MyoD and miR-206 and miR-133a expression, leading to a reduced number and diameter of myotubes. PA (100 µM), despite not affecting proliferation, impairs myotube formation by reducing the expression of Myf5 and miR-206 and decreasing protein synthesis. Interestingly, 100 and 150 µM PA-treated myotubes had a higher number of type II fibers than control cells. In conclusion, PA affects negatively myotube diameter, fusion, and metabolism, which may be related to myomiRs. By providing new insights into the mechanisms by which PA affects negatively skeletal muscle, our data may help in the discovery of new targets to treat sarcopenic obesity.Palmitic acid (PA), a saturated fatty acid enriched in high-fat diet, has been implicated in the development of sarcopenic obesity. Herein, we chose two non-cytotoxic concentrations to better understand how excess PA could impact myotube formation or diameter without inducing cell death. Forty-eight hours of 100 µM PA induced a reduction of myotube diameter and increased the number of type I fibers, which was associated with increased miR-206 expression. Next, C2C12 myotube growth in the presence of PA was evaluated. Compared to control cells, 150 µM PA reduces myoblast proliferation and the expression of  A high-lipid diet, mainly containing saturated fatty acids, such as palmitic acid (PA), the second most abundant fatty acid in circulation, is associated with the development of obesity ,2. ExcesA healthy skeletal muscle mass is essential in the reduction of complications of obesity. Lipid overload in muscle appears to affect not only insulin signaling but also muscle mass and muscle regenerative capacity. Previously, we have shown myogenesis is reduced and myomiRs are dysregulated in mouse soleus muscle after 12 weeks of high-fat diet , suggestMicroRNAs are small non-coding RNAs that act on the post-transcriptional regulation of gene expression. The importance of microRNAs in metabolic diseases are well demonstrated by mice specifically lacking dicer in adipose tissue: these mice exhibit a defect in miRNA processing in AT, resulting in a decrease in white AT mass, whitening of brown AT mass, insulin resistance, and dyslipidemia . By the Taking into consideration that chronic exposure to saturated fatty acids is deleterious to skeletal muscle, we chose two-non cytotoxic PA concentrations to better understand how excess PA could impact myotube formation or skeletal muscle mass by evaluating the expression of proteins and microRNAs involved in myogenesis.Herein, we show that PA reduced myotube diameter in 5-day differentiated C2C12 cells, increased the number of type I fibers, and upregulated miR-206 expression. There was no difference in protein synthesis or levels of proteins involved in protein degradation. When C2C12 myoblasts were cultivated in differentiation media containing PA (100–150 µM), both concentrations resulted in smaller myotubes, but only a higher concentration of PA decreased myoblast proliferation. Of note, myotubes formed from PA-treated myoblasts were more glycolytic and had decreased levels of miR-206, as previously described in skeletal muscle of diabetic subjects.Initially, MTT -2,5-diphenyl-2H-tetrazolium bromide) and lactate dehydrogenase (LDH) assays were performed with the purpose of evaluating cell viability of myoblasts treated with PA and vehicle, and to define the concentration that was not toxic for the cells. We first attempted to use 250 μM and 500 μM concentrations; however, these concentrations significantly reduced cell viability after 24 h . Next, we did a time-course using lower concentrations of PA (50–150 μM). From analyses of both MTT and LDH assays A,B, we op > 0.05)  C,D. In c > 0.05) C,D. Howe > 0.05) C,D.Altogether, these data suggest that PA (100 and 150 μM) does not cause cytotoxicity; however, cells treated with 150 μM PA showed a decrease in proliferation compared to control or 100 μM PA. Thus, we set 100 μM concentration to treat differentiated myotubes to avoid death and keep their proliferative capacity similar to the control myotubes.The effect of PA in the two main fiber types (I and II) was evaluated by immunolabeling of differentiated myotubes with anti-Myosin heavy chain (MyHC)-1 A and antHk, Hexokinase), fatty acid oxidation , and/or mitochondrial markers . Treatment with 100 µM PA for 48 h significantly reduced Pdk4 expression but not Ppargc1a (p = 0.08), Cpt1b (p = 0.13), Hk (p = 0.78), or Acad11 (p = 0.55), suggesting C2C12 myotubes did not have a higher oxidative capacity after PA treatment , and miR-23a (microRNA associated with atrophy) in PA-treated and control myotubes . Our resMuscle mass maintenance is a dynamic process, and the skeletal muscle responds to different stimuli reshaping the morphological, biochemical, and physiological state of the myofibers. Muscle regrowth after injury is crucial for the repair of muscular functions, and in order to elucidate if PA affects C2C12 myogenesis, C2C12 were grown for 5 days in differentiation media in the absence (vehicle-control) or in the presence of 100 or 150 μM PA. For those experiments, we included 150 μM PA concentration, because in the initial experiments, this concentration affected C2C12 myoblasts proliferation.MyoD), Myogenic factor 5 (Myf5), Myomesin-2 (Myom2), and Myosin heavy chain 7 (MyH7)) and myostatin (Mtsn), which is a negative growth regulating factor responsible for inhibition of the PI3K/AKT pathway that acts on protein synthesis [MyoD and Myf5, two early differentiation markers of myotubes, PA has a transient impact on their expression. On day one of differentiation, 100 μM PA increases Myf5 expression 4-fold, but it decreases its expression from day two to five. On day two of differentiation, 150 μM PA increases MyoD expression 6-fold but decreases it on the third day  independent of the treatment  and S6k mRNA was reduced compared to the control group (Igf1r) and Ras homolog enriched in brain (Rheb) transcripts levels among the groups, and only 150 µM PA induced Mtor levels  was dissolved in ethanol 100% and diluted in DMEM supplemented with 10% FBS or with 2% horse serum (Life Technologies) depending on the assay, containing 2% fatty acid-free bovine serum albumin (BSA)  to reach desired fatty acid concentrations [Mouse C2C12 myoblasts were purchased from American Type Culture Collection  and used until passage 8. Myoblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose concentration, supplemented with 10% fetal bovine serum (FBS)  and 1% antibiotic  and maintained in a humidified incubator at 37 °C in an atmosphere of 5% COtrations . For theThe effect of PA on the cytotoxicity of C2C12 myoblasts was evaluated after 24–72 h of treatment with PA (0–500 µM). For MTT assay, 400 µL of MTT solution (diluted in DMEM) was added to each well of a 6-well plate, and then, the plates were incubated at 37 °C for a duration of 2.5 h. After this period, the medium containing MTT was removed, and 700 µL of DMSO was added to the wells to solubilize the formazan salt. Then, the solutions were homogenized, and 200 µL was transferred to a 96-well plate for spectrophotometry analysis, and absorbance was measured at 550 nm. For lactate dehydrogenase (LDH) assay , at the end of the period of treatment, the medium was collected, and 30 µL was transferred to a 96-well plate containing 70 µL of LDH solution. The plate was incubated at room temperature for 30 min, and then, absorbance at 492 nm was measured.TUNEL (TdT-mediated dUTP-TMR Nick End Labeling) assay was used to quantify apoptotic cells using In Situ Cell Death Detection Kits (Roche). Myoblasts were grown on 6-well plates until they reached 100% confluence and then were treated with vehicle or PA (100 μM and 150 μM) for 24 h. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 0.05 M phosphate-buffered saline (PBS) containing 0.1% sodium citrate. Subsequently, the cells were incubated with the enzyme terminal deoxynucleotidyl transferase (TdT) and deoxyadenosine triphosphate (dATP), labeled with Tetramethyl rhodamine diluted 1:5 (GIBCO) and DAPI , at 37 °C for 1 h. The cells were washed with phosphate buffer saline and visualized with microscope fluorescence (Carl Zeiss) using AxioVision 4.8 software .Cells were seeded in 6-well plates and grown to 100% confluence. The cell layer was scratched using a sterile pipette tip (p200), and the distance of the resulting gap was measured initially (time 0 h) and after 2, 12, and 16 h of the injury. Cells were photographed using a camera  coupled to an inverted light microscope . Translocation (net distance by a cell during the course of the experiment) and speed of migration were evaluated. The distance of the gaps was measured using the AxioVision 4.8 software.Myoblasts were cultured and treated with PA during 5 days of differentiation into myotubes, or 5-day myotubes were treated for 48 h with PA. In both conditions, cells were washed 1x with PBS and fixed with methanol for 20 min. Subsequently, cells were washed 3x with PBS and incubated with 5% blocking solution (1× PBS + 5% serum + 0.05% Triton X-100) for 2 h at room temperature. After washing 3x with PBS, cells were incubated with anti-MyHc1  or anti-MyHC2  overnight at 4 °C (1:250), washed with PBS (3 times for 10 min), and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:500 dilution Sigma-Aldrich) and DAPI for 2 h at 4 °C, washed with PBS, and then examined immediately in fluorescence microscope (Carl Zeiss). Images from positive myotubes for anti-MyHC1 and anti-MyHC2 were captured at 200X magnification using an Axiocam MRC Zeiss digital camera. For each well of a 6-well plate, four regions were used, and at least thirty myotubes were analyzed per well and quantified using AxioVision 4.8 software. Both cell diameter and length were measured.