vs. dendrite pruning. Interestingly, endogenous FasII is indeed only expressed in γ axons and excluded form dendrites and cell bodies, which could, in theory, account for the observed phenotype of JNK mutants. However, even strong transgenic FasII overexpression, which was also localized to dendrites, did not inhibit dendrite pruning , output neurons (MBONs), and modulatory neurons that are mostly dopaminergic (DANs). MBONs and DANs innervate the KC lobes in a compartmentalized fashion thus dividing the MB lobes to discrete and functionally relevant sub-axonal zones  neuron were shown to undergo developmental remodeling in the same timeframe. Moreover, cell-autonomous inhibition of γ-KC pruning disrupts APL pruning. This coordination relies on γ-KC activity, and Calcium/Calmodulin signaling within the APL neuron. Interestingly, artificially increasing γ-KC-to-APL adhesion by ectopically expressing FasII is sufficient to inhibit pruning of both neuronal types wiring of complex and stereotypic circuits. In the MB, Dpr12 and DIP-δ mediate formation of the γ4/5 axonal zones during γ-KC regrowth , but therebellum .Drosophila. These are likely to transform our approach to similar mechanisms of neuronal remodeling and (re)wiring in other systems and organisms, in both physiological and pathological contexts.Due to its awesome genetic power and the wide array of biochemical and imaging techniques, we strongly anticipate breakthroughs in our undertesting of the roles of CAMs in spatiotemporal control of remodeling to arise from HM and OS wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cdk5 gene (referred to as DS-CDK5-KD mice) and investigate its role in vivo. DS-CDK5-KD mice exhibited deficits in locomotor activity and disturbances in activity/rest behavior. Additionally, Golgi staining of neurons in the DS revealed that CDK5 deletion reduced dendrite length and the number of functional synapses, which was confirmed by significant downregulation of MAP2, PSD-95, and synapsin I. Correlated with this, DS-CDK5-KD mice displayed reduced phosphorylation of Tau at Thr181. Furthermore, whole-cell patch-clamp recordings of green fluorescent protein-tagged neurons in the striatum of DS-CDK5-KD mice revealed a decreased frequency of spontaneous inhibitory postsynaptic currents and altered excitatory/inhibitory synaptic balance. Notably, anterograde labeling showed that CDK5 KD in the DS disrupted long-range projections to the secondary motor cortex, dorsal and ventral thalamic nuclei, and basolateral amygdala, which are involved in the regulation of motor and circadian rhythms in the brain. These findings support a critical role of CDK5 in the DS in maintaining the striatal neural circuitry underlying motor functions and activity/rest associated with circadian rhythms that are perturbed in neurodegenerative disorders.Dysfunction of striatal dopaminergic circuits has been implicated in motor impairment and Parkinson’s disease (PD)-related circadian perturbations that may represent an early prodromal marker of PD. Cyclin-dependent kinase 5 (CDK5) negatively regulates dopamine signaling in the striatum, suggesting a critical role of CDK5 in circadian and sleep disorders. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to produce mice with a dorsal striatum (DS)-specific knockdown (KD) of the The online version contains supplementary material available at 10.1186/s13041-022-00939-2. Neurodegenerative disorders, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), are increasing in prevalence with the aging of the population. In addition to the classical motor symptoms of neurodegenerative disorders in older adults, high rates of sleep disorders, such as insomnia, hypersomnia, sleep apnea are likely caused by damage to sleep-controlling regions of the brain. PD is a devastating neurodegenerative disorder characterized pathologically by the loss of dopaminergic neurons in the substantia nigra (SN) pars compacta (SNpc) . The dor1 and D2  and mounted on a stereotaxic apparatus . Mice were randomly divided into wild-type (WT), LV-NC-sgRNA (CDK5-NC), and LV-Cdk5-sgRNA (CDK5-KD) groups, and the respective LV vectors were bilaterally microinjected into the DS. For microinjection, we used the following two bilateral sites in the DS  with stereotaxic coordinates (in mm) according to the modified stereotaxic mouse atlas of Paxinos and Franklin . Tissue dissection was performed in the same time window for all groups. For western blotting, solubilized proteins (50 µg per sample) were resolved on SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes . The membranes were blocked with 5% non-fat milk diluted in TBST at room temperature for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-CDK5 mouse monoclonal antibody , anti-MAP2 rabbit polyclonal antibody , anti-synapsin 1 mouse monoclonal antibody , anti-PSD-95 rabbit polyclonal antibody , anti-phospho-Tau (Ser202) rabbit polyclonal antibody , anti-phospho-Tau (Thr181) rabbit polyclonal antibody , anti-Tau mouse monoclonal antibody , and anti-β-actin mouse monoclonal antibody . The membranes were then washed with TBST and incubated with