We analysed 2006–2016 national influenza surveillance data in Japan with regards to age-, sex-, and predominant virus-related epidemic patterns and the prevalence of serum influenza virus antibodies. We found a significant increase in influenza prevalence in both children (≤ 19 years old) and adults (≥ 20 years old) over time. The influenza prevalence was higher in children (0.33 [95% CI 0.26–0.40]) than in adults (0.09 [95% CI 0.07–0.11]). Additionally, the mean prevalence of antibodies for A(H1N1)pdm09 and A(H3N2) was significantly higher in children than in adults, whereas the mean prevalence of antibodies for B lineages was relatively low in both children and adults. There was a biennial cycle of the epidemic peak in children, which was associated with a relatively higher prevalence of B lineages. The female-to-male ratios of the influenza prevalence were significantly different in children , adults , and older adults . The significant increase in influenza prevalence throughout the study period suggests a change of immunity to influenza infection. Long-term surveillance is important for developing a strategy to monitor, prevent and control for influenza epidemics. In Japan, the usual influenza epidemic starts around the beginning of January and peaks within a few weeks8. The influenza epidemic curve in Japan shows exponential growth toward the peak week and immediately decreases thereafter. This recurrent pattern has been also found in other respiratory viruses, such as the COVID-19 epidemic, in winter9. Furthermore, the spread of the epidemic is similarly located in regions over time for different predominant influenza viruses8. For example, during the pandemic caused by the novel swine influenza variant A(H1N1)pdm in 2009, epidemic clustering and transmission similar to that of seasonal influenza was observed17. This event highlighted the importance of understanding epidemic trends to estimate the risk of an epidemic. Similarly, continuous and long-term surveillance is important for the prevention of and preparation against emerging and re-emerging influenza epidemics18. In Japan, influenza surveillance has been conducted across the country by local health centres and public health institutes and infectious disease surveillance centres of the prefectural governments under the guidelines of the Ministry of Health, Labour and Welfare. Surveillance was widely expanded in the 1980s, with an increased number of sentinel clinics that report the weekly number of outpatient incident cases5.Seasonal influenza epidemics occur every winter in mild temperate regionsIn this study, we analyzed the recent trends of influenza epidemics, including the age- and sex-related prevalence and the seasonal predominant virus with respect to the prevalence of influenza antibodies, using national surveillance data obtained between 2006 and 2016 in Japan.5. The surveillance data were collected at 5000 sentinel clinics across Japan 5. The number of sentinel clinics was decided according to the population size of the administrative sector of the local health centres, making the surveillance similar nationwide. The majority of sentinel clinics have been settled, and neither the number and location of the sentinel clinics in the surveillance system nor the estimated coverage of the population by the sentinel clinics changed much over this time. In addition, the population coverage of sentinel clinics was similar for children and adults . The NESID data are available online on the NIID website6. For the observed timeframe, pathogen surveillance was conducted using polymerase chain reactions (PCR)7 of nasal swab specimens collected in approximately 10% of patients with ILI across the country. PCR for virus testing were conducted at the prefectural public health institute or NIID, and the results were uploaded onto the NIID website7.During influenza surveillance, physicians report incident cases to the local health centre via fax according to the clinical diagnosis for influenza-like illnesses (ILIs). The diagnosis of ILI by a physician is usually made using a rapid diagnostic testing kit for patients who presented with any of the four major symptoms of ILI . Local health centres report the weekly number of incident cases to the prefectural public health institutes or infectious disease surveillance centres, which are responsible for entering the incident cases  in the online database of the National Epidemiological Surveillance of Infectious Diseases (NESID), National Institute of Infectious Diseases (NIID)19. Samples of the serology surveillance were randomly collected from approximately 6000 Japanese individuals each year. We compared the influenza prevalence rate and the prevalence rate of serum influenza virus antibody.Separately, surveillance for serum antibodies against each influenza virus subtype and strain was conducted at designated hospitals across the country using the hemagglutination inhibition (HAI) assayStatistical analysis was performed using IBM SPSS version 21 . The level of significance was set to 5%.We calculated the yearly influenza prevalence rate, which is the rate of total ILI cases within the estimated coverage population by sentinel clinics, for each age group . The estimated coverage population by sentinel clinics was calculated for each age group using the national census data in 2010 as the coverage percentage of sentinel clinics within the total medical clinics , influenza antibody testing was implemented.  The HAI assay was conducted at all 47 prefectural public health institutes or the NIID, and the results were uploaded onto the NIID website19, by age group using the data of serology surveillance between 2006 and 20167 for five influenza virus: A(H1N1), A(H1N1)pdm09, A(H3N2), B/Yamagata-lineage, and B/Victoria-lineage. The difference in the prevalence of influenza antibody between children (≤ 19 years old) and adults (≥ 20 years old) was assessed using paired t-tests for each influenza virus. For multiple comparisons, the P value was corrected by Bonferroni’s correction. To assess the relationship between the previous infection and the prevalence of antibodies, Pearson’s correlation between the prevalence rate of the predominant virus strain and the prevalence of antibodies in children and adults was calculated separately.The virus subtypes and strains for the HAI assay were selected according to the vaccine virus subtypes and strains for the year: A(H1N1) subtype [A(H1N1)pdm09 subtype from 2009 onwards], A(H3N2) subtype, and the B lineage of the selected vaccine subtypes and strains, plus another B lineage that was not selected for the vaccine. We calculated the average prevalence with 95% confidence intervals (CIs) for an influenza antibody titer ≥ 1:40, which is considered sufficient to prevent infectionWe compared the sex ratio  of the influenza prevalence rate by age groups. We also compared the difference in sex ratios among children (≤ 19 years old), adults (20–59 years old), and older adults (≥ 60 years old) using a one-way ANOVA. For multiple comparisons, the P value was corrected using Bonferroni’s correction.All methods were performed in accordance with relevant guidelines and regulations.The influenza prevalence rate was relatively higher in children aged 0–5 and 6–14 years old. The influenza prevalence rate in children younger than 20 years old was highest during the 2009–2010 season with the emergence of the novel swine influenza virus A(H1N1)pdm09 Fig. .Figure 1P = 0.036) and adults  subtype was replaced by the A(H1N1)pdm09 subtype, which became the predominant strain almost biennially Fig. . The preP < 0.05, excluding epidemic seasons with emergence of the novel swine flu virus [between the 2009–2010 and 2011–2012 seasons]). In children, the influenza prevalence rate increased significantly during high-prevalence seasons . The prevalence of the B/Yamagata-lineage increased gradually in most recent seasons. Epidemic peaks in the influenza prevalence rate in children appeared biennially, except for the 2009–2010 season Fig. . This biOverall, in all age groups, the prevalence rates of serum antibody (HAI titers ≥ 1:40) did not largely change over time  in children (0.57 [95% CI 0.51–0.64]) than in adults . The mean prevalence of antibodies for B/Victoria-lineage was significantly lower (P = 0.001) in children (0.25 [95% CI 0.18–0.32]) than in adults (0.32 [95% CI 0.25–0.40])  between children (0.36 [95% CI 0.30–0.42]) and adults (0.37 [95% CI 0.31–0.44]). There was no correlation between the prevalence rate of the predominant virus strain and the prevalence of antibodies in children or adults.On average, the mean prevalence of antibodies for A(H1N1)pdm09 was significantly higher .In children, the difference of the influenza prevalence rate between girls and boys was largest in those aged 15–19 years (1.17 [95% CI 1.12–1.21]). In adults, the difference of the influenza prevalence rate between women and men was largest in those aged 30–39 years  between 2005 and 2010 was 0.09 (95% CI 0.06–0.11), which was the same as that in the present study21. Although the predominant influenza virus varied by year, including the seasons in which the A(H1N1)pdm09 subtype emerged, the sex ratio of the influenza prevalence rate was consistent among all age groups. We also found a significant increase in the influenza prevalence in both children (≤ 19 years old) and adults (≥ 20 years old) over time. The reason for this increase is unknown; however, the results suggest a change in immunity to seasonal influenza infection. Because the mean prevalence of antibodies for B lineages was relatively low in both children and adults, one factor may be the increase of predominant B lineages, especially the B/Yamagata-lineage. Prevention with respect to these epidemic trends, such as improved vaccination, i.e. matching the vaccine strain and timeliness, as well as meeting a sufficient rate of vaccination coverage for age and sex groups with a higher rate of prevalence, should be sought4.We found that the prevalence rate of influenza in children in Japan remained relatively high compared with adults, which is in line with former studies25. Similarly, there was no increase in the influenza prevalence rate in adults during the emergence of the A(H1N1)pdm09 subtype, suggesting that adults may have protective immunity against this subtype. Conversely, the results suggested that children are more susceptible to emergent virus subtypes and strains. The relatively higher prevalence of antibodies against A(H1N1)pdm09 could be attributed to the timing of the sample collection for the HAI assay in 2009, which occurred after the epidemic wave of A(H1N1)pdm09 started in August26. Considering the timing of the serum sampling, which mostly occurred before vaccination for the upcoming epidemic season, the observed prevalence of antibodies is thought to be derived from natural infection in the previous epidemic season; the increase in the prevalence of antibodies against A(H1N1)pdm09 over time may suggest residual antibodies from previous influenza infections. Although the HAI assay was conducted for vaccine virus strains, further data on antibodies using the Enzyme-Linked Immuno Sorbent Assay and whole influenza virus for antibody testing should be considered27. Additionally, our data of serum influenza antibodies included no information regarding the individual vaccination status.Although the serum antibody prevalence rates against the vaccine strains were relatively low in adults (except those aged 20–29 years), the influenza prevalence rate in adults was lower than in children. This observation may indicate the existence of immunity in adults from previous infection by different virus subtypes and strains17. Regarding the predominant virus subtypes, it has been found in many countries that after 2009 the A(H1N1) subtype was replaced by the A(H1N1)pdm09 subtype30. Meanwhile, our study illustrated that the prevalence of serum antibodies against influenza viruses was relatively low in children (0–5 years old), including antibodies against both the B/Yamagata and B/Victoria lineages. The biennial increase in the influenza prevalence rate in children in Japan during the study period appears attributable to epidemics caused by B lineage viruses. After 2010, the B/Yamagata-lineage has predominated over the B/Victoria lineage globally 35. In some countries, the number of infections by B lineage strains was increased in children35. Similarly, in Japan, the prevalence of the B/Yamagata-lineage has been increasing over time.National surveillance data for influenza over a long period, including data for age, sex, predominant virus subtypes and strains, and serum antibodies, are relatively limited37. Despite this relatively high vaccination coverage, the prevalence rate of influenza has remained similar over time, including a relatively high influenza prevalence rate in children. Therefore, further research to match vaccine virus strains with the predominant epidemic virus strains and increase the effectiveness of vaccinations in each season is needed, particularly with respect to B lineages40.In Japan, current influenza vaccination coverage is estimated at approximately 80% in school-age children and 50% in older adults45. Differences in incidence according to age and sex are thought to correspond to the immune status46. For example, women of reproductive age may have a stronger inflammatory response to infection than men of that age; thus, the rates of hospitalization for influenza infection may be higher in women than in men47.Both the incidence surveillance and serology surveillance were based on data collected from the outpatients, and thus, the data may have reflected healthcare-seeking or vaccination behavior or accessibility to testing in children and adults. However, concerning differences in the influenza prevalence rate by age and sex, previous studies  reported findings similar to those in our study, including higher incidence rates in male children than in female children and higher incidence rates in female adults than in male adults5. Furthermore, the surveillance network was immediately available for the active surveillance of COVID-1950. Long-term surveillance is important to develop and improve the strategy of monitoring, preventing and controlling future influenza epidemics.The surveillance of all infectious diseases was consolidated in Japan over decades and operated at the level of sentinel clinics to all clinic networks based on the surveillance disease category under the Infectious Disease LawSupplementary Figures.Supplementary Tables.