® reagent , according to the manufacturer’s instructions. Isolated RNA was quantified using a BioDrop µLite spectrophotometer . RNA was stored at −80 °C prior to analysis by RT-qPCR. For miRNA qPCR, the RNA samples were quantified as previously described, and 50ng/reaction of total RNA were transcribed into cDNA using a Taqman® microRNA assay system , according to the manufacturer’s instructions. The expression of miRNA miR-1, miR-206, miR-133a, miR-208b, miR- 23a, and miR-499 was measured by qPCR, using ABI Prism 7500 equipment , following the universal protocol of amplification: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min, followed by dissociation curve. Gene expression was quantified using the 2−ΔΔCT method. The housekeeping genes U6 and sno202 were used as normalizers for myoblasts and myotubes, respectively. For mRNA expression, cDNA was synthesized from 500 ng total RNA extract using SuperScriptTM III RT RNase H (Life Technologies), according to manufacturer’s instructions. The quantification of mRNA expression of genes involved in metabolism was performed by RT-qPCR in a total volume of 10 μL, containing diluted cDNA template (1/10), forward and reverse primers (200 nM each), and SYBR Green Master Mix (Life Technologies). Gene expression was performed using the 7500 Real Time PCR System (Applied Biosystems), following the universal protocol of amplification: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Dissociation curves were performed to test primers specificity. Relative gene expression quantification was determined by 2−ΔΔCT method. Hprt1 or Rpl0 was used as a reference gene for myoblast and myotubes, respectively. Primers sequences are shown in Total RNA from C2C12 cells grown for 1–5 days in the absence or presence of PA and from differentiated myotubes treated or not with PA for 48 h was extracted using TRIzolProteins from 7-day differentiated myotubes treated with PA were extracted by homogenizing the myotubes in radioimmunoprecipitation assay (RIPA) iced buffer containing 1% protease inhibitor cocktail, and concentration was determined by Bradford method. Samples containing 50 μg of proteins in the sample buffer were separated by electrophoresis using 8% or 10% SDS-PAGE gels and transferred to a nitrocellulose membrane (250 mA for 2 h). Membranes were blocked in 5% non-fat milk diluted in TBS-T buffer for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: MurF1 , atrogin-1 , and GAPDH . Detection was performed by a C-Digit Imager  after incubation with peroxidase-labeled secondary antibody  for 2 h at room temperature, using a Pierce ECL Western blotting substrate detection system (Thermo Scientific). Signals were quantified using NIH ImageJ 1.63 Software using GAPDH as a reference.g for 1 min, supernatant was removed, and protein was measured using the Bradford method. The Western blot procedure was used to quantify puromycin incorporation using an anti-puromycin antibody . Signals were normalized to total protein content stained with ponceau.First, 1 µM puromycin diluted in water was added directly to the cells previously treated with PA and incubated for 15 min. Cells were washed twice with ice cold PBS and cells were lysed with sucrose buffer. Cells were centrifuged at 10,000× t-test, one-way or two-way ANOVA followed by Tukey’s post-test. For two-way ANOVA, when there was no interaction between the two factors analyzed (time and PA treatment), the factors were considered separately, and when interaction was statistically significant, Tukey’s post-test was performed. Statistical analyses were performed using GraphPad Prism version 8.0 for Windows .The results are expressed as mean ± standard error of the mean (SEM) and analyzed by an unpaired two-tailed


Handgrip strength, seated medicine ball throw and countermovement jump maximal velocity peaked at-PHV . In general, maximal velocity spurts did not differ in their timing, with the velocities reaching a peak concurrent with PHV and PWV or within 6 months of its attainment. Basketball coaches, as well as strength and conditioning trainers, should consider individual differences in strength development and be aware of rapid periods of growth in stature when planning and designing muscular strength training regimes.Although successful performance in basketball requires high levels of muscular strength during adolescence, its development is confounded by the effects of normal growth. We examine the timing, intensity and sequence of muscular strength according to biological age (years from peak height velocity (PHV)) and hypothesize that young basketball players attain their peak muscular strength spurts around PHV. A total of 160 adolescent male basketballers, aged 11–15 years, were followed bi-annually over 3 consecutive years. The years from attainment of PHV and peak weight velocity (PWV) were estimated and five muscular strength measures  were aligned to years from PHV in 3-month intervals. Strength velocities were estimated using a non-smooth mathematical model. The mean ages at-PHV and at-PWV were 13.90 ± 1.40 years and 13.90 ± 1.79 years, respectively. Maximal velocity in sit-ups was attained 6 months prior to attainment of PHV (intensity = 10.69 repetitions·year The adolescent growth spurt, in height, an index of biological maturation, varies substantially in timing, intensity and duration between individuals . Within Basketball is a highly dynamic and intermittent sport requiring remarkable physical attributes to excel in training and competition . It is c−1) in a group of boys from Portugal [−1) in boys from Brazil [−1) in boys from Canada [−1) [Published reports on developmental spurts in muscular strength mostly focus on non-athletic populations and vary in timing and intensity when aligned to years-from-PHV. For example, in standing long jump (a marker of explosive leg strength) the peak spurt occurred 12 months before-PHV  . A similada [−1) ,12,13,14ada [−1)  identifiada [−1) . Furtherada [−1) . It has ada [−1) . Notwithada [−1) , that yoIn Search of Excellence—a Mixed-longitudinal Study in Young Athletes (INEX) study. The study involves a 3-year mixed-longitudinal approach consisting of five age-cohorts from five team sports (https://www.inex-cifi2d.pt). The purpose of the INEX study was to investigate the interactions among individual characteristics and environmental factors affecting growth, physical and specific-skills performance, game proficiency and psychological development.The data presented in the current paper comes from the n = 31), we followed players consecutively from 11 to 13.5 years, in cohort 2 (n = 40) from 12 to 14.5 years, in cohort 3 (n = 32) from 13 to 15.5 years, in cohort 4 (n = 33) from 14 to 16.5 years, and in cohort 5 (n = 24) from 15 to 17.5 years. Participants were recruited from a population of 1256 adolescent male basketball players. All trained regularly 4.5 to 6.0 h·week−1 with 20 of the 25 clubs in the Porto Basketball Association. Participants were randomly selected by coaches and club coordinators to take part in data collection. Baseline measurements were initiated in June of 2017 and repeated through to December of 2019. All assessments occurred during the same time-periods (June and December) within a time window of 15–20 days. Written informed consent was obtained from parents or legal guardians, as well as players’ individual assents. The study was approved by the Ethics Committee of the lead institution (CEFADE 13.2017). The Porto Basketball Association gave formal permission for data collection.In the present study, data were collected bi-annually over 3 consecutive years from a sample of 293 male adolescent basketballers. Altogether, 160 players fulfilled the condition of having complete data on 5 to 6 time points for anthropometry and muscular strength measures. In cohort 1  with a precision of 100 g. Measurements were assessed following the International Working Group on Kinanthropometry protocols [Height (cm) was measured without shoes and with the participant’s head positioned in the Frankfurt plane using a Harpenden stadiometer  with a precision of 0.1 cm. Weight (kg) was measured using a bio-impedance scale (Tanita Muscular strength was assessed using standardized tests: (1) Abdominal muscular strength and endurance (sit-ups): the maximum number of sit-ups (repetitions (reps)) during 60 s were recorded ; (2) StaA four-step procedure was used: (1) anthropometric measurements were performed by experienced anthropometrists; (2) an in-field reliability study was performed using a random sample of three-to-five participants per day; (3) reliability estimates were computed and the technical error of measurement (TEM) for height was 0.2 cm and for weight was 0.1 kg, whereas the ANOVA-based intraclass correlations (R) values for the muscular strength tests ranged from 0.90 (sit-ups) to 0.99 ; (4) data cleaning was undertook to control for errors in data entry and the presence of outliers.Maximum velocities for height, weight and muscular strength tests were fitted using a modified non-smoothed polynomial method. This method was initially proposed by Van’t Hof et al.  and subs−1 at 18 months before-PHV to 7.99 cm·year−1 at-PHV. The velocity then rapidly decreased to 2.86 cm·year−1 at 18 months after-PHV. Weight velocity increased from 4.98 kg·year−1 at 18 months prior-PHV to 6.89 kg·year−1 at-PHV and thereafter dropped to 5.31 kg·year−1 at 18 months post-PHV. The timing and intensity of height, weight and muscular strength peak spurts are shown in The mean ages at-PHV and at-peak weight velocity (PWV) were 13.90 ± 1.40 years and 13.90 ± 1.79 years, respectively. Height velocity increased from 5.12 cm·year−1 at 18 months before-PHV to 5.65 reps·year−1 at 12 months before-PHV. Subsequently, there was an acceleration with a peak (10.69 reps·year−1) 6 months before-PHV and then dropped to 4.74 reps·year−1 at 9 months after-PHV. Thereafter, velocities slightly increased to 5.37 reps·year−1 at 12 months after-PHV, decreased to 4.79 reps·year−1 