To investigate and compare the lens phosphoproteomes in patients with highly myopic cataract (HMC) or age-related cataract (ARC). 2 enrichment and liquid chromatography-mass spectrometry. The potential functions of different phosphopeptides were assessed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.  In this study, we undertook a comparative phosphoproteome analysis of the lenses from patients with HMC or ARC. Intact lenses from ARC and HMC patients were separated into the cortex and nucleus. After protein digestion, the phosphopeptides were quantitatively analyzed with TiO In total, 522 phosphorylation sites in 164 phosphoproteins were identified. The number of phosphorylation sites was significantly higher in the cortex than in the nucleus, in both ARC and HMC lenses. The differentially phosphorylated peptides in the lens cortex and nucleus in HMC eyes were significantly involved in the glutathione metabolism pathway. The KEGG pathway enrichment analysis indicated that the differences in phosphosignaling mediators between the ARC and HMC lenses were associated with glycolysis and the level of phosphorylated phosphoglycerate kinase 1 was lower in HMC lenses than in ARC lenses.  We provide an overview of the differential phosphoproteomes of HMC and ARC lenses that can be used to clarify the molecular mechanisms underlying their different phenotypes. Cataract is an age-related degenerative disease and the principal cause of blindness worldwide . It freqLens proteins are some of the most long-lived proteins in the body and are the targets of numerous posttranslational modifications . These mIn this first comparative study of the differential phosphorylation status of HMC and ARC lenses, we quantified the phosphoproteomes of the lens cortex and nucleus separately. This was followed by Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially phosphorylated proteins to determine the implications of phosphorylation to the unique phenotype of HMC.In this study, we analyzed the phosphoproteomes of HMC and ARC lenses to detect differences between HMC and ARC. The workflow of this study is shown in In total, three HMC lenses and three age-matched ARC lenses were collected from patients, who provided informed consent before they underwent extracapsular cataract extraction surgery at the Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China. The research strictly adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Eye and ENT Hospital, Fudan University. Patients with an axial length of ≥26 mm were diagnosed with high myopia. In all lenses, the nucleus and cortex were separated by coring through the visual axis with a 4.5 mm diameter trephine. The lens tissues were divided into four groups: ARC lens cortex (ARC-C), ARC lens nucleus (ARC-N), HMC lens cortex (HMC-C), and HMC lens nucleus (HMC-N).μL of lysis buffer , disrupted with agitation using a homogenizer, and boiled for 5 min. The samples were ultrasonicated and boiled again for another 5 min. Insoluble material was removed by centrifugation at 12,000 g for 15 min. The supernatant was then collected for protein digestion. The protein concentrations were quantified with a BCA Protein Kit .Each tissue was lysed with 200 μg of each sample with the filter-aided sample preparation procedure described by Wisniewski et al. [μL of uric acid (UA) buffer  was used to remove the detergent, dithiothreitol, and other low-molecular weight components with repeated ultrafiltration. To block the reduction of cysteine residues, 100 μL of 0.05 M iodoacetamide in UA buffer was added and the samples were incubated for 20 min in the dark. The filter was washed three times with 100 μL of UA buffer and then twice with 100 μL of 25 mM NH4HCO3. The protein suspension was then digested with 3 μg of trypsin in 40 μL of 25 mM NH4HCO3. The mixture was incubated at 37°C overnight, and the resulting peptides were collected as the filtrate.We digested the proteins in 250 i et al. . Briefly2 protocol [μL of loading buffer , and 2% trifluoroacetic acid (TFA)). The TiO2 beads were added and then agitated for 40 min and centrifuged for 1 min at 5,000 × g. The supernatant was mixed with another TiO2 bead, resulting in the second beads which were collected as before. The beads were washed sequentially with 50 μL of washing buffer I  and 50 μL of washing buffer II  three times to remove the remaining unabsorbed material. The phosphopeptides were finally eluted with 50 μL of elution buffer  [The phosphopeptides were captured according to the TiOprotocol , adapted% NH4OH) . The eluμL of 0.1% (v/v) TFA. MS experiments were performed on a Q Exactive mass spectrometer coupled to an Easy nLC™ liquid chromatography (Thermo Fisher Scientific). The phosphopeptide extract (5 μg) was injected onto a C18 reversed-phase column  in buffer A  and separated with a linear gradient of buffer B , at a flow rate of 250 nL/min over 60 min. The most abundant precursor ions from the survey scan (300–1800 m/z) for higher-energy C trap dissociation (HCD) fragmentation. The target value determination was based on predictive automatic gain control. The duration of dynamic exclusion was 25 s. Survey scans were acquired at a resolution of 70,000 at 200 m/z, and the resolution of the HCD spectra was set to 17,500 at 200 m/z. The normalized collision energy was 30 eV, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at the maximum fill time, was defined as 0.1%. All MS experiments were performed in triplicate for each sample.The peptides from each sample were desalted with a C18 Cartridge (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL; Sigma), concentrated with vacuum centrifugation, and equilibrated with 40 All the raw data were identified with the MaxQuant software (version 1.3.0.5.) and screened against the UniProt human database, containing a total of 156,914 entries. The datasets were searched with a mass tolerance of 6 ppm. The search followed the enzymatic cleavage rule: trypsin/P, allowing two missed cleavages; tolerance on a mass measurement of 20 ppm; fixed modification; and carbamidomethylation of cysteines. Protein N-terminal acetylation and methionine oxidation were defined as variable modifications. The cutoff for the global false discovery rate for peptide and protein identification was set to 0.01. Label-free quantification was performed with MaxQuant, as previously described . Proteinhttp://www.genome.jp/kegg/) was used for pathway annotation. The GO terms and KEGG pathways with computed p values < 0.05 