Wound healing-associated difficulties continue to drive biotechnological creativeness into complex grounds. The sophisticated architecture of skin wound sites and the intricate processes involved in the response to the use of regenerative devices play a critical role in successful skin regeneration approaches and their possible outcomes. Due to a plethora of complications involved in wound healing processes as well as the coordination of various cellular mechanisms, biomimetic approaches seems to be the most promising starting ground. This study evaluates the behavior of a crosslinked, porous collagen scaffold obtained by lyophilization and dehydrothermal reticulation (DHT). We address the key physio-chemical and mechanical factors, such as swelling, density and porosity, mechano-dynamic properties, SEM and TG-DSC, as well as important biological outcomes regarding scaffold biocompatibility and cellular metabolic activity, cytokine expression in inflammation, apoptosis and necrosis, as well as hemocompatibility and biodegradation. The mechanical and visco-elastic behavior are correlated, with the samples found to present similar thermal behavior and increased rigidity after DHT treatment. High biocompatibility rates were obtained, with no inflammatory stimulation and a reduction in necrotic cells. Higher percentages of cellular early apoptosis were observed. The hemocompatibility rate was under 2%, coagulation effects expressed after 4 min, and the DHT scaffold was more resistant to the biodegradation of collagenase compared with the untreated sample.  Our skin is an intricate mantle with remarkable capabilities, with its appearance generally reflecting the health and effectiveness of its underlying structures. At a glance, it seems frail and slender, but its importance in our homeostasis is more than skin deep. Being the largest organ of our body, and a key component of the innate immune system, it plays the role of infantry in numerous cases, either against common invaders, such as UV radiation , superfiThe microenvironment of a wound is highly refined by its own regulatory factors and its rich extracellular matrix (ECM), which determine the process of wound healing ,9. The cProduction of reproducible three-dimensional (3D) scaffolds represents one of the most important challenges in tissue engineering. Adequate biomimetic supports for cell migration and infiltration do not come easily, especially when they are required for inducing skin regeneration. In this case, the biomaterials should possess excellent biocompatibility, a suitable microstructure , controllable biodegradability and suitable mechanical properties. Currently, in skin tissue regeneration biotechnologies, natural and synthetic polymers are sought after , as well as poly  . Their iOne of the main advantages of collagen scaffolds is that they benefit wound healing by providing a skeleton of matrix for tissue types that are low in cellularity and have difficulty regenerating on their own after being affected by various external factors. The high porosity and generous cellular attachment sites of collagen in lyophilized scaffolds allow for the infiltration of additional cells, which aid remodeling. Porous scaffolds of lyophilized collagen may also present unique binding sites to cells that signal a remodeling response . The mecOther important advantages include the possibilities for customization of collagenic scaffolds in order to facilitate healing of numerous types of wounds, from superficial to chronic ones. By modifying each phase of fabrication based on the specific need of the tissues, bioscaffolds can be designed with improved therapeutic effects .However, controlling collagen biodegradation and improving its mechanical properties remain challenging, and are current barriers to its implementation within large-scale production and clinical trials. Therefore, our study aims to assess the biomimetic behavior of a collagenic scaffold in an in vitro simulated tissue microenvironment. All chemicals used were of analytical grade. The bovine Achilles tendon was provided by a local slaughterhouse, collected and stored in optional conditions for extraction. The other materials were sourced as follows: pepsin from porcine gastric mucosa ; acetic acid glacial ; hydrochloric acid w/w pepsin solubilized in 0.02 M HCl solution, pH 1.8), in accordance with the methodology described in the literature [Type I collagen was isolated from the bovine Achilles tendon (a by-product of the food industry). Collagen was isolated by the method of solubilization with non-specific enzyme  under vacuum (150 to 50 mbar), ranging the temperature during two days from 60 °C to 135 °C for a certain period of time and then cooled slowly to room temperature under vacuum, as shown in According to Xuefei et al. , a colla4/TiO2 as catalysts, converting N to NH3, which was distilled and titrated. The percentage of protein in a sample was calculated according to the Equation (1):b is the volume of the 0.1 N sulfuric acid used in sample titration (mL); a is the volume of 0.1 N sulfuric acid used in blank titration; Ne is the expected normality of the sulfuric acid solution (0.1 N); f is the correction factor of the sulfuric acid normality obtained by using sodium carbonate solution; Ws is the weight (g) of the sample; n is the conversion sfactor of total nitrogen to protein (6.25). Samples of each kind were measured in triplicate.The Kjeldahl method was used for total nitrogen content determination, using a conversion factor of total nitrogen to protein (6.25). The sample was digested in sulfuric acid  using CuSOThe moisture content was determined using Kern Moisture Analyzer DBS  and an aqueous solution of urea (aq. urea) (6 M) at 30 °C for 5 days. In order to discourage bacterial proliferation, the immersion liquids were changed every day. After wiping the immersion liquid off the surface with a filter paper, the wet weight of the samples was measured. The samples were air-dried for 24 h to obtain the weight in a dry state. The swelling ratio was calculated using Equation (2): W) were immersed for 5 min in a cylinder containing 10 mL of ethanol (V1). The volume of the ethanol containing the sample was measured (V2) and the volume of the liquid was measured again after the removal of the scaffold (V3). The porosity of the scaffold was calculated using the Equation (3), while the density was calculated using the Equation (4):The liquid displacement method was used to measure the porosity and density of the scaffolds. Ethanol was used as the displacement liquid. Collagen sponge scaffolds with a known weight  at room temperature for at least 5 min to reach equilibrious swelling. Subsequently, the sponge was removed for weighing after being gently blotted with a filter paper. The fluid uptake ability of the sample was calculated according to the following Equation (5):m0 is the weight of swollen sponge and m1 is the weight of sponge at 30 min, and at 1, 2, 3, 4, 5, 6, 7 and 8 h. Samples of each kind were measured in triplicate.The porous scaffolds were soaked in ultrapure water, and the swollen sponge was kept at 37 °C and 35% relative humidity in an incubator. At regular intervals of time, the scaffolds were removed and weighed. The weight remaining was calculated according to the following Equation (6):–1 and were accumulated for 400 scans at 4 cm−1 resolution at 22 °C. FTIR spectra of collagen scaffold samples were recorded with an Agilent Cary 630 FTIR spectrophotometer  in ATR mode, in the mid-infrared region, measuring the wavenumbers in the range between 4000–6500 cmAnalysis of surface morphological characteristics of samples Col-NT and Col-DHT was performed by scanning electron microscopy (SEM) analysis using a Zeiss Gemini 500 . Stress–strain dependence was recorded using the DMA 242 Artemis apparatus from Netzsch , equipped with a sample holder for compression (pushrods made of fused silica), in compression mode, at room temperature, under a linear increase of compression force of 50 μN/min, at a total load varying between zero and 8 mN. DMA investigation was performed on the same apparatus, equipped with a sample holder for sharing , on samples of 15 mm diameter and 5 mm height. Flexural moduli  and the dissipation factor (tan δ) were measured in temperature scan mode, under heating between −100 and +280 °C with a linear increase of 2 °C/min. The mechanical oscillations were induced at a frequency of 1 Hz, under a dynamic stress of 65 kPa .−1 from room temperature up to 900 °C, under a flow of 50 mL min−1 dried air. An empty alumina crucible was used as reference.The thermal analysis TG-DSC for the Col-NT and Col-DHT samples was performed with a Netzsch STA 449C Jupiter . The samples were placed in an open crucible made of alumina and heated with 10 K·min2. After 24 h, cells were washed with PBS , harvested using trypsin (Sigma-Aldrich) and counted using Trypan Blue (Sigma-Aldrich) and a hemocytometer. The seeding density for the MTT assays was optimized at 4 × 105. Cells seeded at 4 × 105 density in a clear 24-well cell culture plate were treated with collagenic samples and controls and incubated for 24 and 48 h at 37 °C and 95% humidity with 5% CO2. After 24 and 48 h of exposure to tested compounds, cells were incubated for 4 h with MTT reagent  at 37 °C and 95% humidity with 5% CO2. After incubation, cells were treated with MTT solvent (Roche) for 15 min at room temperature. Absorbance was measured using a spectrophotometric microplate reader  at OD = 570 nm. With the LDH Cytotoxicity Detection Kit (Roche), LDH activity was measured in culture supernatants using a spectrophotometric microplate reader (ELISA reader) at 492 nm with a 600 nm wavelength reference.HEp-2 human epithelial cells (adenocarcinoma) (ATCC-American Type Culture Collection) were selected as the model for cytotoxicity assessment. MTT assay was used for measurement of cellular metabolic activity and as an indicator of cell viability, proliferation and cytotoxicity. HEp-2 cells were cultivated in DMEM culture medium  supplemented with 2 mM Glutamine (Sigma-Aldrich), 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich) and 1% Pen/Strep  for 24 h at 37 °C and 95% humidity