at 15 months after-PHV and increased again to 8.52 reps·year−1 at 18 months after-PHV.For sit-ups, the mean velocity decreased from 10.19 reps·year−1 at 18 months before-PHV and 6.85 kgf·year−1 at 3 months before-PHV. Thereafter, the velocity increased attaining a peak (8.47 kgf·year−1) coincident with PHV. The velocity then declined to 5.72 kgf·year−1 at 15 months after-PHV but increased again to 6.64 kgf·year−1 at 18 months after-PHV.For handgrip, the average velocity curve showed a fluctuation between 6.07 kgf·year−1 at 18 months before-PHV to 0.64 m·year−1 at 12 months before-PHV but declined right after to 0.49 m·year−1 at 9 months before-PHV. It then accelerated reaching a peak (0.75 m·year−1) coincident with PHV. Subsequently, the velocity decreased to 0.55 m·year−1 at 9 months after-PHV and fluctuated until 0.65 m·year−1 at 18 months after-PHV.For seated medicine ball throw, the velocity rose from 0.44 m·year−1 at 6 months after-PHV. Then, the curve fell to 2.77 cm·year−1 at 9 months after-PHV and fluctuated until 3.10 cm·year−1 at 18 months after-PHV.In squat jump, the velocity curve displayed a fluctuation between 18 and 3 months before-PHV, followed by a plateau until 3 months after-PHV. Thereafter, the velocity increased resulting in a maximum peak velocity of 3.93 cm·year−1) coincident with PHV, followed by a decline to 3.32 cm·year−1 at 12 months after-PHV and a plateau thereafter until 18 months after-PHV.For countermovement jump, the velocity curve decreased from 18 months before-PHV until 9 months before-PHV. It then accelerated reaching a peak  to identify peak spurts in muscular strength in young basketballers, we limit comparisons to Philippaerts et al. , the onl−1) was relatively lower than those reported by Šprynarová [−1), Philippaerts et al. [−1), and Bell [−1). The maximum height velocity in basketballers was relatively lower than in samples of non-athletes from the UK, Sweden, Belgium, and Switzerland at 10.3 cm·year−1 [−1 [−1 [−1 [−1).The average age-at-PHV was 13.90 ± 1.40 years, which did not differ to the 13.8 ± 0.8 years estimated in a sample of soccer players from Belgium  but relarynarová   was relatively lower than those reported by Šprynarová [−1), Bell [−1) and Philippaerts et al. [−1). Similar to the age-at-PWV, maximum weight velocity found in our basketballers was relatively lower than the 9.8 kg·year−1 reported in non-athletic boys from the UK [−1 from Sweden [−1 from Belgium [The mean age-at-PWV for our basketballers was 13.90 ± 1.79 years. This value was relatively similar to the 13.8 ± 0.8 years estimated in soccer players from Belgium , but relrynarová  (10.1 kg1), Bell  , whereas in soccer players from Belgium, the peak for 30 s sit-ups was coincident with PHV with an intensity of 2.7 reps·year−1 [−1) in a sample of boys from Canada, whereas Yagüe and De La Fuente [−1) in boys from Spain, even though there was a second period of rapid development 4 months after-PHV (intensity = 4.6 reps·year−1). The differences in timing may be explained by, at least, five factors: (1) tests do not have the same time length (30 s versus 60 s), (2) samples vary in their size and comprise athletes and non-athletes, (3) the use of a different mathematical method to estimate age-at-PHV and corresponding spurts, (4) the time lag between studies, and (5) the country of origin. In any case, the most striking results concern spurt intensities. For example, the intensity of soccer players from Belgium was about half that of non-sporting youth from Spain in spite of their training schedule (3 h·week−1) plus a competitive game during the weekend. Apparently, training and competition did not have enough stimuli to optimize levels of abdominal muscle strength and endurance in young soccer players. On the other hand, the peak intensity in basketballers in Portugal was double that of their non-sporting peers in Canada. The coaches in Portugal emphasize developing a stronger core, that is frequently linked to correct posture, stability, balance, support of the lower back, and reduced injury risk. They also develop a solid base to improve other muscular strength components that are important for game actions like jumping, throwing, sprinting, shuffling or changes of direction [For the 60 s sit-ups, the peak was identified 6 months before-PHV (intensity = 10.69 reps·years·year−1 . Ellis es·year−1  identifia Fuente  found a −1). Comparable results were only available for non-athletic boys from Portugal and Brazil. Guimarães et al. [−1, respectively), whereas Silva et al. [−1, respectively). Similar timings, after-PHV, were reported in other populations of adolescent males, although using different static strength markers [The handgrip strength spurt coincided with PHV , which contrasts with Yagüe and De La Fuente [−1). Nonetheless, our results are consistent with reports from the Ghent Youth Soccer Project [−1). It is possible that distinct test protocols may explain this difference, although in both studies a medicine ball with equal weight (3 kg) was used. Young basketballers had to throw it while seated on the floor with legs fully stretched and back against the wall, whereas in the study conducted in Spain it is not known which test protocol was used . It should be noted, however, that basketballers continued to develop their explosive arm strength after attaining peak spurt. In fact, the velocity curve reaches twice an intensity of 0.65 m·year−1 (12 and 18 months after-PHV), in contrast to the sudden drop in velocity (0.40 m·year−1 at 16 months after-PHV) observed in non-athletic boys from Spain. It is possible that this continued improvement reflects stimuli and neuromuscular adaptations promoted by repeated actions of shooting, passing, controlling and dribbling during basketball trainings and games. In addition, it may be that basketball coaches invest in strength and conditioning workouts that enhance synchronization and coordination of upper body segments, given its contribution to a more effective skill domain in ball throwing [The peak spurt in 3 kg seated medicine ball throw occurred coincident with PHV  and coincident with PHV (intensity = 5.59 cm·year−1), respectively. While we were unable to locate any study reporting these two types of jump, our results are in part comparable with data from athletes and non-athletic boys from Belgium. Using a vertical jump test in which participants were instructed to bend their knees and swing their arms before jumping as high as possible, that is, benefiting from the stretch-shortening cycle as basketballers in the countermovement jump, Philippaerts et al. [−1), whereas Beunen et al. [−1). Furthermore, a growth spurt in lower body explosive strength was identified a year after-PHV by Yagüe and De La Fuente [Peak spurts in squat and countermovement jump were attained 6 months after-PHV (intensity = 3.93 cm·years et al.  reportedn et al.  found ita Fuente , but wita Fuente . MoreoveThe pattern in muscular strength peaks was similar to that observed in young athletes from Belgium . When alThis study is not without limitations. The first concerns the sample size and representativeness. From the original sample of 293 male adolescent basketball players, PHV was only identified in 159 out of the 160 basketballers who had complete data on 5 to 6 time points. Moreover, depending on data availability, the valid sample used to estimate peak spurts varied across muscular strength components. Although we acknowledge that this is a challenging issue, we are aware that this is a common weakness in this type of research. In the Philippaerts et al. ,13. SecoWe argue that the information presented in this study may be helpful for basketball coaches to make decisions when planning strength training. It is important to clearly understand the variation in growth, maturation and development, namely across puberty, and its effects on young athletes’ responses to training programs. Since basketball is a team sport, youth team coaches must consider individual changes in growth that represent differences in performance among players. Furthermore, they should be aware that youngsters pass through periods of rapid growth that constitute great windows of opportunity to improve physical condition. If workloads are applied at the right time, adaptations to training within these sensitive periods may be optimized.In conclusion, peak spurts for muscular strength components were attained coincided with the attainment of PHV and PWV or occurred within 6 months of its attainment in basketballers. This finding suggests that the timing of spurts in weight and muscular strength occurs relatively early in adolescent athletes. Additionally, it suggests that young players’ maximal gains in muscular strength are somewhat related to increases in weight and muscle mass as occurs with their non-athletic peers. Moreover, basketballers reached higher spurt intensities in most strength tests, which suggests that systematic and regular basketball training appear to boost the development of muscular strength levels in young basketballers. Our novel data enhance understanding of how strength develops in youth basketballers. Based on our findings, basketball coaches as well as strength and conditioning trainers should consider individual differences in strength development and be aware of rapid periods of growth when planning and designing muscular strength training regimes. Finally, we recommend that in the future, researchers should include information on training experience, hormonal and possible genetic markers, as well as environmental factors, to provide a more encompassing understanding of spurt timings, intensities and sequences. Moreover, researchers should investigate the effects of the application of training loads during these putative sensitive periods, as well as where a lack of adherence may have a limiting effect on athletic development.