were considered significantly enriched.The GO program Blast2GO was used to annotate the differentially phosphorylated proteins and to create histograms of the GO annotations, including cell components, biological processes, and molecular functions. The KEGG database , a two-fold change was used as the cutoff to screen for differentially phosphorylated proteins. The level of phosphorylation was higher in the cortex than the nucleus, in both the HMC and ARC lenses. Twenty-six phosphopeptides were hyperphosphorylated in the HMC-C, whereas 10 were underphosphorylated compared with the HMC-N. In the ARC lenses, we detected 104 more abundant phosphopeptides and eight less abundant phosphopeptides in the cortex relative to the nucleus. A comparison of the HMC and ARC lenses revealed that the level of phosphorylation was higher in the HMC-N group than in the ARC-N group. We identified 58 phosphopeptides that were significantly altered in the HMC-C group: 23 were more abundant and 35 were less abundant than in the ARC-C group Figures . Among tS-formylglutathione hydrolase and glyceraldehyde-3-phosphate dehydrogenase was greater in the HMC-C than in the HMC-N. Meanwhile, in comparisons of ARC-C and ARC-N, a total of 33 phosphosites were exclusively detected in ARC-C and the phosphorylation degree of β-crystallin A3 (phosphorylation at site Y36) was much higher in ARC-C, with a different ratio of 34.04. Other significantly overabundant phosphosites were also detected in phakinin, β-crystallin B, and filensin (p < 0.05)  .γ-crystallin D, which was phosphorylated at Y29  were predominantly extracellular exosome proteins Figures .p < 0.05 are shown in We also performed KEGG pathway enrichment analysis to identify the biological pathways associated with the differentially phosphorylated proteins. The 20 most abundant enrichment terms with As shown in 2 enrichment combined with LC-MS/MS. For the first time, two parts of the lens with known histological differences, the lens cortex and nucleus, were compared separately. Proteomic differences were detected in the different regions of the lens, and by quantifying the differences in the phosphorylated proteins between HMC and ARC, we clarified the different pathogeneses in these two phenotypes.To improve the efficiency of identifying the phosphorylation sites in this study, we used TiOα-Crystallin A and α-crystallin B are the most abundantly phosphorylated proteins in the porcine lens [We identified 522 phosphorylation sites in 164 phosphoproteins in this study. Previous studies have reported 73 phosphorylation sites and 32 phosphoproteins in normal and cataractous lenses, using immobilized metal affinity chromatography and nano-LC-coupled MS/MS . α-Crystβ-crystallin, α-crystallin, phakinin, and filensin.In this study, the number of phosphorylation sites was significantly greater in the lens proteins of the cortex than in those of the nuclear region, in both HMC and ARC. One possible explanation to this finding is that the lens epithelium cells immediately adjacent to areas of the cortex are metabolically relatively active and metabolites decrease from the lens cortex toward the lens nucleus. Protein enzymatically phosphorylated in the outer cortex could gradually dephosphorylate nonenzymatically in the metabolically inactive nucleus. Among the phosphoproteins in these groups, a high percentage of the differentially phosphorylated proteins were crystallins and lens structural proteins, including α-Crystallin is a small heat shock protein that maintains the transparency of the lens. Phosphorylation is considered to change its chaperone activity by inducing a change in the protein's structure and altering the subunit exchange dynamics [α-crystallin B has been shown to regulate the protein's activity in both lenticular and extralenticular tissues [α-crystallin B are S19, S45, and S59. We detected these phosphopeptides in the lenses of patients with both HMC and ARC. When we compared the S19 site between these two groups, the level of phosphorylation was higher in the HMC-C than in the ARC-C. However, the pathological significance of these proteomic changes requires further analysis. There was a slight difference in the phosphorylation at S59 between the ARC-C and ARC-N and between the HMC-N and ARC-N. The phosphorylation of α-crystallin B at S59 is thought to be associated with actin nucleation and the migration of lens epithelial cells [dynamics . The pho tissues , 18–23. al cells –27. As pal cells . As the al cells .In the HMC group, the phosphosites that differed strongly in their phosphorylation between the cortex and nucleus predominantly comprised enzymes involved in glutathione synthesis, including GSS and S-formylglutathione hydrolase. Our KEGG pathway analysis also showed that the largest proportion of phosphoproteins was associated with glutathione metabolism.GSS and S-formylglutathione hydrolase catalyze key steps in glutathione synthesis. Glutathione is an essential antioxidant that protects the lens from oxidative damage . The levBy comparing the catalogues of differentially phosphorylated protein in the cortical regions of HMC and ARC, results showed that the degree of phosphorylation of GSS and GSR, the key enzymes of glutathione synthesis, was higher in HMC than in ARC. However, the precise roles of GSR and GSS phosphorylation remain unclear.In our comparison of the two lens tissues in HMC and ARC, the number of phosphopeptides was much higher in the HMC-N than in the ARC-N, which may be associated with the severity of nuclear cataract in patients with high myopia. Truscott  proposed+-K+-ATPase in the lens, and the cascade reaction leads to an imbalance in lens homeostasis.When we examined the glycolysis and metabolic pathways, which were enriched in differentially phosphorylated proteins in HMC and ARC, we found that the degree of phosphorylation of PGK1 was lower in HMC-C and HMC-N than in the ARC-C and ARC-N, respectively. A previous study showed that phosphorylation of PGK1 reduces its activity, thus reducing the glycolytic activity . GlycolyIn summary, we analyzed the phosphoproteomes of the cortex and nucleus of HMC and ARC lenses, while considering the clinical features of the lenses. We found significant differences in the extent of protein phosphorylation and the types of proteins phosphorylated between different regions of the lens. Our results will be valuable for the future investigation of the molecular characteristics and pathological pathways underlying HMC and ARC.