with 5% CO6 colony forming units/mL)—E. coli (ATTC strain) and S. aureus (ATCC). After 24 h of stimulation, either with the collagenic material or with the bacterial strains, the supernatants were collected for cytokine quantification. Cytokine analysis was performed as per the manufacturer’s instructions . The fluorokine beads  were diluted 1:100 in bead dilution buffer. Next, 50 μL of diluted bead was added to 50 μL of the sample in a 96-well filter bottom plate. Loaded 96-well plates were incubated overnight at 4 °C on an oscillating rocker (60 Osc./min). After washing, biotinylated primary antibodies (1:100 dilution) and plates were incubated at room temperature, protected from light (2 h at 60 Osc./min). After washing off the primary antibody, streptavidin-PE (1:100 dilution) was added to each well and plates were incubated . Finally, 100 μL of wash buffer was added to each well and samples were analyzed using a Luminex xMAP200 . Data points are represented as mean fluorescence intensities (MFI).RAW 264.7 murine macrophages were used in order to evaluate the immune response induced by the collagen scaffolds. Cells were grown in DMEM medium supplemented with 10% fetal bovine serum. Positive controls were represented by macrophages stimulated with two bacterial strains . In total, 10,000 cells were analyzed per measurement. Data were analyzed using FlowJo 10.0.7 software . HEK-293 T cells were cultured in DMEM medium supplemented with 10% FBS under standard cell culture conditions . Cell numbers were assessed using a hemocytometer. Trypsin/EDTA  was employed to detach the cells from the flask, either for passaging, heat shock induction or flow cytometry. For induction of apoptosis, cells were exposed to room temperature (22 °C) for a period of 24 h to collagenic scaffolds. HEK-293 T cells (3 × 106) were stained using the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit according to manufacturer instructions. Stained cells were diluted in Annexin V-binding buffer. Suspended cells were used to perform the flow cytometry assay. Determination of early and late apoptosis was performed by flow cytometry using a FACS Alexa cytometer and an Alexa Fluorw/v) at 37 °C for 30 min. Then, they were incubated in the presence of citrate blood and diluted with physiological saline solution (4:5) for 1 h at 37 °C. Subsequently, the whole blood was diluted with distilled water to cause complete hemolysis (positive control) and also with physiological saline (negative control). After incubation, the samples were centrifuged at 5000 rpm for 15 min. The absorbance was measured at 524 nm for the supernatant to record the amount of hemoglobin released. The percentage of hemolysis (HR%) was calculated using the formula:The hemolysis ratio was analyzed using citrated whole blood diluted with physiological saline solution according to Balaji et al. . The sam2) was added onto the sponge surface in a glass dish and incubated for 1, 2, 3, 4 and 5 min at 37 °C. The negative control involved the addition of whole blood to the glass dish. Then, 2 mL of distilled water was added to dissolve the hemoglobin in free hemocytes. The content of hemoglobin on the samples was measured using a UV-Vis spectrophotometer at 542 nm by the following equation: The hemoglobin adsorption assay was conducted according to Yan et al. . A quant2 at 37 °C for 1 h. Then, the biodegradation was performed at 37 °C for 24 h after the addition of 200 U bacterial collagenase Clostridium hystolyticum . Digestion was completed by adding 0.25 M EDTA and cooling on ice. The samples were centrifuged for 15 min at 4 °C, and the hydroxyproline was determined for supernatants by the method described by Kolar [The method was performed according to the protocol described by Sun et al.  with minby Kolar . A standp < 0.05 was considered to be statistically significant.The statistical analysis was performed using GraphPad Prism 9 . For biocompatibility, the data were analyzed using the two-way ANOVA test. All data were expressed as the mean ± SD for triplicates. For hemocompatibility, data were analyzed using ordinary two-way ANOVA with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with individual variances computed for comparison of Col-NT and Col-DHT. For biodegradability (%) the analysis was performed using the unpaired t-test method. A value of Protein concentration was determined for samples of the untreated collagen scaffold (Col-NT) and the dehydrothermally treated collagen scaffold (Col-DHT) using the Kjeldahl method. A nitrogen content of 14.77% was recorded for the untreated collagen scaffolds, leading to a total protein content of 92.35% . The dehThe moisture content was much lower in the case of Col-DHT (5.17%) compared with the Col-NT (13.23%) . The watThe DHT treatment causes crosslinking, which improves the mechanical properties of scaffolds. As a result, it was important to understand the type of crosslinking caused in the treated samples.Protein denaturants such as urea are commonly used to analyze protein stability. Julian at al.  found, bIn a high-concentration aq. urea solution, the protein’s noncovalent hydrogen bonding is broken, and the protein swells significantly . The covThe swelling ratios of the collagen scaffold in DI water and urea, before and after applying the DHT treatment, are shown in the The swelling ratio in DI water for thermally treated collagen was reduced to 2.9, indicating that extra crosslinking was introduced. In contrast, the swelling ratio in urea was reduced considerably to 3.5, almost reaching the same value as the swelling ratio in water, indicating that the crosslinking upon dehydrothermal treatment mostly comprises covalent bonding, without excluding the formation of hydrogen bonding.Products that are to be used as medical devices and applied directly to wounds have to absorb all hematic and lymphatic fluids present in the wound itself. From this point of view, the porosity and the density of the collagen scaffold play important roles. The results of the porosity and density analyses are presented in the The scaffolds were easily wettable by polar solvents such as PBS. They exhibited a high swelling ability; this is because collagen contain a large number of functional groups capable of binding water. This fast-swelling behavior is a characteristic property of hydrophilic and porous materials. PBS solution has a pH of 7.4, which corresponds to the pH of blood. The use of such a solution allows for examination of the behavior of the material after its application inside the body. The percentage of scaffold swelling after immersion in PBS for 1 h, together with the results of the moisture content measurements, are presented in The water holding capacity was evaluated for both scaffolds. Based on the weight remaining percentage over time, it was found that the Col-DHT scaffold lost water more slowly than the Col-NT . After 5−1, moderate absorbance at 1300–1500 cm−1 and high absorbance in the regions 1500–1700 cm−1 and 2800–3500 cm−1. The amide I band associated with the stretching vibration of the carbonyl group along the polypeptide backbone was found at 1653 cm−1. The amide II and III bands were observed at 1550 cm−1 and 1238 cm−1, and are associated with N-H bending vibrations and C-H stretching, respectively. The amide A band was found at 3317 cm−1 and is associated with N-H stretching vibration and hydrogen bands [−1 and is related to the asymmetrical stretching of CH2.The changes in the chemical structure of the collagen that occurred upon DHT treatment were analyzed using Fourier-transform infrared spectroscopy. Significant information can be extracted from −1) spectra shifts to lower wavenumber compared with the Col-NT (1653 cm−1) but does not reach the values corresponding to gelatin (1630 cm−1); this proves that, during the thermal treatment, a series of changes occurs in the collagen triple helix structure, but these do not lead to a complete denaturation.In order to determine the changes in the amide I band during the dehydrothermal treatment of collagen (Col-DHT), the IR spectra for both pure collagen (Col-NT) and gelatin were recorded. Gelatin is a hydrolyzed form of collagen with a similar chemical composition, but which partially or completely loses its native triple helix structure. The variation of the amide I band was clear during the thermo-denaturation process. The center of the amine I band of the Col-DHT  can be observed. The pore size (varying from 54 nm to 265 nm) is visibly modified as compared to Col-NT, appearing condensed and well-contoured, and the rigidity of the scaffold seems to be increased. The physical-mechanical behavior of the dehydrothermically crosslinked collagen scaffolds was studied according to a protocol designed for insulating foams, in two steps: (i) identification of the linear visco-elastic domain by means of a stress–strain experiment, and (ii) determination of visco-elastic properties by subjecting the sample to an oscillating force of a precise value. For both types of experiments, the applied forces and the resulted deformations were accurately measured, and the physical-mechanical properties of the scaffold material were then calculated. The samples exhibited a similar thermal behavior. In the RT-160 °C range, the samples lost ~10% of their initial mass, most likely due to the elimination of residual water molecules  . This process was accompanied by an endothermic effect with a minimum of 87.3 °C. The samples underwent oxidative degradation after 200 °C, losing ~87% of their mass up to 700 °C a. The degradation process occurred in three phases, partially overlapping, as can be seen from the exothermic effects on the DSC curve. In the first part, the oxidation of the lateral groups, labile at ~280 °C, probably occurred, followed by the oxidation of the main chain at ~325 °C. At higher temperatures, the oxidation process would have been continuous, slow and accompanied by a wide exothermic effect between 380–490 °C. Around 625 °C there was a sharp exothermic effect that can be attributed to the presence of sodium in the sample or the burning of carbon dioxide. Detailed analysis of the DSC curve b indicatThe HEp-2 cell line was used to evaluate the biocompatibility of the collagenic scaffolds. An MTT assay was performed to evaluate cellular metabolic activity and an LDH assay to evaluate cytotoxicity and consequently extensive membrane damage. The results obtained by MTT assay  indicateAssessment of cellular behavior after 24 h of contact with each collagenic scaffold using a phase contrast microscope  enabled The evaluation of LDH activity can indicate intra- and extra-cellular values of pyruvate, and quantitatively measure cytotoxicity in response to applied treatments. The results obtained  indicateIn the context of skin wounds, proinflammatory cytokines are among the first factors to be produced in response to barrier damage, and they regulate the functions of immune cells in epithelialization. Proinflammatory cytokines mainly include interleukins (IL)-1β, IL-6 and interferon (IFN-γ), among others, and participate in the inflammation phase of wound healing through activating downstream cascades . CytokinE. coli and S. aureus (as positive controls). The results obtained for IFN-γ  response. PS is located on the inner side of the intact plasma membrane of live cells and these cells do not stain with Annexin V or PI. PS localization is turned over to the outer side of the plasma membrane of apoptotic cells and these cells bind Annexin V on the outside but still exclude PI. Annexin V binds to PS on the plasma membrane and PI is taken up and binds to the DNA . 