Saccharomyces cerevisiae, it is controlled by a system of four methyltransferases  and four demethylases . While the histone targets for these enzymes are well characterized, the connection of the enzymes with the intracellular signaling network and thus their regulation is poorly understood; this also applies to all other eukaryotes. Here we report the detailed characterization of the eight S. cerevisiae enzymes and show that they carry a total of 75 phosphorylation sites, 92 acetylation sites, and two ubiquitination sites. All enzymes are subject to phosphorylation, although demethylases Jhd1p and Jhd2p contained one and five sites respectively, whereas other enzymes carried 14 to 36 sites. Phosphorylation was absent or underrepresented on catalytic and other domains but strongly enriched for regions of disorder on methyltransferases, suggesting a role in the modulation of protein–protein interactions. Through mutagenesis studies, we show that phosphosites within the acidic and disordered N-terminus of Set2p affect H3K36 methylation levels in vivo, illustrating the functional importance of such sites. While most kinases upstream of the yeast histone methylation enzymes remain unknown, we model the possible connections between the cellular signaling network and the histone-based gene regulatory system and propose an integrated regulatory structure. Our results provide a foundation for future, detailed exploration of the role of specific kinases and phosphosites in the regulation of histone methylation.Histone methylation is central to the regulation of eukaryotic transcription. In  Histone methylation is a widespread and dynamic post-translational modification (PTM) that is central to the regulation of eukaryotic transcription . It reguSaccharomyces cerevisiae retains the transcriptionally activating lysine methylation sites on H3K4, H3K36, and H3K79 (S. cerevisiae is controlled by four methyltransferase (MTase) and four demethylase (DMase) enzymes. The SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain MTases Set1p, Set2p, and Set5p methylate histone proteins on their N-terminal tails  and two mass spectrometry fragmentation methods  and electron transfer/HCD (EThcD)), applied to homologously overexpressed and purified versions of enzymes. Seven of all eight of the histone methylation proteins could be analyzed; Set1p was refractory to overexpression in via different methods, thus providing confirmatory evidence for the existence or absence of modification sites. For example, a total of 47 phosphorylation sites were identified in multiple independent mass spectrometry analyses  and LysargiNase-generated  peptides. Similarly, novel phosphorylation at serine 30 of Dot1p was confirmed by exemplar HCD and EThcD fragmentation spectra of chymotryptic  and Asp-N-generated peptides , respectively.To establish the extent to which histone MTase and DMase enzymes are connected to the intracellular signaling network, mass spectrometry data were searched for the presence of phosphorylation. Putative phosphosites were filtered using stringent score and localization probability cutoffs, thus generating a high-confidence library of modification sites. From all analyses, we identified 180 non-redundant phosphopeptides  mapping Fig. S2e.g., Set5p, Rph1p, and Gis1p) are potential integrators of signaling information, while enzymes with few sites  are likely to be regulated through other means.With respect to the number of phosphorylation sites present on each enzyme, some striking trends emerged. Histone MTases Set1p, Set2p, Set5p, and Dot1p are all phosphorylated to similar degrees, harboring between 14 and 26 sites each . By contWe then explored the relative stoichiometry of phosphorylation for the enzymes, as purified from the yeast cell. This was done by label-free quantification of phosphopeptides and their unmodified counterparts . Our quaIn regard to the relative amount of phosphorylation that is present at specific sites, we identified some modest but appreciable differences between histone MTase and DMase enzymes. Rph1p and Gis1p, histone DMases with the largest number of phosphosites , also haHaving identified and collated a large number of phosphorylation sites on yeast histone MTase and DMase enzymes, we next examined their potential regulatory role by contextualization within relevant sequence and structural features. Firstly, phosphosites were mapped onto known protein domains  and disotop panel). Although histone MTases are still mostly ordered, their phosphorylation sites reside predominantly within disordered stretches. This enrichment of phosphorylation within disordered sequences on histone MTase enzymes is of functional relevance as such regions are known to modulate protein–protein interactions  (bottom panel). Consistent with total protein values, the majority of phosphorylation sites (>70%) on DMase enzymes are located in ordered sequence contexts.Our analysis, importantly, revealed that histone MTases and DMases have demonstrably different disorder profiles; in terms of both total protein disorder and phosphosite distribution. This may reflect distinct modes of action and/or regulation. With the exception of Set5p, histone MTases are predicted to be more disordered than their DMase counterparts, ranging between 29% (Dot1p) and 47% (Set1p) sequence disorder  . Of its ; Set1C) , 46 and S. cerevisiae , have, at least partially, been resolved (A). Given that phosphorylation is known to regulate multimerization of some yeast protein MTases . This phosphosite is surface exposed and located on a spatially accessible protrusion distal to the catalytic JmjC domain.To determine whether phosphorylation is associated with interaction interfaces, phosphosites were projected onto three-dimensional crystal structures of histone MTases and DMases. Structures for four enzymes from resolved . The strresolved A. Given ., Hmt1p ), phosph., Hmt1p B. This pC). Notably, two phosphorylation sites previously reported on Set1p . The region on Swd1p that physically contacts Set1p is rich in negatively charged aspartate and glutamate residues, thus Set1p phosphosites could impede Swd1p binding and subsequent Set1C assembly.Set1p is the catalytic constituent of the multi-subunit COMPASS, or Set1C, H3K4 MTase complex, for which the molecular stoichiometry and three-dimensional configuration are known C. Notablon Set1p , at threon Set1p C, inset.A), with the exception of the yeast-specific MTase, Set5p. Strikingly, of the 19 phosphorylated amino acids in yeast that are conserved at the sequence level in human, six are known to be phosphorylated in vivo (A) (B).We explored the evolutionary conservation of phosphoresidues on histone MTase and DMase enzymes in eukaryotic orthologs. To determine the degree of conservation in mammals, pairwise sequence alignments were performed between all yeast histone MTases and DMases and their equivalent human proteins A, with t in vivo A 49, 50, 50A, wivivo (A) . More brvivo (A) B.Figure There is a paucity of kinases known to phosphorylate specific sites on histone MTase and DMase enzymes in yeast. As such, our understanding of the biological function of phosphosites and the network context of their cognate proteins is fragmentary. Using publicly available phosphoproteomic datasets, interaction databases, and prediction tools, we modeled possible connections between the histone methylation enzymes and cellular signaling pathways . Our drae.g., Rim15p, Sch9p, and Tpk2p). Interestingly, our network analysis revealed a highly interconnected and integrated regulatory structure whereby kinases may phosphorylate multiple sites on the same or different enzymes. For example, seven observed phosphosites on Set5p are predicted to be regulated by the sphingolipid signaling kinase, Pkh1p, and five sites on Set1p are likely phosphorylated by Hsl1p, a mitogen-activated protein kinase , that may function cooperatively with phosphorylation to regulate Set2p activity through an electrostatic mechanism. Accordingly, multiple phosphorylation sites at the N-terminus of mammalian heterochromatin protein 1α are known to regulate its recruitment to chromatin . By comparison, a set2Δ negative control showed complete loss of all H3K36 methylation states , the overall H3K36 methylation of each SET2 mutant yeast strain was compared with the wild-type control. This revealed significant differences for triple phosphonull (p = 2.0 × 10−16), triple phosphomimetic (p = 2.2 × 10−9), and S8D (p = 1.1 × 10−4) Set2p phosphomutants when compared with the wild-type control . These changes were characterized by a lower level of H3K36 trimethylation and a higher level of unmodified H3K36. Other mutants, however, did not show any statistically significant differences in H3K36 methylation.For functional characterization of Set2p, phosphonull  and phosphomimetic (serine-to-aspartate) mutations of phosphosites were engineered into the bination . Six strtrometry . In yeastrometry B. By comB). This observation suggests that there is a minimum charge requirement within this region for Set2p to retain wild-type levels of activity. With respect to phosphomimetic substitutions, each confers a single, but not double negative charge. Accordingly, single-site mutations  did not markedly affect H3K36 methylation, while the triple phosphomimetic substitution  reduced the relative abundance of H3K36 trimethylation to ∼12% . This model highlights the likely importance of charge in the N-terminus of Set2p, as the measured methylation of H3K36 in triple phosphomimetic and triple phosphonull mutations corresponded to the capacity for these mutant proteins to carry negative charge.Given that the degree of negative charge in the N-terminal region of Set2p may regulate its interaction with histones, we examined the net possible charge on amino acids 1 to 15 of Set2p, with and without mutations, and its association with H3K36 methylation. We considered one positive charge each for the N-terminus and lysine 3, six potential negative charges from phosphorylation sites at serine residues 6, 8, and 10, and one positive and four negative charges within acidic patch EDEKE. From this, wild-type Set2p can carry a net charge of −7 in its N-terminal region, while Set2p phosphomutants have a reduced capacity to hold negative charge . With reFig. 