IgE mediates type I hypersensitivity reaction and can be found in the mucosa of organs affected by allergy. Acute appendicitis (AA) is a common disease, but its etiology remains poorly understood. Here, we investigated IgE deposition in histological sections of AA samples to test the hypothesis that an allergic reaction may substantially contribute to the pathophysiology of AA.  In a retrospective study, we assessed the presence of IgE in appendicular specimens of histologically confirmed appendicitis and in the control group, comprised of negative appendicitis and incidental appendectomies, using a monoclonal antibody against human IgE. Samples from 134 appendectomies were included: 38 phlegmonous and 27 gangrenous appendicitis from the study group and 52 incidental appendectomies and 17 negative appendicitis from the control group. The slides were visualized by light microscopy, and a standard procedure was used to manually count the positive IgE staining cells. p = 0.005; p < 0.0001 when adjusted for age and gender). No difference was found for gangrenous appendicitis. Discussion. The presence of IgE supports the contribution of an allergic reaction for the pathophysiology of phlegmonous appendicitis. The reduced number of IgE staining cells in gangrenous appendicitis can be due to tissue destruction, or, as been claimed by others, gangrenous appendicitis is a distinct entity, with different etiology.  IgE staining was present in the cells of all but 5 appendicular specimens. We found a significantly increased number of IgE-positive cells in phlegmonous AA (median = 28) when compared to incidental appendectomy (median = 17) ( In this study, phlegmonous appendicitis had the highest number of IgE-positive appendicular cells. These findings suggest that an allergic reaction can contribute to the pathophysiology of AA, opening a novel possibility for preventive measures in a disease that typically requires surgery. Acute appendicitis (AA) is the most frequent abdominal surgical emergency. Its etiology remains poorly understood, but it is assumed that endoluminal obstruction is the primary event . HoweverType I hypersensitivity reaction, or allergy, is the clinical manifestation of an immune response against allergens, which are inducers of immunoglobulin E (IgE) synthesis . Every dUsing a monoclonal antibody against human IgE, we have measured the presence of IgE in appendicular specimens of patients with clinical diagnosis of AA and in incidental appendectomies. If AA is the result of an allergic reaction, it is expected that IgE tissue deposition is higher in AA.The objective of this paper is to measure the presence of IgE in the specimens of patients with clinical diagnosis of AA and in incidental appendectomies.The study was performed in a 600-bed tertiary public hospital, which provides medical care to a population of 280,000 urban habitants. Both surgical and pathology departments are Associate Academic Centres of Faculdade de Medicina da Universidade de Lisboa.During the study period, all of the patients with the clinical diagnosis of AA were included as potential candidates. The only exclusion criteria were patients aged under 18 years old that were in the paediatrics department care.Two groups of patients were studied for the presence of IgE in appendicular specimens. The study group consisted of patients that presented to the emergency department with abdominal right lower pain between April 2016 and June 2017 and had histologically confirmed AA. The control group included patients that presented to our department in the same time period with clinical diagnosis of AA, were submitted to appendectomy, but had normal histological findings (NHF), classified as negative appendicitis, as well as patients that were submitted to incidental appendectomy (IA)  defined as the removal of a normal appendix along with the treatment of another pathology, between January 2010 and June 2017.The study protocol was approved by the Ethics Committee of Hospital Garcia de Orta (Reference 7/2016) and conducted in accordance with the Helsinki Declaration.muscularis propria, and acute gangrenous appendicitis (AGA) was defined as necrosis of appendicular wall in a background of transmural inflammation [Acute phlegmonous (suppurative) appendicitis (APA) was defined as neutrophilic infiltration in the ammation . Appendiammation .muscularis propria [The presence of neutrophils in the mucosa was considered a normal variant without clinical significance . The spe propria , 11.All the surgical specimens have been proceeded according to our department protocols. The original histology reports were not used. A dedicated GI pathologist reviewed all cases without having previous information from the clinical data or the pathological reports (HC). The evaluation of IgE-positive cells is not part of the pathology lab routine, so this was exploratory. Usually, there are no specific fields for evaluation, as during the most common diagnosis there is no special procedure for the microscopic evaluation of any specimens. Nevertheless, when a new analysis was needed, we would cut new paraffin sections and perform the selected method.