0, i.e., the initial contact point   . These fIn correlation with the fluid uptake ability obtained for the Col-NT (35.61 ± 2.54) and Col-DHT (47.74 ± 1.04), the lower HR rates for the DHT sample can be explained by its ability to release hemoglobin being conditioned by the applied treatment, and, consequently, by the rigidity obtained for the fibrous scaffold. p < 0.0001) and reached a coagulation effect similar to Col-NT after 4 min (p > 0.05).The rate of hemoglobin adsorption by the collagen sponges is shown in p < 0.0001). Therefore, Col-DHT was more resistant to the biodegradation of collagenase.Biomaterial biodegradation is vital for their use both as implantable materials and externally  because it affects cell growth, cell vitality and host response . BiodegrCollagen is the most common natural polymer, being the most abundant protein on earth, and can be easily extracted from both animal and plant sources. Type I bovine collagen, extracted from tendons, skin and bones, is the most common form, being widely used as a biomaterial. The intermolecular crosslinking of collagen can be achieved by the esterification of carboxylic groups or by amide group formation, collagen being a protein with a high number of hydroxyl, carboxyl and amino groups.Without crosslinking, collagen is frequently subject to problems related to its weak mechanical properties, cracking and high swelling ratio. Its stability can be increased with the formation of an extensive network of stable intermolecular crosslinks. Dehydrothermal (DHT) treatment, a physical crosslinking method, involves the exposure collagen to high temperatures for a certain period of time under a vacuum in order to remove the water and form new crosslinks. Removing the water from a collagen scaffold may cause damage to the original hydrogen bonds within the collagen. In this research, the most favorable DHT conditions were employed, with a highest temperature of 135 °C for 3 h.In the present study, we successfully produced a crosslinked collagen sponge scaffold using DHT treatment with a highly porous structure. Porosity and density are two very important parameters that define porous scaffolds. The reduction in the porosity of the sponge upon DHT crosslinking was counteracted by an increase in density, which would favor cell growth, proliferation, and nutrient and metabolite exchange, due to the larger surface accessible to cells .SEM analysis depicted the sample’s progression from a frail, fibrous and high porosity scaffold to a denser, rigid and condensed structure after the DHT treatment. As for the mechano-dynamic assessment, three domains can be identified: the first two following a conventional pattern, and the unusual third one, the latter probably associated with the progressive rupturing of the elongated fibers. The smallest feasible strain falling within the linear domain of the stress–strain curve was 65%. The thermally induced evolution of the main visco-elastic parameters of the scaffold material was observed in the domain between −50 … +100 °C, where the “melting” of the triple-helical structure of collagen occurs, at around 60 °C.TG-DSC analysis of samples indicated similar behavior for both of them. Upon close inspection, the DSC curve obtained indicated the presence of a weak endothermic effect at 220 °C, corresponding to the transition from triple-helix to an accidentally twisted chain. This transition is specific to collagen, and occurs when hydrogen bonds are broken by rising temperatures, turning collagen into gelatin.Evaluation of cellular biocompatibility in an in vitro stimulated tissue microenvironment provides great overview of the behavior of a medical device, especially in wound healing applications, where the physiopathology of the wound further complicates the affected site. The results obtained by MTT and LDH assays, performed after 24 h of sample contact with HEp-2 cells, indicate desirable biocompatibility for the sample Col-DHT, and decreased viability rates for Col-NT, further indicating the positive outcome of the DHT treatment. The monolayer exhibited better proliferation and adherence in the presence of the Col-DHT sample. The decreased viability for the sample Col-NT may be due to the different degradation process and the formation of small peptidic chains that suffocate cells and do not promote proper proliferation. As other authors have mentioned, biodegradation plays a critical role in obtaining a desirable 3D scaffold ,49.Cytokines are critical mediators that oversee and regulate immune and inflammatory responses via complex networks and serve as biomarkers for many diseases. Quantification of cytokines has significant value in both clinical medicine and biology, as the levels provide insights into physiological and pathological processes, especially in the development of novel medical devices of clinical interest. Cytokines and their clinical significance can be characterized from the perspective of their pro- and anti-inflammatory effects . ProinflE. coli and S. aureus (as positive controls), we observed lower levels of 1β and IL-6 for the collagen samples in comparison with the positive controls; as for IFN-γ, similar values for both collagenic scaffold samples were obtained, with decreased expression levels in comparison to E. coli and S. aureus controls. Conversely, stimulation with bacterial strains led to enhanced production of IL-1β, IL-6 and IFN-γ. A clear distinction between the two types of collagenic scaffolds was not observed, suggesting that the dehydrothermal reticulation did not impact cytokine expression in any way. Since IFN gamma is a pro-apoptotic effector [After exposing RAW murine macrophages to samples of Col-NT and Col-DTH, as well as effector , the resThe evaluation of early/late apoptosis and necrosis provided great insights into the cellular behavior after treatment with the collagenic scaffolds. The evaluation at the point of initial contact presented higher rates of viability (over 90%). However, after 24 h, cellular viability decreased significantly, and early apoptosis occurred. These findings may indicate some limitations of the MTT and LDH assays; as we know, these provide colorimetric end-point measurements. Annexin V and Propidium Iodine labelling paint a more detailed picture of how cells are affected at the metabolic level for both early apoptosis pathways (extrinsic and intrinsic) , and falThe evaluation of hemocompatibility for scaffolds intended to be used as implantable devices is a crucial assay. The results obtained indicate a HR of 1.39 ± 0.06% for Col-NT and 1.04 ± 0.19% for Col-DHT. According to ASTM F 756–2000 requirements, both samples are non-hemolytic. These results correlate with the higher fluid uptake ability of the Col-DHT samples as compared to the Col-NT. Moreover, Col-DHT starts from a significantly lower absorption rate and reaches a coagulation effect similar to Col-NT after 4 min.The biodegradability of samples was assessed using collagenase. Collagenase is an enzyme responsible for cleaving the triple helix. It specifically catalyzes the hydrolysis of peptide bonds with glycine . The resCompared with intact human skin, the mechanical and visco-elastic behavior of the studied scaffold registers modest values (with at least ten-fold less stress accommodated at the same applied strain) . HoweverAn adapted DHT treatment with different conditions of time and temperature was employed to obtain the most appropriate collagenic scaffold that is intended to be used in due course as a medical device for wound healing. The formation of covalent crosslinking bonds was observed by analyzing the swelling ratio of collagen scaffolds in DI water and 6M aq. urea. The IR data confirm that the DHT treatment did not lead to the loss of the native triple helix structure of the collagen scaffold. SEM, TG-DSC and mechano-dynamic assessment further confirmed this and presented a detailed depiction of the scaffolds’ behavior. Furthermore, we focused on analyzing the biomimetic behavior of the collagenic scaffold attained. The satisfactory biocompatibility results obtained, with no inflammatory stimulation, decreased necrotic cells and low HR rates, suggest considerable scope for further studies. Higher percentages of cellular early apoptosis were observed. Collagenase degradation rates were consistent with literature reports regarding the influence of crosslinking methods on collagen scaffold degradation. Notwithstanding the favorable results obtained so far in this study, due to complex and comprehensive requirements from legislative parties, there is a long way to go before even attempting to market this type of product. Besides the biotechnological challenges, clinical trials and CE marking approval are among the ones that still lie ahead.