7Having identified extensive post-translational phosphorylation of yeast histone MTases and DMases, we sought to establish the extent to which enzymes are subject to other modification types. Akin to our systematic approach to phosphosite discovery, combinatorial mass spectrometry data were queried for the presence of acetylation, methylation, ubiquitination, SUMOylation, ADP-ribosylation, and crotonylation. Altogether, we identified 92 acetylation sites across all enzymes that were analyzed and discovered two ubiquitination sites on Jhd1p . Our anaS. cerevisiae is modest. Similar to the distribution of phosphorylation across enzymes, different histone MTases and DMases are also acetylated to markedly different extents. Three-quarters of all acetylation sites are found on just three enzymes: Set2p, Set5p, and Jhd2p, which harbor 37, 23, and 15 sites, respectively. Mapping of sites onto linear protein sequences revealed that acetylation generally occurs outside of MTase and DMase domains. Of the 101 acetylation sites identified to date, 27 (26.7%) are located within catalytic or regulatory domains  confirms previous reports; however, the number of proteome-scale studies of protein acetylation in  domains . It is i domains  but show domains . Conversin vivo, suggesting that there may be interplay between two competing modifications of the same residue. More broadly, it will be interesting to determine whether closely positioned modifications of different types participate in crosstalk on histone MTase and DMase enzymes. For example, three acetylation sites are proximal to phosphorylation at serine 572 of Set2p and may affect kinase recognition . PhosphHistone methylation enzymes are phosphorylated to strikingly different extents. They are thus likely to be regulated through different mechanisms. This was particularly apparent for the two evolutionarily related pairs of histone DMases in yeast. The paralogous H3K36 DMases, Rph1p and Gis1p, are heavily phosphorylated, harboring 36 and 26 phosphosites, respectively, suggesting regulation at the post-translational level by upstream signaling pathways. Rph1p and Gis1p also have the highest average relative occupancy of phosphorylation across their cognate sites, thus furthering evidence for their extensive phosphoregulation. By contrast, the Jumonji domain-containing DMases, Jhd1p and Jhd2p, receive relatively little information from the intracellular signaling network, containing just one and five phosphosites, respectively. We note that the small number of sites was not just due to differences in protein overexpression or small protein size. The phosphosites on Jhd1p and Jhd2p may function as molecular switches to activate/inactivate enzyme function in response to a specific signal, or alternatively, transcriptional  and poste.g., Rad53p, Tpk2p, Cdc28p, and Rim15p) may have the capacity to transmit information to multiple histone methylation enzymes, raising the possibility of coordinated regulation. While evidence for kinases acting on multiple MTases and/or DMases in the histone methylation network has not been demonstrated in yeast, there are several instances of this in the human cell. For example, Akt kinase phosphorylates H3K36 MTase NSD2   , 71. Morusculus) , 73. TheThe molecular interactions of histone MTases may be regulated by phosphorylation. Sites on MTases were strongly enriched within disordered regions and at interaction interfaces but largely absent or under-represented on domains. This is functionally important as intrinsically disordered regions are known to mediate protein–protein interactions  and assoGiven the aforementioned differences in the extent to which histone methylation enzymes are phosphorylated, their cognate phosphosites are likely to have different functional roles. In the case of extensively phosphorylated proteins, such as Set5p, Rph1p, and Gis1p, an obvious question emerges: do individual phosphosites carry out distinct functional roles (switch), or does the cumulative effect of multiple phosphosites fine-tune enzyme activity ? There is evidence emerging in support of both regulatory processes. Several targeted studies into histone DMases Rph1p and Gis1p have shown that specific phosphorylation events can have switch-like effects on function. For example, phosphorylation at serine 652 of Rph1p triggers its dissociation from chromatin in response to DNA damage , while pin vivo. Charge-based interactions are known to regulate the nucleosomal association of S. cerevisiae histone methylation enzymes (Chaetomium thermophilum Set2 have shown that Set2 makes extensive contact with histone proteins H2A and H3 (via phosphorylation. This model contrasts with the downstream acidic patch in Set2p (residues 31–39) that is sequence encoded and thus not tuneable nor signal responsive.Through mutagenesis studies, we have shown that a phosphorylation cluster at serine residues 6, 8, and 10 of Set2p, within an acidic and intrinsically disordered N-terminal region, affects its H3K36 MTase activity  enzymes , 82. An  enzymes . Our res enzymes , that stA and H3 ; howeverin vivo  between phosphosites reported here and those identified previously in large-scale phosphoproteomic screens, which is strong evidence that our sites reflect those in natively expressed proteins. For our novel phosphorylation sites, many of these were likely not reported previously as they are either of low phosphorylation stoichiometry, making them difficult to detect in native phosphoproteomic analyses, or because they were found using proteases other than trypsin. We also note that there is a precedent for using overexpression to identify functionally important PTMs; our previous systematic analysis of overexpressed MTases   as per the manufacturer’s instructions, except that SapI  endonuclease digestion was uncoupled from the T4 DNA ligase (New England Biolabs) reaction (GAL1 promoter and the URA3 selectable marker for auxotrophic selection in yeast (S. cerevisiae haploid strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) using the lithium acetate method with URA3 selection   were a keviously  into thereaction . All ovein yeast . Resultain yeast  and tranelection .SET2 was amplified from S. cerevisiae genomic DNA and cloned into the p426 shuttling vector using the Gibson Assembly cloning kit (New England Biolabs). Site-directed mutagenesis of p426-SET2 was performed using relevant mutagenic oligonucleotides  supplemented with glucose (0.68% (w/v) yeast nitrogen base with ammonium sulfate , 0.4% (w/v) synthetic dropout medium without uracil (Sigma Aldrich), 2% (w/v) D-glucose) were grown at 30 °C and 200 rpm shaking to an optical density at 600 nm of 0.8. To alleviate repression of GAL1 by glucose, cells were harvested by centrifugation, washed twice with sterile deionized water before resuspension in 150 ml SC-ura medium. Recombinant protein expression was then induced by the addition of 75 ml of 3× yeast extract peptone galactose medium (3% (w/v) yeast extract, 6% (w/v) bacteriological peptone, 6% (w/v) galactose) and carried out overnight at 30 °C and 200 rpm shaking. Following induction, yeast cells were harvested and stored at −80 °C until lysis and purification. Protein extraction and affinity purification of hexahistidine-tagged enzymes were performed as per  using Amicon Ultra-4 10K centrifugal filters  to a volume of 80 μl.Hexahistidine-tagged proteins were overexpressed in eviously , with thd as per , with th4HCO3), LysargiNase , Asp-N , or chymotrypsin , 10 mM CaCl2) for 16 h in 100 μl reactions. Digests with trypsin, LysargiNase, and Asp-N were at 37 °C, while chymotryptic digestion was at 25 °C. Resultant peptides were extracted from gel bands and dried down in a SpeedVac  before resuspension in 0.1% (v/v) formic acid, as described previously (Lysates (10 μl) and eluates  were separated by SDS-PAGE and prepared for liquid chromatography mass spectrometry according to , with thargiNase  .Peptide samples were separated and ionized by nano-electrospray liquid chromatography