μm) were stained. The primary antibody was 1/400 dilution  rabbit anti-IgE (RP022), Diagnostic Biosystems, antigen recovery buffer . The same appendicular specimen was used as external positive control in all slides.IgE detection was performed by immunocytochemistry (IHC) with the “Optiview DAB IHC Detection Kit,” Ventana® (Roche), detection system on a Ventana® BenchMark ULTRA, according to the manufacturer's protocol. Formalin-fixed, paraffin-embedded appendix sections . We counted the positive IgE cells with bright-brown membrane or cytoplasm . Diagram 1 tried to represent the aleatory, but reproducible, way; we evaluated all specimens to avoid counting errors and to reproduce the same method in all slides of all section's specimens. First, we scanned at a low magnification and then at high magnification, which was at ten high power fields  . We thenFor continuous variables, an assessment of the data distribution was performed using histograms, as well as using machine learning techniques, such as random forests, to determine the best distribution family for downstream regression analyses. This type of analysis not only is robust and flexible but also can account for confounding factors . For cathttps://cran.r-project.org), using ggplot2 [Statistical analysis was performed on R .The study population included 134 cases .There was 52 IA, including 20 patients in the context of bladder cancer, 18 in the context of ovarian cancer, and 14 miscellaneous causes.p < 0.001). In regard to gender, there were no statistically significant differences among groups. A personal history of allergy was present in approximately 15-17% of each group.Based on a regression analysis with a normal distribution, an age difference between patients with IA and the remaining groups was uncovered, with this patient being older than the remaining types of appendicitis . The distribution of IgE was higher in the APA group than that in the other groups with an IgE median (Q1-Q3) level of 28.0 (15.8–61.8). In pairwise comparisons .There were important differences among the groups  while, apart from APA-AGA (p = 0.0314) and IA-NHF (p = 0.0409), the others remain nonsignificant. This regression outperforming the univariate one using only appendicitis type (AIC multiple = 1209.311 vs. AIC univariate = 1214.901), suggesting age and gender should be taken into consideration.There were no differences between the other groups: IA versus NHF, There is still considerable discussion about the nature of the etiology of AA . HistoloIf AA is mediated by an allergic reaction, it would be expected that the IgE levels on the appendicular tissue, the target organ, are higher in AA than that in the control group, IA, and NHF. A significant difference was found between the 4 groups, APA, AGA, NHF, and IA. In the pairwise analysis, the difference was maintained between APA and IA. These results are compatible with an allergic reaction hypothesis for AA. In fact, in IA, no clinical and histologic features of appendicitis are present, and the number of IgE-positive cells in appendicular tissue is lower than that in the APA group.No difference was found between APA and NHF. It is well known that a substantial proportion of histologically normal appendices had clear evidence of an inflammatory molecular response in the form of increased cytokine expression, including TNF alpha and IL-2 mRNA . Also, wIt is possible that patients with pain in the right iliac fossa and no histologic criteria of AA are at an initial phase of AA, and as the neutrophil infiltrate has not yet occurred, the histological diagnosis of AA cannot be made.No difference was found between AGA and NHF or IA. Tissue destruction in AGA is so extensive that IgE can also be destroyed, and so, no anti-IgE fixation can be documented , 23. IncAppendicular abscess and appendicular perforation are clinical diagnoses, having been classified as AGA. In fact, it is well known that intraoperative assessment of the appendix frequently does not concur with histologic findings, and so, the appendicitis classification was based on histology, a more objective criterion , 26.lamina propria infiltration by IgA, IgM, and IgG, but IgE was not studied [IgE-positive nasal cells in allergic rhinitis consist mainly of mast cells. In our study, no specific coloration was used for evaluating different cell types; it was assumed that most of the cells that fixed anti-IgE at membrane were mastocytes and at cytoplasm were plasma cells Figures . An expe studied , 27. Nea studied . Again, To the best of our knowledge, this is the first study assessing IgE in appendicular specimens. The difference of IgE levels in APA and IA are in accordance with a hypersensitivity local reaction responsible for local inflammation in appendices. At a time when antibiotics can prevent some patients from undergoing appendectomies , our resRecent evidences suggest the presence of an allergic reaction in AA –32. A clA cohort study of 605 children undergoing appendectomy showed that those with IgE-mediated allergy had a 3-fold lower risk of complicated appendicitis compared with those without allergy .A case-control study using a national sample cohort showed an increased risk of appendectomy in asthma patients .In our study, the prevalence of allergy was evaluated according to patient clinical history and medical records, although no difference was observed between groups. At least 20% of the Swedish population suffers from one or several of the clinical manifestations of atopic allergy . UnfortuWe have straight and uniform definitions of APA, AGA, NHF, and IA. Using a methodology never applied in appendicular mucosa, anti-IgE antibody staining, we herein show that a mediated IgE reaction is present in inflammatory appendicular tissue, and so, AA could be the result of an allergic reaction.Study performed in a single center, with a reduced number of cases. These results should be confirmed in larger cohorts and by others.In this study, the higher number of IgE-positive cells in appendicular specimens was found in acute phlegmonous appendicitis, suggesting that an allergic reaction can be involved in the pathophysiology of acute appendicitis. A novel opportunity for preventive measures in a disease that typically requires surgical intervention is introduced.