PANGEA allows GO analysis to be performed on different sets of GO annotations, for example excluding high-throughput studies. Beyond GO, gene sets for pathway annotation and protein complex data from various resources as well as expression and disease annotation from the Alliance of Genome Resources (Alliance). In addition, visualisations of results are enhanced by providing an option to view network of gene set to gene relationships. The tool also allows comparison of multiple input gene lists and accompanying visualisation tools for quick and easy comparison. This new tool will facilitate GSEA for Drosophila and other major model organisms based on high-quality annotated information available for these species.Gene set enrichment analysis (GSEA) plays an important role in large-scale data analysis, helping scientists discover the underlying biological patterns over-represented in a gene list resulting from, for example, an ‘omics’ study. Gene Ontology (GO) annotation is the most frequently used classification mechanism for gene set definition. Here we present a new GSEA tool, PANGEA (PAthway, Network and Gene-set Enrichment Analysis;  Modern genetics and genomics owe much to work done using common model organisms. These models continue to make a significant contribution to the understanding of development, metabolism, neuroscience, behaviour and disease. With the onset of the ‘big data’ era has come a need for analysis platforms that deconvolute complex data from multispecies studies. Model organism databases (MODs) are knowledgebases dedicated to the curation, storage and integration of species-specific data for their research community. The past decade has seen a number of efforts aimed at pulling together model organism and human data to facilitate a more interdisciplinary approach; examples include MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) , Gene2FuAlthough these integrated resources allow search and comparison across certain data classes, large-scale data analysis remains in the domain of stand-alone bioinformatic tools such as DAVID , TermMapDespite the abundance of tools, we found that they do not fully meet community needs, primarily because they were overly focused on human gene data and were not using the most up to date data on other species. For example, Reactome pathway annotation is based on computational predictions derived from manually curated human pathways. There are a few organism-targeted analysis tools; the prokaryote-centred GSEA FUNAGE-Pro  is one eD. melanogaster, the nematode worm C. elegans, the zebrafish D. rerio, the mouse M. musculus, the rat R. norvegicus and human H. sapiens. For annotations based on a controlled vocabulary arranged in hierarchical structure, such as gene group and phenotype annotations from FlyBase, gene-to-gene set relationships were assembled after the hierarchical structure was flattened. An exception was made for GO annotations, which were assembled in two ways, with and without being flattened, allowing users to choose which output is used in the analysis. GO annotations include evidence codes that indicate the type of evidence supporting the annotation. For example, ‘IDA’ means that an annotation was supported by a direct assay, whereas ‘ISS’ means that the annotation was inferred from sequence or structural similarity. Using such evidence codes, GO gene sets were built with additional configurations: 1.) subsets based on experimental evidence codes, i.e., excluding annotations only based on phylogenetic, sequence or structural similarity and other computational analyses ; 2.) subsets excluding annotations only supported by high-throughput (HTP) evidence codes ; 3.) subsets of GO generic terms (GO slim) provided by the GOC (http://geneontology.org/docs/download-ontology/#subsets); 4.) subsets of very high-level GO term classifications used by FlyBase and the Alliance originally generated to support GO summary ribbon displays. For Drosophila phenotype annotations from FlyBase, we assembled the gene-to-phenotype association using the “genotype_phenotype_data” file available in the FlyBase Downloads page, in which phenotypes are associated with individual genotypes and controlled vocabulary identifiers are indicated. This allowed us to extract only those genotypes where we could be certain that the phenotype was associated with the perturbation of a single Drosophila melanogaster gene . Because different resources use different gene or protein identifiers, we used an in-house mapping program to synchronise IDs to NCBI Entrez gene IDs, official gene symbols and gene identifiers of species-specific resources, such as MGI and FlyBase . A program was implemented in the Python programming language to identify genes expressed at a substantially higher levels in one tissue versus any other tissue. This program first groups the RNA-seq datasets based on tissue. For example, all data related to the nervous system are grouped together. It then calculates the average reads per kilobase per million mapped reads (RPKM) expression values for each gene in each tissue group. Genes were identified as preferentially expressed in a given tissue group if their average expression in the tissue group is three-fold or higher than the average expression in any other tissue group. Genes with average RPKM value lower than 10 were excluded. Genes defined in this way as ‘tissue-specific’ then get annotated with the relevant tissue to generate the tissues expression classification set.To study the diversity and dynamics of the Drosophila transcriptome, the modENCODE consortium sequenced the transcriptome in twenty-nine dissected tissues  and the https://www.flyrnai.org/) via download of a file of all available public screen ‘hits’ . RNAi reagents of optimal design were selected. The criteria for optimal design were no CAN or CAR repeat, fewer than 6 predicted OTEs  and a single gene target. RNAi reagents were mapped to current FlyBase gene identifiers using a DRSC internal mapping tool. Screens focused on major signalling pathways were selected for PANGEA analysis    and highHypergeometric testing is performed to calculate P values for GSEA using the PypeR function in R. Bonferroni correction for multiple statistical tests, Benjamini-Hochberg procedure for false discovery rate adjustment, and Benjamini-Yekutieli procedure for false discovery rate adjustment were performed using the p.adjust function in R.https://www.flyrnai.org/tools/pangea/) and is built following a three-tier model, with a web-based user-interface at the front end, the knowledgebase at the backend, and the business logic in the middle tier communicating between the front and back ends by matching input genes with gene sets, doing statistical analysis and building visualization graphs. The front page is written in PHP using the Symfony framework and front-end HTML pages using the Twig template engine. The JQuery JavaScript library is used to facilitate Ajax calls to the back end, with the DataTables plugin for displaying table views and Cytoscape and VegaLite packages for the data visualisations. The Bootstrap framework and some custom CSS are used on the user interface. A mySQL database is used to store the knowledgebase. Both the website and databases are hosted on the O2 high-performance computing cluster, which is made available by the Research Computing group at Harvard Medical School.PANGEA is a SaaS (Software as a Service) web tool  and human. For example, pathway annotations allow users to identify metabolic or signalling pathways that are over-represented in a gene list and help understand causal mechanisms underlying the observed phenotype from a screen. Pathway annotations from KEGG, PantherDB, and Reactome, as well as manually curated Drosophila gene sets, such as FlyBase Signalling Pathways and the DRSC PathON annotation  from organism-specific resources (human and fly), protein complex annotations for multiple organisms from the EMBL-EBI Complex Portal  and COMPIn summary, we have assembled more than 300,000 different gene sets that can be used in PANGEA to assess the enrichment of particular biological features in an input gene list.GSEA can be computationally intensive because of the number of gene sets being tested and potentially large number of genes entered by users. Therefore, the step of pre-processing user’s input by mapping the input gene identifiers to the gene identifiers used for gene set annotation, is set up as a standalone ID mapping page (accessed by clicking ‘Gene Id Mapping’ on the top toolbar) instead of combining it with the analysis step. Gene identifiers supported by PANGEA include Entrez Gene IDs, official gene symbols and primary gene identifiers from MODs. Users might need to analyse lists of other identifiers such as UniProtKB IDs and Ensembl gene IDs. Users can use “Gene Id Mapping” tool and select an organism of choice to map IDs. As gene annotation is an on-going process, the gene identifiers as well as gene symbols might change over time. Even with the same type of gene identifiers such as FlyBase gene ID, the IDs used by users might be from a different FlyBase release. Therefore, ID-mapping step is an optional but recommended first step to ensure that the entered IDs are synchronized with the IDs used by PANGEA gene set annotation. Users of FlyBase may also directly export a ‘HitList’ of genes generated in FlyBase to the tool by selecting the ‘PANGEA Enrichment Tool (DRSC)’ from the dropdown ‘Export’ menu. An option for users to upload a background gene list for the analysis is provided; this may be useful when analysing hits from a focused screen using a kinase sub-library instead of a genome-scale library, for example. PANGEA identifies all relevant gene sets and provides enrichment statistics such as p-values, adjusted p-values, and fold enrichment, as well as the genes shared by the input gene list and gene set members. Users have the option to set different p-value cut-offs and visualise the results using a bar graph, the height and colour intensity of which can be customised . In addiAn under-appreciated use of GSEA tools is that researchers often use them as simple gene classification tools, for example, asking “which genes in my list are kinases?” to help inform further computational or experimental analysis. Having diverse classification sets is important because depending on the type of data/experiment being analysed, different gene sets may be more useful than others. It is often useful to be able to compare similar gene sets from different sources to help evaluate the evidence for support. In addition, PANGEA not only reports genes in an enriched gene set but also reports genes not covered by the gene set category selected, which may be interesting because of their lack of characterisation. This feature can help user answer question like “which genes in my list are not covered by KEGG annotation?”