using established methods  and thenRS node was incorporated into processing workflows after database searching to provide localization probabilities for putative modification sites in the search output (ptmRS Best Site Probabilities column). False discovery rates (FDRs) for peptide-spectrum matches (PSMs) were determined by a decoy database search, and PSMs for modified peptides (FDR < 0.01) were filtered by ion score (≥30), expectation value (p < 0.05), and site localization probability (≥95%) and tabulated in To identify putative modification sites on histone MTases and DMases, mass spectrometry data were analyzed on Proteome Discoverer 2.2 (Thermo Fisher Scientific) using basic PTM analysis processing and consensus workflows with primarily default settings . Data wem/z of observed phosphorylated peptides, and their unmodified counterparts, were used to generate extracted ion chromatograms (XICs) in the Thermo Xcalibur Qual Browser 2.2, within a mass range window of 10 ppm. The ICIS algorithm was used to determine the area under the curve of XIC peaks, corresponding to either phosphorylated or unmodified versions of a peptide. The relative amount of phosphorylation present at a given site was then calculated by dividing the abundance of its cognate phosphopeptide by the total amount of peptide detected (phosphorylated + unmodified). Where phosphosites occur proximally to one another, isobaric phosphopeptides were differentially quantified by their unique chromatographic retention times. Specific details regarding phosphorylation quantification are summarized in For phosphopeptide abundance measurements, label-free quantification was performed as described previously . Brieflyhttps://visquant.cmri.org.au/). PTMs reported in the literature and on modification databases are summarized in http://www.interactivepeptidespectralannotator.com/PeptideAnnotator.html, (http://bioinf.cs.ucl.ac.uk/psipred/). For three-dimensional contextualization of phosphosites, the following yeast protein structures were retrieved from the Protein Data Bank (RCSB): Dot1p  from peptide sequences identified in database searches using an in-house script and visualized in GraphPad Prism . Modification sites were mapped, to scale, onto linear protein representations using PTM VisQuant , while D: 1U2Z, ), Rph1p D: 1U2Z, ), and SeD: 1U2Z, ). StructCaenorhabditis elegans), common fruit fly (Drosophila melanogaster), zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and red junglefowl  were retrieved from UniProt (2020_04 release) and aligned by multiple sequence alignment in Geneious Prime using an identity cost matrix. Phosphorylation sites on mammalian enzymes were collated from the PhosphositePlus metadatabase  using a global alignment with free end gaps. For extended conservation analysis across Eukarya, sequences for relevant homologues of yeast enzymes in nematode  were included in the regulatory network. Kinase–substrate interaction data from Ptacek et al.  were collated from the Yeast Kinase Interaction Database  was use .SET2 mutant yeast strains were grown in triplicate 15 ml cultures to an optical density at 600 nm of 0.8 in yeast extract peptone dextrose medium (2% (w/v) D-glucose, 2% (w/v) bacteriological peptone, 1% (w/v) yeast extract). Cells were lysed in 300 μl HEPES lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesolfonic acid (HEPES), 100 mM NaCl, 2 mM DTT, 2 mM EDTA, 0.5% (v/v) Triton-X-100, pH 7.5), supplemented with 1× protease inhibitor cocktail (Roche), by glass-bead homogenization, as done previously  using collision-induced dissociation as per established methods K(propionyl)PHR, m/z = 539.97; monomethylated, sequence K(propionyl)SAPSTGGVK(methyl+propionyl)K(propionyl)PHR, m/z = 544.65; dimethylated, sequence K(propionyl)SAPSTGGVK(dimethyl)K(propionyl)PHR, m/z = 530.64; trimethylated, sequence K(propionyl)SAPSTGGVK(trimethyl)K(propionyl)PHR, m/z = 535.31. Label-free quantification was performed using the area under the curve of XIC peaks, as described above for phosphopeptide abundance measurement. The relative amount of methylation present at H3K36 was calculated by dividing the abundance of each methylation state by the total amount of peptide detected (unmodified + monomethylated + dimethylated + trimethylated).To quantify H3K36 methylation from mass spectrometry data, XICs were generated in the Thermo XCalibur Qual Browser 2.2 using the thylated , the expSET2 mutant was compared with the wild-type control using an ordinal logistic regression model with the four possible methylation states as response variables and the SET2 mutation as a categorical covariate (A). This regression makes the proportional odds assumption, assuming that the odds of moving between adjacent categories  are equal. Using a likelihood ratio test, we found that fitting a more complex adjacent category model (B), which does not make the proportional odds assumption, explained significant variation in the data (p = 0.01). However, a comparison of the two regression models revealed that their respective outputs were not sensitive to the proportional odds assumption, and thus we have reported the common odds ratio and its statistical significance, per mutant, from the ordinal logistic model in Figure 7B for ease of interpretability.For statistical analysis, the overall H3K36 methylation level for each ovariate A. This rry model B, which via the PRIDE (All data described in this study are contained within the article and its supporting information, with the exception of the mass spectrometry proteomics data, which has been deposited to the ProteomeXchange Consortium he PRIDE  partner The authors declare that they have no conflicts of interest with the contents of this article.


Lactobacillus casei–infected Hermetia illucens larvae (HIL) against Salmonella species. The result demonstrates that the extract is a powerful natural antibiotic and preservative.In this investigation, we found antimicrobial activity of the extract of Hermetia illucens, were significantly increased by pathogen or stimulant induced innate immunity activation. We immunized H. illucens fifth instar larvae with five different Lactobacillus species, that is, Lactobacillus acidophilus, L. brevis, L. casei, L. fermentum, or L. delbrueckii, to induce the mass production of AMPs and selected optimal immune inducers. Antimicrobial activities in hemolymph and H. illucens larvae (HIL) extract were evaluated against three salmonella species . Highest antimicrobial activity was shown by the hemolymph of HIL immunized by L. casei and its activity was closely linked with the inductions of cecropin 1 (HiCec1) and defensin 1 (HiDef1) gene expressions. Furthermore, antimicrobial activity in hemolymph was stable to heat and pH and the growth of three Salmonella species were dramatically suppressed by HIL hemolymph and extract after immunization with L. casei. The minimal inhibitory concentration (MICs) of L. casei-immunized HIL extract against Staphylococcus aureus, Escherichia coli, and Salmonella species ranged from 100~200 µg/100 µL and no cytotoxicity to CaCo-2 and L929 cells were observed in the concentration range 100~40,000 µg/100 µL. Taken together, the present investigation demonstrates that L. casei-immunized HIL extract is a powerful natural antibiotic and preservative that can prevent contamination by Salmonella species.The expressions of antimicrobial peptides (AMPs) in the larvae of the black soldier fly,  Antibiotics are used to treat infectious diseases and prevent infections in humans and animals and to promote animal growth and thus have played leading roles in the intensive farming of livestock. However, abuse of in-feed antibiotics has caused many problems, such as the appearance of antibiotic-resistant bacteria, antibiotic residues in livestock products, weakening of disease resistance in livestock, and ecosystem pollution ,2,3. To Hermetia illucens, have evolved innate immune systems that produce potent antimicrobial peptides (AMPs) to protect them from pathogen invasion, and these AMPs are viewed as strong natural antibiotic candidates  was mixed with each bacterium and hardened in a 100 mm square plate. Then, we prepared 3.5 mm-diameter of well onto underlay gel and 10 µL of hemolymph or extract of HIL was injected into the well. The underlay gel was held at 37 °C for 3 h and then covered with a sterilized overlay gel (6% TSB and 1% low electroendosmosis agar). Gel plates were incubated for 18 h at 37 °C, and antimicrobial activities were assessed by measuring the widths of clear zones.The antimicrobial activities of HIL extracts were assessed using a radial diffusion assay (RDA) using Gram-positive S. aureus and E. coli were used to determine the MICs of extracts obtained after immunization with L. casei. Determination of MICs was also used for evaluation of antimicrobial activity against Salmonella species. Bacteria, such as S. aureus, E. coli, S. pullorum, S. typhimurium, and S. enteritidis, were inoculated into liquid medium, respectively, and grown at 37 °C for 18 h in a shaking incubator (shaking speed: 200 rpm), and then further cultured to 4 × 106 cfu/mL over 2.5 h under the same conditions. A total