Bifidobacterium animalis ssp. Lactis Bb-12 and Lactobacillus rhamnosus was studied in sheep milk ice cream. There was no effect of the apple fiber and the type of bacteria on the number of bacteria in the probiotics after fermentation. As a result of freezing, in the mixture containing Bifidobacterium animalis ssp. Lactis Bb-12, there was a significant reduction in the bacteria from 0.39 log cfu g−1 to 0.46 log cfu g−1. In all of the ice cream on the 21st day of storage, it exceeded 10 log cfu g−1, which means that the ice cream retained the status of a probiotic product. The Lactobacillus rhamnosus ice cream showed a lower yellow color compared to the Bifidobacterium Bb-12 ice cream. The overrun of the sheep’s milk ice cream was within the range of 78.50% to 80.41%. The appearance of the sheep’s milk ice cream is influenced significantly by the addition of fiber and the type of bacteria and the interaction between the type of bacteria and the addition of fiber, and storage time and fiber.The aim of the study was to assess the effect of the addition of inulin and the replacement of part of the inulin with apple fiber on the physicochemical and organoleptic properties of ice cream. Moreover, the survival of  The growing awareness of consumers and their expectations regarding healthy and good quality food has contributed to increased demand for functional food production. Bioactive ingredients added to food positively affect the product’s characteristics and quality, and positively affects human health . The tre6–109 CFU (colon forming unit) is required [The popularity of ice cream consumption and its availability contributed to developing functional ice cream recipes with increased nutritional value, enriched with probiotic bacteria and prebiotics . Accordirequired .Bifidobacterium animalis ssp. Lactis Bb-12 (Bifidobacterium Bb-12) and Lactobacillus rhamnosus (Lb. rhamnosus) are among the most commonly used probiotics from the group of lactic acid bacteria. Probiotics have antioxidant, anti-inflammatory, antibacterial, and antiviral effects [Lactobacillus and Bifidobacterium among others including inulin, pectin, galactooligosaccharides (GOS), fructooligosaccharides (FOS) [Bifidobacterium Bb 12 and Lb. rhamnosus in sheep milk ice cream. effects . Moreove effects ,8. Howev effects ,10. In s effects ), accele effects ,14,15 afes (FOS) . Inulin es (FOS) , obtainees (FOS) . After cThe material for the production of the ice cream was raw sheep’s milk , with the following chemical composition: 5.34 ± 0.2% protein, 6.20 ± 0.3% fat, 5. 01 ± 0.12% lactose determined on a Bentley milk and milk product analyzer  and pH 6.8 ± 0.12 .Bifidobacterium Bb-12 and Lb. rhamnosus .For the production of the ice cream, the following were used: white sugar , mango–passion fruit flavor essence  with the following composition: natural and identical to raw mango and mango–passion essence, citric acid E330 and mango juice, inulin , apple fiber  composed of 100% micronized apple fiber. To inoculate the sample, probiotic bacteria  were used: Bifidobacterium Bb-12 (Abb12: sample with 4% inulin and Bifidobacterium bb12) and Lb. rhamnosus . Inulin (2.5%) and apple fiber (15%) were added to the second batch, and they were also divided into two groups, AFbb12  and BFLr . The milk with the additives was mixed and homogenized using a homogenizer  with a pressure of 20 MPa at 60 °C. After, the milk was pasteurized at 85 °C for 30 min and cooled to 37 °C. The groups Abb12 and AFbb12 were inoculated with a monoculture of Bifidobacterium Bb-12, while the groups Blr and BFLr were inoculated with Lb. rhamnosus. Each prepared milk sample was inoculated with a previously activated starter culture according to the method of Mituniewicz–Małek et al. [−1 of bacteria, which was added to the milk in the amount of 5%. Prepared mixtures were fermented in an incubator  at 37 °C for 10 h, then cooled to 5 °C and conditioned at this temperature for 12 h. Prepared ice cream mixes were frozen in a freezer  for 40–50 min. The produced ice cream was packed in 100 mL plastic containers and stored at −22 °C for twenty-one days.Sheep’s milk (85%), sugar (11%), and mango–passion essence (0.1%) were mixed and divided into two batches. Inulin (4%) was added to the first batch and divided into a sample containing k et al.  with som−1 [The chemical composition of ice cream and ice cream mixes were determined using a Bentley B-150 milk and dairy analyzer . Measurement of the pH value of the milk, ice cream mixes, and ice cream was performed with a FiveEasy pH meter . Lactic acid content was determined by titration of the samples with 0.1M NaOH using phenolphthalein as an indicator. The results are expressed in g L−1 . Ice cre−1 . The melBifidobacterium Bb-12 and Lb. rhamosus were determined according to the method of Lima et al. [−1.The number of probiotic bacteria a et al. . The inoa et al. . The colThe color of the ice cream was determined by the instrumental method using a colorimeter  using the CIE LAB system . The folThe organoleptic evaluation was carried out by 20-person panel experts. The parameters were assessed on a 9-degree scale with structures and definitions.p < 0.05.The results from two independent studies were expressed as the mean and standard deviation in Statistica v. 13.1 . One, two, and three-way ANOVA was performed, and the differences between the mean values were verified with the Turkey test, with The chemical composition of ice cream mixes based on sheep’s milk before and after fermentation is presented in The carbohydrate content of ice cream blends before fermentation ranged from 19.45% in AFbb12 to 19.50% in Abb12 and was not significant. As expected, the carbohydrate content after fermentation decreased by 1.3–1.4%. There was no significant effect of the apple fiber and the type of bacteria used for fermentation on the carbohydrate content in sheep’s milk ice cream mixes. Also, in the study by Góral et al. , no signp ≤ 0.05). Ice cream mixes with