.Often users need to analyse multiple gene lists and compare the results; however, majority of current web-based tools only allow the analysis of a single input list (plus background). Thus, users have to perform comparisons manually or using different tools. To address this need, PANGEA allows users to input multiple gene lists and compare results directly via a heatmap or a dot plot visualisation. For example, users might input gene hits from different phenotypic screens and compare what pathways, gene groups or biological processes that are common or different among results .Drosophila S2R+ cells. Submitting the combined list of 75 interacting proteins from the four baits via the ‘Search Single’ option at PANGEA (accessed by clicking ‘Search Single’ on the top toolbar) for Fly and performing GSEA over phenotype, SLIM2 GO BP as well as protein complex annotation from COMPLEAT (literature based) enrichment, we identified mRNA metabolic process (GO:0016071), protein folding (GO:0006457), abnormal sex-determination (FBcv:0000436), abnormal neuroanatomy (FBcv:0000435), CCT complex and Spliceosome complex among the top enriched gene sets with the most significant p-values   . Next, w<1×10−5) . We also<1×10−5) . Enrichm<1×10−5) . These G<1×10−5) ,1C, whicWe further analysed the protein complexes associated with each individual subunit using the ‘Search multiple’ option at PANGEA (accessed by clicking ‘Search Multiple’ on the top toolbar) and inputting the interacting protein lists for each bait, then compared the enrichment result using a heatmap visualisation . The resDrosophila. At the DRSC, datasets generated from more than one hundred screens are publicly available  screening is a powerful method for functional studies in vailable . We selevailable .These use cases of PANGEA for phenotype screening data as well as proteomics data demonstrate the value of the tool in validating screen results as well as generating new hypotheses for further study.GSEA is a computational method used to identify significantly over-represented gene classes within an input gene list(s) by testing against gene sets assembled based on prior knowledge. Input gene lists are typically from high-throughput screens or analyses. Here we present PANGEA, a newly developed GSEA tool with major model organisms as its focus, that includes gene sets that are usually not utilized by other GSEA tools, such as expression and disease annotations from the Alliance, phenotype annotations from FlyBase, and GO subsets with different configurations. PANGEA is easy to use and has new features such as allowing enrichment analyses for multiple input gene lists and generating graphical outputs that make comparisons straightforward for users. In addition to the use cases presented here, i.e., analysing phenotypic screening and proteomic data, we anticipate that the tool will also facilitate analysis of gene lists from other types of data. For example, analysis of single-cell RNA-seq datasets at PANGEA might help users identify pathways and biological processes that are characteristic of various cell types. Users will also be able to answer questions on classification such as, “which genes in this list are kinases?”. PANGEA is designed to accommodate a wide range of biological data types and questions, providing users with a web-based analysis tool that is easily accessible and user-friendly.We also note that gene classifications are not static, and the generic design of the tool means that it will be easy to update or expand PANGEA for more gene set classification and/or more species. In developing PANGEA, we sought to improve the effectiveness of GSEA by 1) providing multiple collections of genes classified by their function in different ways (classified gene sets); 2) ensuring the data underlying the classification of gene function was up to date; and 3) improving the visualization so that results from multiple gene sets or multiple gene lists could be compared easily.Supplement 1


Computational and Mathematical Methods in Medicine has retracted the article titled “Research on Classroom Online Teaching Model of “Learning” Wisdom Music on Wireless Network under the Background of Artificial Intelligence” [ligence”  due to cFollowing an investigation conducted by the Hindawi Research Integrity team , signifi


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Fusarium graminearum. The Gpmk1 mgv1 Fghog1 triple mutants had severe growth defects and was non-pathogenic. It was defective in infection cushion formation and DON production. Conidiation was reduced in the triple mutant, which often produced elongated conidia with more septa than the wild-type conidia. The triple mutant was blocked in sexual reproduction due to the loss of female fertility. Lack of any MAPKs resulted in an increased sensitivity to various abiotic stress including cell wall, osmotic, oxidative stresses, and phytoalexins, which are likely related to the defects of the triple mutant in environmental adaptation and plant infection. The triple mutant also had increased sensitivity to the biocontrol bacterium Bacillus velezensis and fungus Clonostachys rosea. In co-incubation assays with B. velezensis, the Gpmk1 mgv1 Fghog1 mutant had more severe growth limitation than the wild type and was defective in conidium germination and germ tube growth. In confrontation assays, the triple mutant was defective in defending against mycoparasitic activities of C. rosea and the latter could grow over the mutant but not wild-type F. graminearum. RNA-seq and metabolomics analyses showed that the MAPK triple mutant was altered in the expression of many ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes and the accumulation of metabolites related to arachidonic acid, linoleic acid, and alpha-linolenic acid metabolisms. Overall, as the first study on mutants deleted of all three MAPKs in fungal pathogens, our results showed that although MAPKs are not essential for growth and asexual reproduction, the Gpmk1 mgv1 Fghog1 triple mutant was blocked in plant infection and sexual reproductions. It also had severe defects in responses to various abiotic stresses and bacterial- or fungal-fungal interactions.Mitogen-activated protein kinase (MAPK) cascades are activated by external stimuli and convert signals to cellular changes. Individual MAPKs have been characterized in a number of plant pathogenic fungi for their roles in pathogenesis and responses to biotic or abiotic stresses. However, mutants deleted of all the MAPK genes have not been reported in filamentous fungi. To determine the MAPK-less effects in a fungal pathogen, in this study we generated and characterized mutants deleted of all three MAPK genes in the wheat scab fungus The online version contains supplementary material available at 10.1007/s44154-021-00025-y. Fusarium graminearum is a causal agent of Fusarium Head Blight (FHB), a destructive disease of wheat, barley, and other grain cereals worldwide  and zearalenone  and MEK kinase (MEKK) genes are non-pathogenic and sterile, and have severe cell wall defects  kinases play important roles in activating cellular responses to extracellular signals, including host and environmental stimuli in plant pathogenic fungi , abundant infection cushions were formed by the wild type on wheat lemma at 2 days post-inoculation (dpi)  and deletion of ant Fig. a. TherefFghog1 mutant and apical swelling in the triple mutant. Nevertheless, irregular swellings were not observed in the Gpmk1, Gpmk1 mgv1, and mgv1 Fghog1 mutants -enrichment analysis, the top 500 up-regulated DEGs in the triple mutant were not enriched in any biological process. However, the top 500 down-regulated DEGs were significantly enriched for transmembrane transport, carbohydrate transport, interspecies interactions between organisms, and oxidation-reduction process .F. graminearum. In comparison with the wild type, 10 up-regulated and 19 down-regulated ABC transporter genes were identified in the triple mutant  and major facilitator superfamily (MFS) transporter genes in ant Fig. b. For diant Fig. b. These FAC1 adenylate cyclase gene was higher in the triple MAPK mutant than the wild type (Table S2). When intracellular cAMP level was measured, the triple mutant also showed an elevated cAMP level in comparison with the wild type  and three MEK  genes had no significant changes in their expression levels in the triple mutant. The expression levels of genes encoding the Gα, Gβ, and Gγ proteins also were normal in the tripe mutant, suggesting that the expression of well-conserved upstream G-proteins, MEKKs, and MEKs are not affected by deletion of these three MAPKs. In addition, deletion of three MAPKs resulted in no significant effect on the expression levels of the well-conserved STE12, MCM1, SWI6, RLM1, and ATF1 orthologs that are downstream transcription factors of MAPK signaling in the budding yeast  and 1017 metabolites with no significantly changes between the wild type and triple mutant  in our RNA-seq data, CON1 expression was affected (down-regulated 5-fold) in the triple mutant, likely related to the defects in conidiogenesis. In addition, deletion of three MPAKs may increase the intracellular cAMP level, which in turn activates the cAMP-PKA signaling pathway. The hyper-activation of cAMP-signaling may partially rescue some defects of the MAPK triple mutant but result in additional defects in conidiogenesis.The roles of individual MAPKs in regulating developmental and infection processes, as well as stress responses have been characterized in a variety of fungal pathogens that differ in host ranges and infection mechanisms  and AbHog1 are activated by camalexin, an indolic phytoalexin structurally related to brassinin  in the triple mutant were enriched in the fast evolving subgenome, which is under the control of heterochromatin and likely contains many genes important for adaption and infection  in stress response are not clear in F. graminearum. Because the Gpmk1 mgv1 FgHog1 triple mutant displayed similar sensitivity to CR as the mgv1 Fghog1 double mutant, indicating that Gpmk1 fails to function as a MAPK important for regulating responses to cell wall stress when MGV1 and FgHOG1 are deleted. Indeed, the role of Gpmk1 orthologs in cell wall integrity varies among different fungi. Whereas deletion of ChMK1 resulted in hypersensitivity to CR in Colletotrichun higginsianum  have showed that MAPKs can be activated by microbe-associated molecular patterns (MAMPs) or interactions with bacteria . Complete medium (CM) cultures grown at 25 °C were used for assaying growth rate and colony morphology , respectively. The resulting PCR products were purified and connected to the neomycin resistance gene cassette by overlapping PCR and transformed into protoplasts of the Gpmk1 mutant as described , and then connected with the neomycin resistance gene cassette by overlapping PCR. The resulting PCR product was transformed into protoplasts of the Gpmk1 mgv1 double mutant. Transformants resistant to hygromycin, geneticin, and fludioxonil were verified by PCR for the deletion of FgHOG1  resuspended in YEPD medium. After incubation at 25 °C for 6 h, germination rates were counted and analyzed. After incubation at 25 °C for 12 h or 24 h, germlings were examined with an Olympus BX-51 microscope. Each experiment was repeated at least three times independently.The final concentration of 200 μg/ml CR, 0.05% H5 spores ml− 1 in sterile double-distilled water (DDW). Wheat heads of 6-week-old cultivar XiaoYan 22 were inoculated with 10 μl of conidium suspensions at the fifth spikelet from the base  glutaraldehyde, and coated with gold-palladium before examination with a JEOL 6360 scanning electron microscope as described (Boenisch and Schafer 5 spores ml− 1) over the wound sites  were inoculated 2.5 cm apart on the opposite side of CM medium. After incubation at 25 °C for 3 days, inhibition of colonial growth was examined. To assay its inhibitory effects on conidium germination and hyphal growth, 3 ml B. velezensis (OD600 = 1.0) was mixed with fungal conidia and then resuspended in liquid YEPD (105 spores ml− 1). After incubation at 25 °C for 6 h or 12 h, germination and germlings were examined with an Olympus BX-51 microscope. Tip or intercalary swelling was observed after 24 h incubation. Each experiment was repeated at least three times independently.To assay fungal-bacterial interactions, C. rosea strain CanS41 . RNA-seq reads were mapped to the PH-1 reference genome using HISAT2 with its two-step algorithm  system  as described  and 2203 down-regulated (green dots) DEGs in Gpmk1 mgv1 Fghog1 triple mutant compared to the wild type.Additional file 4: Fig. S4. GO enrichment analysis of the down-regulated DEGS in the triple mutant. BP, Biological Process; CC, Cellular Components; MF, Molecular Function.Additional file 5: Fig. S5. Multidimensional scaling plot of the metabolomic profiles of the wild type and triple mutant.Additional file 6: Fig. S6. A proposed model for the functions and crosstalk of three MAPK pathways. a. Roles of F. graminearum MAPKs in fungal development, pathogenicity, DON production and abiotic stress responses. b. Roles of F. graminearum MAPKs in fungal-bacterial interaction and fungal-fungal interaction.Additional file 7: Table S1. Primers used in this study.Additional file 8: Table S2. Profiles of the differentially expressed genes in the wild type and Gpmk1 mgv1 Fghog1 triple mutant.