of 90 µL (1 × 106 cfu/mL) of bacterial culture mixtures were transferred to a 96-well plate and 10 µL of serially diluted extracts were then added to wells and incubated at 37 °C for 18 h. Absorbances were measured at 600 nm to determine MICs. Melittin was purchased from Sigma-Aldrich; Merck KGaA  was used as control.Firstly, we determined cfu/mL of each bacterial by serial tittering and then measured absorbance of each cfu/mL at 600 nm. Then, Lactobacillus-infected HIL were held at RT for defined times and then total RNA was extracted. First-strand cDNA was synthesized using a high capacity reverse transcription kit , and the cDNA obtained was assessed for the gene expressions of cecropin 1 (HiCec1) and defensin 1 (HiDef1). Primers for HiCec1 and HiDef1 were designed using the Primer 3 program  and gene expressions were normalized versus the Drosophila melanogaster actin 5C (DmAct5C) gene. Primer sequences are provided in Lactobacillus-immunized HIL extract using CaCo-2 human intestinal cells and L929 mouse fibroblasts. Cells were seeded in a 96-well plates and allowed to attached for 24 h in a 5% CO2 atmosphere at 37 °C. Then, culture medium was removed and then cells were treated with 180 µL of various concentrations of Lactobacillus-immunized HIL extract (0~5000 µg/100 µL) or melittin (0~32 µg/µL) for 24 h. Cells were then administered 20 µL of 5 mg/mL MTT reagent and left for 4 h in the dark. Media were then carefully discarded and 200 µL of DMSO was added to each well to dissolve the formazan. Absorbances were measured at 540 nm using an ELISA reader.We investigated the cytotoxicity of t-test and one-way analysis of variance. Experiments were independently performed at least three times, and results are presented as means ± standard deviations (SDs). p-value of less than 0.05 was regarded as statistically different.The analysis was performed using the Student’s Lactobacillus to activate the innate immune system of H. illucens. RDA results showed normal HIL hemolymph did not exhibited antimicrobial activity against E. coli or S. aureus (Lactobacillus species (data not shown). In contrast, although the induction of antimicrobial activity was depended on Lactobacillus species type, increased antimicrobial activities to E. coli and S. aureus were observed in the hemolymph of Lactobacillus-immunized HIL  or Serratia marcescens (KACC 11961) challenged larvae (L. casei was chosen as an activator of the innate immune system of H. illucens.We used five types of . aureus . Furtherized HIL . Highestd larvae  23]. Th. ThLactoL. casei infection in a concentration-dependent manner (L. casei infection. As shown in H. illucens) peaked at 24 h  for 24 h and over the pH range 2~10   A,B.L. casei-immunized HIL extract in CaCo-2 and L929 cells . No cytotoxic effect was observed on against CaCo-2 and L929 cells at extract concentrations from 100 to 4000 µg/100 µL or from 100 to 2000 µg/100 µL, respectively  as determined by RDA and MIC assays  for 24 h and the pH range (pH 2–11), respectively. In conclusion, the present investigation reveals that L. casei-immunized HIL extract is a potent natural antibiotic and preservative with potential applications the medical, food, and livestock industries.Summarizing, we show that


Given their high degree of identity and even greater similarity at the amino acid level, Kat3 coactivators, CBP (Kat3A) and p300 (Kat3B), have long been considered redundant. We describe the generation of novel p300 S89A knock-in mice carrying a single site directed amino acid mutation in p300, changing the highly evolutionarily conserved serine 89 to alanine, thus enhancing Wnt/CBP/catenin signaling . p300 S89A knock-in mice exhibit multiple organ system, immunologic and metabolic differences, compared with their wild type counterparts. In particular, these p300 S89A knock-in mice are highly sensitive to intestinal injury resulting in colitis which is known to significantly predispose to colorectal cancer. Our results highlight the critical role of this region in p300 as a signaling nexus and provide further evidence that p300 and CBP are non-redundant, playing definite and distinctive roles in development and disease. Differential usage of Kat3 coactivators, CBP and p300, by β-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by β-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution. The vertebrate radiation, which was initiated approximately 450 million years ago, ushered in a major lifestyle change with a significant increase in adult lifespan ,2 and wiTo further investigate the role that p300 serine 89 (S89) plays in vivo, we have generated p300 S89A knock-in mice. p300 S89A knock-in mice, albeit born at sub-Mendelian ratios, appear to be relatively normal. Nevertheless, these mice exhibit multiple organ system, immunologic and metabolic differences, compared with their wild type counterparts. We now initially report on the generation of these mice and their high sensitivity to intestinal injury, which is apparently related to a complex interplay between aberrant epithelial differentiation, gut immunity and changes in their intestinal microbiota and metabolites and which results in colitis, a significant risk factor predisposing to colorectal cancer ,28.loxP site to correct the orientation to activate, and then excise the wild type fragment flanked by lox2272 to inactivate it. However, we found that mice homozygous for the knock-in construct displayed early embryonic stage lethality, similar to that of p300 knockout mice [Animal studies were approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) as per protocol #11023. The S89A knock-in point mutation in exon 2 of the mouse p300 gene, via site-specific mutagenesis, was generated using the flip-excision (FLEx) switch construct. This mutation removes the highly conserved phosphorylation site at S89. The construct included five segments: the 5′ homology arm, a point-mutated exon 2 in inverted orientation, a PGK-Neo selection cassette, the wild type exon 2, and the 3′ homology arm. The design of the construct, cloning of the targeting vector, electroporation into mouse ES cells, screening of the 129 ES cells, injection into blastocysts, and screening of the chimeric mice were performed by Ozgene Pty Ltd. . In principle, transcription from the mutant exon 2 should have been activated via Cre recombinase in two steps. First, Cre recombinase would invert the mutated fragment flanked by the g in a tabletop centrifuge for 5 min. Supernatant was aspirated, and the cell pellet was re-suspended in basal media containing 5% FBS and then centrifuged at 100× g for 5 min, after which supernatant was removed and samples were frozen at −80 °C until further analysis.Crypt cells from ileum were isolated based on a previously described protocol  with min150–200 mg of intestinal crypt cells were re-suspended in CERI buffer  containing 5 mM DTT and 1X protease inhibitor cocktail, using a dounce homogenizer. After re-suspension in CERI buffer, the procedure for nuclear extraction was performed based on manufacturer protocol. Protein concentration of nuclear extract was performed using the Protein Assay Dye Reagent . 100–500 µg of nuclear protein was diluted in CoIP buffer ) to a final volume of 1000 µL. 2 µg of CBP , p300 , 14-3-3-Ɛ , or normal IgG  antibody was added and mixture incubated overnight at 4 °C on a tube shaker/rotator. 20 µL of Dynabeads Protein A  was added and mixture incubated for 1 h at 4 °C on tube shaker/rotator. The magnetic beads were washed three times with 500 µL of CoIP Buffer each time, using the magnetic stand. 20 µL of 2× Laemmli buffer was added and mixture vortexed. Beads were boiled in 2× Laemmli buffer for 10 min. Supernatant containing proteins were separated from the magnetic beads, using the magnetic stand. Protein samples were subjected to electrophoresis on a 4–20% Teo-Tricine SDS-PAGE gel . Proteins were transferred onto PVDF membrane. Proteins of interest on the PVDF membrane were detected by incubating with β-catenin antibody  or p300 antibody  as the primary antibody, and subsequent incubation with CleanBlot  as the secondary antibody, followed by application of chemiluminescent reagent ECL Plus  and imaging on the ChemiDoc Imaging System (Bio-Rad), after which relative protein concentration was determined by densitometry.g for 15 min, after which supernatants were collected. Protein concentration was determined using the Protein Assay Dye Reagent . 