the appropriate peroxidase-conjugated secondary goat anti-rabbit/mouse IgG  for 1 h at room temperature. An ECL detection kit  was used, and immunoreactive bands were quantified on an imaging system . β-actin was used as the loading control.Cdk5-sgRNA injection, the AAV-hSyn-mCherry-IRES-WGA-Cre was bilaterally microinjected into the center of the original virus injection points . Animals were anesthetized throughout surgery with pentobarbital sodium . Following craniotomy, a microsyringe was lowered into the brain using the above coordinates, and 500 nL of AAV-hSyn-mCherry-IRES-WGA-Cre were injected over a 10-min period  using a vibrating tissue slicer . Coronal slices were submerged for 30 min at 32 °C in artificial cerebrospinal fluid (ACSF)  at 295–300 mOsm/L. All reagents were from Sigma-Aldrich  unless otherwise indicated. Following this recovery period, slices were transferred to room-temperature ACSF until recording.Mice were anesthetized with pentobarbital sodium , and brains were removed. Striatal slices (300 μm) were cut in ice-cold cutting solution . The pH of the pipette solution was adjusted to 7.3 with 1 mM CsOH, and osmolarity was adjusted to 285–290 mOsm/L. A low-power objective (4×) was used to identify the DS region, and a 40× water immersion objective  coupled to an infrared differential interference contrast microscope with a fluorescence system and a CCD camera was used to visually identify, patch, monitor, and record CDK5-KD neurons in the DS. MSNs were identified according to previously determined membrane characteristics and firing properties . Taken together, the immunostaining and western blot analyses showed that LV/Cas9-sgRNA efficiently knocked down CDK5 in DS neurons by day 14 after injection.We generated DS-CDK5-KD mice using the CRISPR–Cas9 system, and the efficiency of LV/Cas9-p < 0.001] and the total velocity [Fig. p < 0.001] were significantly reduced in DS-CDK5-KD mice. In addition, the activity heatmap revealed that DS-CDK5-KD mice preferred to travel in the peripheral area more than the central area of the arena  and shorter activity duration during the day and night . It is well known that the α/ρ ratio [the ratio of running duration (α) to rest duration (ρ)] is positively correlated with the circadian period . By concentric circle (Sholl’s) analysis, we found a significant decrease in dendritic branching in DS-CDK5-KD mice  compared with that in WT  and DS-CDK5-NC  mice [Fig. p = 0.0011]. CDK5 KD in the DS also impaired MSN spine generation and significantly decreased dendritic spine density. Representative sections of dendritic spines are shown for each group in Fig. n = 15 segments) compared with that in WT  and DS-CDK5-NC  mice [Fig. p < 0.0001].CDK5 is required for radial neuronal dendrite and spine maintenance , and CDKing Fig. A. Dendrip = 0.0001], MAP2 [Fig. p = 0.0016], and synapsin 1 [Fig. p = 0.007]. Next, we examined Tau protein and its phosphorylation sites Thr181 and Ser202 -mediated signaling in D2-MSNs [1-PKA-induced phosphorylation of DARPP-32 at Thr34 [1-MSNs. Consistent, the selective involvement of CKD5 in D1-MSNs has been documented in the other studies [1 receptors, which play a key role in sleep–wake behavior [In the current study, we did not examine whether CDK5 dysfunction is involved in night-time sleep disturbances associated with neurodegenerative disorders, such as PD or AD, and this remains to be addressed in future studies. However, in an animal model of PD, in which the neurotoxin MPTP is used to selectively induce the neurodegeneration of DA neurons in the SNpc, higher levels of CDK5 activity are detected . Furtherar level , 59, sti D2-MSNs –62. Impoat Thr34 , 17. We  studies . The strbehavior , 44. TheCdk5 gene in the DS produced major changes in dendrite structure at most E/I synapse of MSNs in the striatum. These changes impacted communication between the DS and other brain regions, resulting in the dysregulated initiation of sleep–wake disturbance behaviors  Cell morphology in the DS area was detected by HE staining. The striatal neuron in DS-CDK5-KD mice were morphologically normal and showed no significant loss. Scale bars = 20 μm. (B) Left panel: detrimental effects in the DS area of CDK5-KD were detected by Nissl staining. Right panel: quantitative profiles of Nissl bodies in the DS area of WT, DS-CDK5-NC, and DS-CDK5-KD mice. Nissl staining revealed no detrimental effects induced by the delivery of LV/Cas9-CDK5-sgRNA into the DS . Arrows: Nissl body. Scale bars = 0 μm. (C) Left panel: cell apoptosis in the DS area was detected by TUNEL staining. Right panel: quantitative profiles of TUNEL-positive cells in the DS area of WT, DS-CDK5-NC and DS-CDK5-KD mice. Quantitative analysis for the TUNEL-positive cells showed there were no significant difference among in WT, DS-CDK5-NC, and DS-CDK5-KD mice . Arrows: TUNEL-positive cells. Scale bars = 20 μm. The TUNEL-positive cells showed the typical morphological features of apoptosis such as chromatin condensation, cytoplasmic budding and apoptotic bodies. Data are represented as the mean ± SEM, n = 15. One-way ANOVA, Tukey’s multiple comparisons test.