The Reading the Mind in the Eyes test (RMET) is a widely applied test of social cognition, based on mental state judgments in response to photographs of human eyes, which can elicit impairment in patients with numerous psychiatric and neurological disorders. However, interpretation of task performance is limited without the use of appropriate control tasks. In addition to a matched task requiring age judgments of the RMET stimuli, it was recently shown that a mental state judgment task of comparable difficulty, could be developed using photographs of domestic cat eyes. The current study aimed to further develop a Non-human Animal RMET (NARMET) by testing additional stimuli in the form of photographs of domestic dog eyes. A variety of additional tasks were used alongside the eyes test stimuli in a large sample of healthy young adults, to explore how alexithymia, schizotypal features, and autistic tendencies may differentially influence mental state attribution in response to cat, dog, and human eyes test stimuli. The resulting NARMET features both cat and dog trials, depicting a similar range of complex mental states to the human RMET. It shows favorable psychometric properties as well as being well matched to the RMET in terms of linguistic variables, length and difficulty. However, reading measures predicted performance on the RMET, but not on the NARMET. Although further testing is required in samples with a higher proportion of males, future application of the NARMET in neuropsychiatric populations exhibiting cognitive and behavioral difficulties could offer enhanced assessment of social cognitive skills. The Reading the Mind in the Eyes test (RMET) is a widely applied test of social cognition, involving selection of complex mental states to match photographs of the human eye region . Use of In one recent study, it was shown that a counterpart measure of comparable difficulty to the human RMET could be developed using photographs of domestic cat eyes . Cats weIt was previously found that higher accuracy on both the RMET and cat eyes test was positively associated with ratings of liking dogs on a simple animal preference questionnaire . Given tA variety of additional tasks were used alongside the eyes test stimuli to explore whether psychological factors may differentially influence performance across the human and non-human animal versions of the tasks. Because vocabulary and verbal fluency can be related to RMET performance , 28, meaAfter ethical permission was granted for the study by University of Birmingham, 210 undergraduate students without any current psychiatric or neurological diagnoses, or cat/dog phobia, volunteered to participate for course credit. Five had missing data and 1 demonstrated below chance performance on the eyes tests. A further three participants had incomplete data on a few questionnaires but were included after imputation of missing values based on group mean . The finParticipants completed the tasks individually in a lab at the University. They provided demographical information , and completed the Hospital Anxiety, and Depression Scale (HADS), Test of Irregular Word Reading Efficiency (TIWRE), and Test of Word Reading Efficiency (TOWRE). Participants then completed the three computerized eyes tasks, which were presented using Presentation  software, after being provided with Baron-Cohen et al.’s glossary of mental state terms. The order of presentation of each stimuli set  was counterbalanced across participants. These were followed by computerized questionnaires: Interpersonal Reactivity Index (IRI), Toronto Alexithymia Scale (TAS-20), Revised Social Anhedonia Scale (rSAS), Autism Spectrum Quotient (AQ50), Emotional Contagion Scale (ECS), Individual Differences in Anthropomorphism Questionnaire (IDAQ), Mind Reading Motivation scale (MRM), and Oxford-Liverpool Inventory of Feelings and Experiences (O-Life).Participants read out loud 108 regular words and then 39 irregular words , 31 withThe IRI , 33 contThe TAS assesses alexithymia and has good reliability and construct validity –36. TherThe revised SAS  containsThis self-report measure  containsThis scale , 41 contThis self-report measure  was deveThis scale assesses tendencies toward thinking about mental states rather than ability . The devThis scale  assessesThe AQ  consistsThe RMET  were detn = 18) was selected to match to the RMET for difficulty, and to achieve sufficient reliability and internal consistency. Correct answers were based on consensus (majority response for each item in the current study were no different to our previous study). The dog eyes test stimuli were developed for the current study following the same process as for the cats eyes stimuli i.e., 36 images (plus 1 for practice) without re-use restrictions were selected from the internet, based on visual similarity to the human stimuli , with a view to matching each individual RMET trial. Each eyes test commenced with onscreen instructions to pick “the word that best describes what the person/cat/dog in the image is thinking or feeling.” Images were approximately 28 cm × 9 cm high , with response options in Arial 22 point (approximately 1 cm high) outside the corners of the image, mapped to the numeric keypad which was used to respond . The first trial began after the participant pressed the space bar. There was no time limit, and a valid button response initiated the next trial.The development of the cats eyes test was described previously . Trials n = 228), as was done previously  and TAS-20 (n = 20), although more participants scored above the cut-off for the rSAS (n = 44).Accuracy scores (%) and mean reaction times  for the human , cat , and dog  stimuli were well matched. Mean accuracy for all cats plus all dogs was 72.17 ± 0.15, RT 3.33 ± 1.05 s. Descriptive statistics for the full set of measures can be found in r = 0.423, p < 0.0001; cats and human RMET: r = 0.483, p < 0.0001; dogs and human RMET: r = 0.468, p < 0.0001. Pairwise t-tests indicated no significant differences between any two stimulus sets . Good performers on one type of eyes stimuli tended to score highly on the other types. Eyes stimuli score distributions and correlation plots are shown in Correlations between the eyes tests were strong and positive, suggestive of convergent validity: dogs and cats: t(203) = −5.334, p < 0.001] and dog trials . There was no difference between cat and dog trials . In real terms, this amounts to just a few more seconds required to complete the entire human RMET in comparison to the entire set of cats plus dogs  was 0.67 for the human RMET, 0.54 for cats and 0.59 for dogs; while Fleiss’ Kappa for inter-rater agreement was: human RMET = 0.44; cats = 0.39; dogs = 0.44 .SE = 0.76%), with no significant difference for accuracy .To further test reliability of the newest stimuli i.e., the dog eyes test, extra data was collected for this task plus the human RMET in an additional sample of 228 undergraduate students . Accuracy for the dogs eyes stimuli was 72.45% (SE = 0.93%), and for the human RMET was 72.86% . Stepwise linear regression models were then run with the score for each eyes stimuli set as DV, and scores for any correlated variables  as IVs.R2-values with all predictors making a significant contribution to the model. Human RMET  scores were predicted by TAS EOT , TOWRE-A , and IRI fantasy scores . There were two predictors in the best model for the cat eyes stimuli  which were TAS EOT  and MRM scores , and for the dog stimuli set , which were EOT , and IDAQ non-anthropomorphic attribution  scores.The best models were identified based on highest F = 9.474, p < 0.001, adj R2 = 0.111], i.e., TAS EOT , MRM , and IDAQ non-anthropomorphic attributions .Combining the 18 trial cats eyes test with the set of 18 dog eyes trials creates the Non-human Animal Reading the Mind in the Eyes Test NARMET: , which iThe correct answers for the NARMET and the RMET see  were comBuilding on the development of the cat eyes test , this stGiven that it was possible to create the NARMET, this may tell us something about the processes involved in these eyes tasks. Individuals appear to share a common interpretation of a fairly complex level of psychological state as reflected in the eyes of both humans and at least domesticated non-human animals such as cats and dogs. This ability seems unlikely to be a specialist skill, according to the degree of consensus seen across a few different samples during the development of the NARMET. It is still debated whether the RMET measures Theory of Mind, or emotion/mental state recognition, with difficulties posed by either interpretation. For example, Theory of Mind is often a more appropriate label when the cues are more abstract than visual . On the other hand, visual cues may be more likely to prompt a recognition matching process. The issue with this latter interpretation is that we do not know whether the mental states that are being attributed to the eyes stimuli are indeed correct, and this is the case for both the RMET and NARMET stimuli, as correct responses are simply based on consensus. In the current study, response times were recorded, although most studies do not record this data. It took little more than 3 s of presentation time to per trial to elicit an accurate response, and this was consistent for both the human and non-human animal trials. This implies a fast and automatic underlying process, and perhaps reliance on a more instinctive mirroring type mechanism versus a more reasoned abstract perspective taking process, as the latter would likely take more time due to careful evaluation of each of the verbal labels in turn. Performance on the NARMET suggests that there are some fairly reliable visual cues  that individuals may use to draw conclusions about likely mental state, and that these cues may be more crude, and/or widely perceptible, than previously thought.Although the range of mental states thought to be attributable to the cats and dogs was slightly less wide ranging than for humans, it was still quite extensive. This aligns with research showing that humans can attribute secondary emotions such as jealousy and guilt to dogs . While sMental state attribution in relation to human, cat and dog stimuli were all negatively associated with externally oriented thinking. When cats and dogs were combined into a single task, NARMET scores were predicted by mind reading motivation and non-anthropomorphic attributions, while human RMET performance was predicted by fantasy and word reading efficiency. Therefore, while NARMET performance may be more clearly associated with motivational factors, it is perhaps less susceptible to confounds related to abstraction, language or verbal skills, which can influence RMET performance . It may In conclusion, using the NARMET alongside the RMET , and theThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University of Birmingham Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.CE conceived and designed the study, prepared materials for the data collection, performed statistical analysis, interpreted the findings, and wrote the first and final draft of the manuscript.