40 µg protein mixed with 4× Laemmli sample buffer were incubated at 37 °C for 15 min and subjected to SDS-PAGE. After overnight transfer onto PVDF membrane , membrane was incubated overnight with primary antibody at 4 °C. Membrane was washed and then incubated with secondary antibody for 1 h, followed by application of chemiluminescent reagent ECL  and imaging on the ChemiDoc Imaging System (Bio-Rad), after which relative protein concentration was determined by densitometry. The primary antibodies used were DUOX2 , GAPDH . The secondary antibody was mouse IgG kappa binding protein conjugated to horseradish peroxidase .Flash-frozen mouse ileum was resuspended in RIPA buffer , containing protease inhibitors . Tissue was homogenized and then centrifuged at 12,000× −ddCt method. PCR primer sequences used for OTU217 were as follows: F: 5′-TACCGCATAAGCCTGCTGTG-3′, R: 5′-ATCGTTGTCTTGGTAGGCCG-3′. PCR products were resolved by agarose gel electrophoresis.Total mRNA was extracted by TRIzol reagent  according to the manufacturer’s protocol. cDNA was synthesized using qScript cDNA Synthesis Kit . cDNA template was used for qPCR with SYBR Green detection method utilizing the CFX Connect Real-Time PCR Detection System . The PCR primer sequences used for mouse cells were as follows: Duox2 ; Duoxa2 . Housekeeping gene PCR primer sequences for mouse Gusb were . Relative expression levels for genes of interest were calculated using the 2Stool samples from mice were collected and genomic DNA was extracted using the QIAAmp Fast Stool Mini Kit  as per manufacturer instructions. DNA samples were sent to Research and Testing Laboratory  for processing and analysis based on a previously described protocol ,32. 16S Organoids were derived from mouse intestines and cultured based on a previously described protocol . Brieflyp-value, i.e., “q-value” < 0.05 or multimodel p-value < 0.05. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) (Qiagen).RNA-seq was performed on mouse intestine and intestinal organoids at the USC Core Lab, based on a previously described protocol . BrieflySamples were prepared and subjected to proteomic analysis by 2DICAL as previously described . Methanop < 0.05) and were subjected to Ingenuity Pathway Analysis (IPA)  to identify top canonical pathways associated with S89A mutation in mice.A total of 2836 proteins were detected in both wild type and p300 S89A mice. The proteomic data were quantile normalized and subjected to differential expression analysis. 93 proteins showed significant changes (fold change (FC) ≤ −1.2 or FC ≥ 1.2, and Mice were treated starting on day 1 for up to 5 days with low (2%) dose of dextran sodium sulfate (DSS) administered via the drinking water, based on a previously described protocol , after wp-values < 0.05 were considered significant.Numerical data were expressed as the means ± standard deviation (s.d.) and Student’s t-test was performed, unless otherwise noted. We previously utilized in vitro CRISPR/Cas9 editing of a highly conserved insertion in the N-terminus of p300 (aa 61–70) to demonstrate its importance in regulating Wnt/β-catenin/nuclear receptor interactions [We previously reported that phosphorylation at S89 of p300 enhanced the association of β-catenin with p300 and mutation of serine 89 to alanine abrogated this phosphorylation dependent increase in vitro . To confAlthough p300 S89A mice did not exhibit obvious homeostatic defects under normal feeding and housing conditions, we decided to evaluate their response to insult. Inflammatory bowel disease (IBD), including Crohn’s Disease and ulcerative colitis , may ariPrior to DSS treatment, the colonic epithelium of S89A mice was normal, essentially the same as WT mice. Prior to treatment, in both S89A and WT mice, crypt architecture was intact and there was no evidence of neutrophil mediated epithelial injury or histological features suggestive of ongoing chronic mucosal injury. Alcian blue staining demonstrated the presence of intact goblet cells B. HistolPerturbations of host–microbiota homeostasis induced by the host genetics and/or environmental factors can fuel inflammation at mucosal surfaces ,38. S89ADUOX2, ERO1l, GSR and MPTX2 up-regulated and BCMO1, PMP22, PRELP, SLC13A1 and SST down-regulated)  A being cl mucosa ,40. p = 5.00 × 10−5) and intestinal organoids (1.5-fold p = 7.99 × 10−6). We further confirmed increased expression of Duox2 and Duoxa2 at both the message  in intestinal organoids. Among the significantly down-regulated genes in the ileum was REG3A (~50% p = 5.00 × 10−5), an antibacterial C-type lectin, which is constitutively generated in the intestine and displays anti-Gram-positive bactericidal activity [IFITM3 (p = 0.047) in intestinal organoids. Differential expression of IFITM3 has been found in endoscopic biopsies from Crohn’s Disease patients [SLC13A1, an FXR transcriptional target was down-regulated 2.5-fold (p = 4.67 × 10−8) in organoids with an approximately 50% reduction in ileum (p = 5.00 × 10−5), consistent with the importance of p300 Ser89 phosphorylation on p300/nuclear receptor interactions [HOXB3 and HOXB5-9 were all down-regulated more than 2-fold in intestinal organoids (p = 7.97 × 10−7 to 4.49 × 10−5). Among the top canonical pathways affected in the IPA analysis of the ileum RNA-seq were, interferon signaling, LPS/IL1 mediated inhibition of RXR function, estrogen biosynthesis and fatty acid and xenobiotic metabolism. 1.4-fold increases in Stat1 (p = 5.00 × 10−5) and Irf8 (p = 5.00 × 10−5), 2-fold (p = 5.00 × 10−5) increases in granzyme b (Gzmb) and immunity-related GTPase family M member 1 (Irgm1) and a 1.6-fold (p = 5.00 × 10−5) up-regulation of the Interferon Inducible Protein 47 gene (IFI47) were observed in S89A mice consistent with the known IFN/STAT1 pathway dysregulation in IBD [SAA1), which demonstrates bactericidal action in vitro, may provide a feedback protective mechanism in S89A mice and was increased 5.3-fold (p = 5.00 × 10−5) [Duox2 expression was significantly increased in both ileum  . BMAL2, similar to its paralog BMAL1, forms a dimer with CLOCK, to activate E-box-dependent transcription thereby playing an active role in circadian-regulated transcription [We performed a targeted proteomic analysis of intestinal proteins associated with CBP and/or p300 in wild type versus p300 S89A mice. Interestingly, the aryl hydrocarbon receptor nuclear translocator-like protein 2  demonstrated increased association with CBP versus p300 in both wild type and p300 S89A mice under both fed or fasted conditions, and although the ratio of CBP to p300 binding did not change significantly in the male mice, fed female p300 S89A mice showed a somewhat decreased Bmal2/CBP versus Bmal2/p300 interaction . BMAL2, cription . Bmal1 rcription . Clock/Bcription  and diffcription  and the cription . The effcription , this arp < 0.05) . Bile acids and their FXR nuclear receptors play important roles in inflammatory response and intestinal barrier function and are involved in IBD pathophysiology [We next undertook global proteomic analysis of intestinal tissue from wild type and p300 S89A mice , which r < 0.05) . IPA ana < 0.05) . Interesysiology . Calretiysiology . Calretiysiology  and beliysiology . InteresThe 14-3-3 protein family of scaffolding chaperones regulates diverse intracellular signaling pathways . We obseDifferential Kat3 coactivator usage by β-catenin is a fundamental regulatory mechanism in stem cell maintenance and the initiation of differentiation and repair ,60. StemTo further investigate mechanisms of signal integration effected by this domain of the Kat3 family, we generated p300 S89A knock-in mice. Based upon our earlier pharmacologic studies, p300 S89 is critical for controlling differential coactivator usage by β-catenin via post-translational phosphorylation in stem/progenitor populations ,20,26 anFurther investigation demonstrated cell intrinsic differences in the intestinal epithelium, based upon RNA-seq of intestinal organoids, as well as microbiome compositional differences and differential immune responses in the intestine. Interestingly, S89A mice separately housed were dramatically more sensitive A than whSLC13A1, an FXR transcriptional target was significantly down-regulated in both organoids and ileum of S89A mice, consistent with the importance of p300 Ser89 phosphorylation on p300/nuclear receptor interactions [p-cresol sulfate and phenol sulfate, potentially toxic intestinal bacterial fermentation products [Global genomic and proteomic analysis showed a number of prominent pathway differences including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. We have found in further analyses of other organ systems  that these are fundamental differences generally associated with differential Kat3/β-catenin coactivator usage. Notably, sulfate transporter ractions ,45. Sulfractions  and veryractions . Interesproducts , were siproducts ,72, furtproducts , whereasproducts ,75,76. Rproducts  has provproducts . Furtherproducts , consistproducts . The diverse array of effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the role of this region in the Kat3 coactivator p300 as a critical signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution. Additional studies related to the fundamental regulation of metabolism via differential Kat3/β-catenin usage and its roles in development and disease will be reported in due course.We describe the generation of novel p300 S89A knock-in mice carrying a single site directed amino acid mutation in p300, changing the highly evolutionarily conserved serine 89 to alanine, thus enhancing Wnt/CBP/catenin signaling . We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly predispose to colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in the intestines of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus in development and disease  and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.