apple fiber (AFbb12 and BFLr) were characterized by a significantly lower pH value than the mixes made only with inulin , titratable acidity or sugar content [In the process of ice cream manufacturing, the ingredients used in the recipe may adversely affect the probiotic by changing the pH . The low pH of the mixtures and the high content of lactic acid contributed to reducing the Bifidobacterium Bb-12. The decrease in bacterial cell counts resulting from freezing was likely due to damage to the bacterial cell walls that led to the bacterial cells’ death [Lactobacillus acidophilus and Bifidobacterium Bb-12, the number of bacterial cells decreased by 1.5- to 2 log CFU. Lb. rhamnosus cells in BLr and BFLr ice cream. The lack of this effect on the number of Lb. rhamnosus cells can be explained by significantly higher pH values and lower lactic acid content. According to Godward et al. [Lactobacillus has a broad cytoplasmic buffering capacity and resistance to pH (3.72–7.74), which enables its stability and resistance to changes in cytoplasmic pH in an acidic environment.When the temperature is decreasing during the freezing of the ice mixtures, changes in the osmotic pressure in the cells result in changes in the microorganisms, causing the loss of their metabolic properties. During the freezing process, the formed ice crystals can mechanically damage cell walls, and the condensation of harmful solutes or dehydration of cells additionally intensify the adverse changes ,38. The acterium ,36, shous’ death , during d et al.  and Tamid et al. , probiot−1 of Lactobacillus acidophilus and 8 log CFU mL−1 of Bifidobacterium bifidum immediately after freezing the ice cream. Akbari et al. [Lactobacillus acidophilus) and by 0.33 (Lb. rhamnosus) log CFU.Also, the 1.5% addition of apple fiber did not significantly affect the number of viable bacterial cells immediately after freezing . Mohammai et al.  reportedBifidobacterium Bb-12 and Lb. rhamnosus  was found during ice cream storage. According to the International Dairy Federation’s recommendations, products defined as probiotic should contain at least 7 log cfu g−1lactobacillus or 6 log cfu g−1Bifidobacterium [Lactobacillus acidophilus cells exceeding 6 log cfu g−1 in probiotic ice cream. Similarly, Akalin and Erisir [In these studies, ice cream storage at −22 °C for 7 and 21 days resulted in insignificant reduction of both hamnosus . Howeverc status . The prod Erisir , a decreacterium . The stuacterium  showed td Erisir  reported−1 is required to obtain a therapeutic  effect.The excellent survival rate of probiotic bacteria cells obtained in these studies ensures that the therapeutic level of synbiotic sheep’s milk ice cream is maintained for at least 21 days. According to Jayamann and Adams , a bacteThe results of the color of the ice cream mixes and ice cream during storage are presented in Lb. rhamnosus, and then in the mix with inulin and Bifidobacterium Bb-12. The lighter color of the Abb12 and BLr ice cream was maintained throughout the storage period. The 1.5% addition of apple fiber decreased the color brightness by about 9 units in the AFbb12 and BFLr blends. After 7 and 21 days of storage, an increase in L* brightness was found in all ice cream samples. Extending the storage time from 7 to 21 days resulted in a further increase in L* brightness, but the differences were not significant. A significant effect of the storage time, the addition of apple fiber and the interaction of these two factors on the brightness of the color of the ice cream was demonstrated  were characterized by a higher red color (+a*), which comes from the phenolic compounds and pectin contained in the fiber ,46. On tLb. rhamnosus throughout the storage period (p ≤ 0.05). These results were confirmed by the analysis of variance, which showed that the type of bacteria, the addition of fiber, and the interaction of these two factors significantly influenced the yellow color intensity. The results for the intensity (C*) and hue (h*) of the color indicate that these color coordinates are dependent on all of the factors tested  and their interactions. The addition of apple fiber decreased the h* color saturation parameter in AFbb12 and BFLr ice cream. The studies of Akalin et al. [Lb. rhamnosus. The analyzed ice cream and ice cream mixture were characterized by a high value of yellow (+b*) due to the mango–passion fruit essence used in their production. A significantly lower value of yellow color was found in the ice cream fermented with n et al.  also foun et al. , Dervison et al. , ice cren et al.  determinn et al. , L* brigThe results in Lb. rhamnosus (BLr and BFLr) had a longer time for the first dropping time after 7 days of storage than those containing Bifidobacterium Bb-12 (The melting rate of ice cream is influenced by many factors, including the total dry matter content, ice crystals, size, and the number of fat globules . The firum Bb-12 . ExtendiA fast-melting product is undesirable, and a too slow melting rate can also be a disadvantage of ice cream . The totFrom a technological point of view, the use of fruit fiber in ice cream production causes significant changes in organoleptic characteristics, improves their taste and texture .The addition of apple fiber significantly changed the appearance of sheep’s milk ice cream. It increased sandiness and intensified the additives taste, and the smell of the expansion of mango–passion fruit essence, both on the 7th and 21st day of freezing storage .Akalin et al.  showed tSheep’s milk ice cream can be a good source of probiotic bacteria and dietary fiber. The addition of 1.5% apple fiber instead of inulin resulted in a change in the ice cream’s physicochemical and organoleptic characteristics. On an industrial scale, when using the addition of apple fiber for the production of probiotic ice cream, attention should be paid to selecting the strain and the survival of probiotic bacteria during the freezing of the mixture and storage of ice cream.


GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 μM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na The GABAergic origin of the measured current has previously been validated, since SNAP-5114 had no effect on the tonic current in the presence of the GABAA antagonist picrotoxin  medium, constant opening of GAT-3 may increase tonic inhibitory currents that were shown to be inhibited by SNAP-5114. In fact, elevated extracellular concentration of GABA was measured in the striatum of Mn2+-exposed rats, suggesting impairment of clearance of or enhancement of release of GABA by GABA transporters [2+ by Cu2+/Mn2+ might also occur at other tripartite synapse targets including various Glu and GABA receptor and transporter subtypes [Another potential pathway to discuss is the activity-dependent release of Zn2+ ,72, co-r2+ . The preus GAT-2  was explAT-2 [2+ ,76. In h effect) ,78. Stabsporters . In prinsubtypes ,85,86,872+ significantly exceeds those recorded with added 20 µM Mn2+ alone or in combination with the GAT-2/3 specific inhibitor SNAP-5114. Furthermore, the significantly higher level of tonic inhibition persists after washout, suggesting that added Cu2+ elicits a long-lasting shift of neuronal excitability. We propose that the tonic inhibitory currents enhancement by 2 µM CuCl2—that substantially exceeds the 20 µM MnCl2 –induced GAT-3 regulated tonic inhibitory currents—can be traced back to some additional GABA release mechanisms. At first approximation, we focused on data linked to PA metabolism. Literature data show that added Mn2+ dose-dependently (1–100 µM) increases putrescine content (3.2–4.5 nmol/mg protein) in human SH-SY5Y cells [2+ induced enhancement of putrescine → GABA catabolism also boosts the putrescine → spermidine → spermine metabolism pathway. Spermine may in turn support astroglial GJC coupling [7 receptor pore [Unexpectedly, tonic inhibitory currents elicited by ten-minute single application of 2 µM Cu5Y cells . Besides5Y cells . It is pcoupling ,31, longtor pore  or via ctor pore . These afor a detailed discussion see [mK values characterizing copper uptake by CTR1 range between 1 μM and 6 μM [2. Our data provide supporting evidence on the gliocentric scheme of copper signaling [Data on depolarization-induced bulk copper release amounts to ≥ 100 μM synaptic transients (sion see ). Synaptsion see ,92. The and 6 μM . These fignaling  by relat2+ is mediated by the astroglial CTR1 that provides copper for CAO. This pathway can be blocked by Ag+. (2) Changes in intracellular copper concentration affect CAO activity. Although copper loading into the CAO active site is considered to be an irreversible process, the rate by which the loading occurs does depend on solute copper concentration. An experimentally validated kinetic model developed by Adelson et al. [2+ is comparable with 30 min loading with 10 fold copper excess [−1) copper binding to a second, reversible, pre-equilibrium “kinetic” site on CAO [a receptor subtype. (5) Cu2+ may also bind to the extracellular Zn2+ binding sites of the GAT-3 transporter, keeping GAT-3 open and therefore facilitating GABA release. (6) On the other hand, putrescine is also used to synthesize spermine which keeps gap junction channels (GJCs) open and enables inter-cellular trafficking of Ca2+ and other messengers. (7) Alternatively, GABA can also spread through the GJCs and released at distant astroglial processes through P2X7 purinergic receptor pores or Cx43 hemichannels. (1) Uptake of extrasynaptic Cun et al.  showed tr excess . The depe on CAO . It was e on CAO ,96. (3) Consequently, astrocytic putrescine metabolism simultaneously participates both in reduction and enhancement of neuronal activity by providing tonic GABA currents and GJC-mediated activity-spreading, respectively.The amount of copper released into the synapse, diffused extra-synaptically and taken up by astroglial copper transporters determines the strength of redox coupling which may vary from physiological to up- or down-regulated. The high extracellular level of copper elicits low surface expression of CTR1 , that ma


Fennel and dill are widely used as food additives owing to their various biological activities, such as antioxidants, antimicrobials, food-preservatives, and seasoning capacities. Herein, the nutritional composition and essential oil (EO) chemical profiles of fruits, umbels, stalks, and roots from one dill and two fennel cultivars were evaluated. The fruits had the highest content of crude protein (≥15%), crude fat (≥8%), and phosphorus (≥0.5%), and exhibited the highest total energy (≥20 MJ/kg) and EO yield (≥2%). Moreover, estragole (86.56% in Fdf), anethole (71.17% in Fhf), fenchone (16.74% in Fhf), limonene (50.19% in Agf), and carvone (42.41% in Agf) were the main components of the EOs generated from the fruits. The chemical profiles of EOs in the roots were significantly different from those of the aerial parts of the fennel and dill; thus, the roots and aerial parts could be distinguished based on myristicin (Variable Importance in Projection (VIP) = 1.90399) and apiol (VIP = 1.85922). The EO components of the aerial parts varied remarkably, and the chemical markers for differentiating these three cultivars were anethole (VIP = 1.36571), estragole (VIP = 1.30292), and carvone (VIP = 1.11947). Overall, our results provide a noteworthy chemical basis for further development of fennel and dill, especially as food additives. Foeniculum vulgare Mill.) and dill (Anethum graveolens L.) are the most vital medicinal and aromatic plants in the Apiaceae family [The Apiaceae plant family comprises some of the most important medicinal and aromatic plants cultivated worldwide . Fennel e family , as theye family ,4,5,6. Me family ,8.Fennel and dill fruits can be eaten as a condiment because of their unique flavor and abundant nutritional profile . FurtherConsumer preference for clean-label products requires the food industry to reformulate products by replacing artificial additives, such as colors, flavors, and preservatives, with natural alternatives . EssentiMany studies have focused on the biological activities of fennel and dill. However, a lack of systematic studies on fundamental chemical information currently limits the application of these plants. Thus, given the immense nutritional value and wide range of biological activities of fennel and dill, this study aimed to investigate the chemical diversity of different parts of the plants and cultivar species. The nutritional composition and EO chemical profiles of different parts of locally grown fennel and dill species were measured. Moreover, a multidimensional exploration regarding EOs was performed in this study. The detailed chemical characterization of different parts of fennel and dill plants will provide baseline data to guide their application as food additives.Foeniculum vulgare “Dwarf” (Fd) and Foeniculum vulgare “High” (Fh), and one dill cultivar of Anethum graveolens (Ag), were collected from Minqin County in Gansu