A subset of CRPC patients demonstrate increased p300/CBP activity based on a novel CTC biomarker assay. With further development, this biomarker suite may be used to identify candidates for CBP/p300 acetylation inhibitors in clinical development.Reduced SIRT2 deacetylation and increased p300 acetylation activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites  in castration-resistant prostate cancer (CRPC). We examined whether circulating tumor cells (CTCs) identify patients with altered p300/CBP acetylation. CTCs were isolated from 13 advanced PC patients using Exclusion-based Sample Preparation (ESP) technology. Bound cells underwent immunofluorescent staining for histone modifying enzymes (HMEs) of interest and image capture with NIS-Elements software. Using the cBioPortal PCF/SU2C dataset, the response of CRPC to androgen receptor signaling inhibitors (ARSI) was analyzed in 50 subjects. Staining optimization and specificity revealed clear expression of acetyl-p300, acetyl-H3K18, and SIRT2 on CTCs . Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced p300 and H3K18 acetylation. In CRPC patients, a-p300 strongly correlated with its target acetylated H3k18 (Pearson’s R = 0.61), and SIRT2 expression showed robust negative correlation with a-H3k18 (R = −0.60). A subgroup of CRPC patients  demonstrated consistent upregulation of acetylation based on these markers. To examine the clinical impact of upregulation of the CBP/p300 axis, CRPC patients with reduced deacetylase SIRT2 expression demonstrate shorter response times to ARSI therapy (5.9 vs. 12 mo;  Androgen deprivation therapy (ADT) is the cornerstone treatment for advanced PC, but most patients develop castration-resistant prostate cancer (CRPC) 36-40 months after initiating ADT . AdditioHistone modifying enzymes (HMEs) result in histone post-translational modifications that alter gene expression and cell behavior . HMEs maCirculating tumor cells (CTCs) have generated interest as minimally invasive, biomarkers for cancer management , 10. CTCIn the current study, employing a novel Exclusion-based Sample Preparation (ESP) technology (9)  to isolate CTCs, we evaluated p300 activity, SIRT2 expression and H3K18 acetylation in CTCs in a series of patients with sensitivity or resistance to ADT. We find a concerted increase in the expression of H3k18 acetylation and acetyl–p300 and downregulation of SIRT2 in CTCs in a subset of CRPC patients. Decreased SIRT2 is associated with shorter time of response to ARSIs. The results from this initial cohort support the integration of these biomarkers into prospective clinical trials.Previously validated antibodies against acetyl-p300, total p300, SIRT2 and acetyl-H3k18 were tested in single-cell staining assays using LNCaP and Du145 initially . PositivWe previously demonstrated that acetyl-p300, acetyl-H3K18, and SIRT2 reflect p300 acetylation enzyme activity in PC cells . The twoThe epigenetic biomarker suite was initially optimized on CTCs generated from several patients. Patient PBMCs (blood monocytes/mononuclear cells) were isolated from blood samples, captured via EpCAM, and then stained with the target biomarkers. PBMCs without captured by EpCAM are used as controls to compare expression intensity between CTCs as described . PBMCs dClinicopathologic characteristics are outlined in We previously found that decreased SIRT2 expression, increased p300 acetylation activity, and elevated histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC [We then tested whether there was a correlation between p300/CBP markers in the CTCs obtained from individual patients . Using Pn = 50). Pearson correlation analysis showed that SIRT2 mRNA levels are significantly correlated with progression-free survival time  (SPOP mutations (who were distributed evenly across the quartiles) because these patients have increased sensitivity to abiraterone [p = 0.03)  . To furt = 0.04) . We exclraterone . Patient = 0.03) . This clAlthough there are multiple available therapies for men with metastatic CRPC, there are currently few molecular biomarkers to help guide optimal treatment choice and predict prognosis in these patients. CTCs have the potential to address this need. The present study is an initial analysis of a biomarker set representing CBP/p300 activity in a series of patient CTCs. We find a strong correlation between p300 acetylation, SIRT2 and its target H3K18 acetylation in patients with advanced CRPC. We also demonstrate in a separate clinical dataset that low SIRT2 levels are associated with a shorter response to drugs that block androgen receptor signaling.We successfully isolated specific CTCs from patient blood samples, and by quantifying the protein expression in actual CTCs performed with immunocytochemistry (ICC), we found increased p300 activity and H3K18 hyperacetylation, and decreased SIRT2 expression in a subset of patients resistant to ADT treatment . We haveP300 activity is critical for the proliferation of PC3 and other CRPC cell lines. Researchers have found that the disruption of p300 transcripts through small interfering RNA inhibited PC cell proliferation both at the basal level and upon interleukin 6 stimulation . In the An important insight from our study is how the CBP/p300 axis is associated with treatment response. In one patient, a sample (575.1) was collected when ADT was initiated, and the PSA dropped to 2.4 ng/ml. A second sample (575.2) was obtained one year later when the cancer became androgen independent, and the PSA increased to 50 ng/ml with bone metastases. Acetyl-p300 and H3k18 were higher and SIRT2 was lower in 575.2 (CRPC) than in the responding hormone-sensitive PC sample (575.1). Extending our findings, in a well-annotated clinical database of mCPRC patients, we find that decreased deacetylase SIRT2 expression is associated with a shorter time of response to ARSI . SIRT2 pOur study has some limitations. Because of the small sample size, we were unable to achieve a statistical significance comparing HSPC and CRPC groups. CTCs demonstrate heterogeneity   and the The identification of a subset of CRPC patients with increased CBP/p300 activity in CTCs, and previous data demonstrating p300 activity correlates with these three epigenetic biomarkers, suggests they may be used to identify candidates for targeted treatment. P300 inhibitors have been examined in the context of advanced PCa patients, and a recent orally available p300/CBP bromodomain inhibitor CCS1477) has been found to decrease AR and MYC signaling in PC tissues [7 has beeThe PC cell lines Du145 is resistant to androgen deprivation therapy, and androgen-sensitive LNCaP were obtained from the ATCC and genotyped for validity. Du145 or LNCaP cells were then exposed to A-485 , a potenPeripheral blood was collected after informed written consent from patients with advanced PC as previously described  under a Sera-Mag (SM) streptavidin-coated magnetic beads (Cytiva) at a concentration of 50 ug per reaction were employed for all experiments. SMs were washed twice and resuspended in 0.1% Tween-20 (Fisher Scientific) in PBS prior to use. SM solutions were then incubated under agitation for 20 minutes with 1 μL (concentration) of a single capture antibody, against EpCAM . Prior to use, bead conjugates were washed an additional three times with 10% FBS.An overview of the CTC capture technology is shown in Supplementary Figure 1. CTCs were captured from PBMCs with antibody labelled SMs. PBMCs were incubated on a rotator with antibody-labelled SMs in a buffer containing 10% FBS for 20 min at 5°C. Cells bound to SMs were isolated with ESP technology using an automated magnetic head attachment for the standard Gilson PipetMax pipetting robot. SM-bound cells were moved through staining and washing wells prior to imaging at 20× with a Nikon Eclipse Ti fluorescent microscope (Nikon) and NIS-Elements AR 4.10 software (Nikon) after a final transfer to glass-bottom chamber slides.For all staining, cells were blocked with 10% FBS prior to extracellular staining, then, if intracellularly stained, cells were treated with Permeabilization Solution (BD) and stained for intracellular markers in Perm/Wash Buffer (BD). Stains: Hoechst, and antibodies against CD45 , CD34 , CD66b , pan-Cytokeratin , SIRT2 , Anti-histone H3-acetyl K18 , p300 , acetyl-p300 .Images were analyzed with NIS Elements software (Nikon). Average background fluorescence was subtracted from each channel then cells were identified and masked based on user-defined Hoechst staining thresholds, then further evaluated for average cellular staining intensity for all other fluorescent antibodies. Data was output and analyzed in Microsoft Excel, and GraphPad Prism.Staining intensity is compared between different cells by graphing each cell as an individual data point on a scatter plot, similar to flow cytometry. When graphed in this way, cells distribute into higher and lower density regions, where cells with similar staining characteristics cluster together . These chttp://www.cbioportal.org/). There were 128 patients in this cohort with metastatic CRPC that have baseline biopsy and matched clinical data and 75 patients of this 128 sub-cohort have gene expression data captured by Poly-A RNA-seq. A subset (55 patients) of this 75 sub-cohort have records of time on either enzalutamide/abiraterone. Five patients of this cohort were excluded because they have SPOP mutations that demonstrate elevated sensitivity to antiandrogen treatment [SIRT2 mRNA distribution, and time on treatment graph were generated by GraphPad Prism v9.5.1. The p-value for time on treatment probability was calculated using Gehan-Breslow-Wilcoxon test.Data was obtained from a 444 PCF/SU2C metastatic prostate cancer patient cohort (18). RNA-seq data and enzalutamide/abiraterone treatment data were downloaded from cBioPortal (reatment . The cor