Province, where the climate is temperate, continental, and arid. The studied plants were grown on soft and sandy soils, and these cultivars were commercially available. Thirty individual samples of each plant were wholly (including roots) harvested at dawn between September and October 2018 and stored at room temperature (20–25 °C). Each plant was divided into four parts: fruit, umbel, stem, and root. Abbreviations corresponding to a total of twelve samples are as follows: Fd fruit (Fdf), Fd umbel (Fdu), Fd stem (Fds), Fd root (Fdr), Fh fruit (Fhf), Fh umbel (Fhu), Fh stem (Fhs), Fh root (Fhr), Ag fruit (Agf), Ag umbel (Agu), Ag stem (Ags), Ag root (Agr).Two fennel cultivars of TM Sampler8420 Automatic Kjeldahl nitrogen analyzer (FOSS) was used to measure the crude protein content. Total fat was measured using an ANKOMXT15 automatic fat analyzer.Dry matter, ash, phosphorus, and calcium contents were determined according to the AOAC procedures . Acid dev/w for 100 g of dried raw material).Air-dried samples were ground and subjected to steam distillation using a Clevenger-type apparatus for 3 h (100 g sample:1000 mL water). The isolated EOs were dried over anhydrous sodium sulfate and stored at 4 °C in an amber bottle before gas chromatography-mass spectrometry (GC-MS) analysis. The EO yields were estimated based on the dry weight of the plant materials  17 library spectra and the retention index (RI) [The EOs were filtered and diluted in n-hexane at a ratio of 1:200. These samples were then detected via GC-MS  analysis with an HP-5MS column . The injector temperature was 250 °C. The oven program was conducted as follows: 1 μL of the sample was injected in a 100:1 split mode. The temperature was maintained at 40 °C for 2 min, and the linear ramp reached 152 °C at a rate of 4 °C·minp values < 0.05 were considered significant. Heatmaps, Spearman’s rank correlation analysis (p < 0.05) results, UpSet plots, Venn diagrams, and principal component analysis (PCA) plots were obtained using the R platform. To further distinguish the differences and relationships among EOs, supervised statistical data treatment was performed using OPLS-DA by SIMCA .Variance analysis was performed to evaluate the significance of differences using SPSS software  and results with As shown in p < 0.05; Species-related factors and different parts of the plants had significant effects on the nutrient composition and total energy parameters  between the varieties and parts. These values ranged from 0.04–3.90% v/w (dry matter). The lowest EO yields were obtained in the stalks and roots (<0.2% v/w), and the highest EO yield was obtained in the fruits (≥2% v/w). The EO yields of fruits from Fd, Fh, and Ag were 2% v/w, 2.9% v/w, and 3.9% v/w, respectively. The EO yield of Fhf was similar to that of a previous study about fennel fruit (~3% v/w) [v/w) [EOs can be extracted from all parts of Fd, Fh, and Ag. The evaluation of EO yields, shown in ~3% v/w) . Meanwhiw) [v/w) . Umbels In the Apiaceae family, the accumulation of EOs is delimited to specialized structures located along with the vegetative and reproductive organs of plants, known as secretory ducts (or oil ducts) and vittae, respectively . In the In this study, twenty-six compounds were identified, accounting for 97.71–99.63% of the total EO components, and the composition of the EOs varied greatly among parts and species. Heatmap analysis further visualized the differences in detail A. Our reMoreover, an UpSet plot was obtained to visualize the distributions of shared and unique components of different parts and species A. The reTo further classify the EOs in the fruits, umbels, stalks, and roots from the three species , two PCA plots were constructed based on the species and different parts, respectively A,B. OverAccording to the dendrogram analysis results C, a supeCuminum cyminum L. [Daucus muricatus L. [Thapsia villosa L. [Carpesium cernuum EOs, whereas only 37 compounds were found in the roots, which is consistent with previous studies. The proportions of the main compounds also vary greatly; for example, α-pinene (35%) and 2,5-dimethoxy-p-cymene (12%) were the predominant compounds in the EOs of shoots, whereas 2,5-dimethoxy-p-cymene (55%) and thymol isobutyrate (9%) were predominant in the EOs of roots [In addition, EO chemical profiles may vary substantially between parts of the same plant species . In partminum L. , Daucus catus L. , and Thallosa L. . Notablyof roots .In conclusion, this study provides fundamental information regarding the nutritional composition and EO profiles of fennel and dill. Multivariate statistical analysis showed that EOs extracted from root parts were significantly different from those obtained from the other parts. Some potential chemical markers  were identified for distinguishing the arterial parts  and roots of fennel and dill. Anethole, estragole, and carvone were identified as vital chemical markers for differentiating the plant species Fa, Fd, and Ag. These results provide a new understanding of the chemical characteristics of different parts of fennel and dill, which is significant for the efficient utilization of fennel and dill as food additives. In the future, to fully utilize the medical and nutritional properties of fennel and dill, the determination of specific chemical compositions from different parts and species is essential. Accordingly, we suggest that fruits are a good source of EOs, and also can be a source of further compounds with food additive relevance, of which the biological activity is worth further exploration. Umbels act as an important by-product of fennel and dill, whose EOs and minerals should be efficiently utilized and worth exploring. Furthermore, stalks have a large biomass and can be used as feed additives, whose nutritional compositions should be further explored. Taken together, the consumption of umbels and stalks would also reduce the amount of waste produced annually from the global food industry.


Levels of overall titin phosphorylation did not differ in the four experimental groups. Maximal Ca2+-activated force (Fmax) of LV and RV cardiomyocytes were preserved in ZDF animals. Ca2+-sensitivity of isometric force production (pCa50) was significantly higher in LV (but not in RV) cardiomyocytes of ZDF rats than in their counterparts in the Lean or Lean + Vard groups. In accordance, the phosphorylation levels of cardiac troponin I (cTnI) and myosin binding protein-C (cMyBP-C) were lower in LV (but not in RV) cardiomyocytes of ZDF animals than in their counterparts of the Lean or Lean + Vard groups. Vardenafil treatment normalized pCa50 values in LV cardiomyocytes, and it decreased pCa50 below control levels in RV cardiomyocytes in the ZDF + Vard group. Our data illustrate partially overlapping myofilament protein alterations for LV and RV cardiomyocytes in diabetic rat hearts upon long-term PDE-5A inhibition. While uniform patterns in cGMP, 3-NT and Fpassive levels predict identical effects of vardenafil therapy for the diastolic function in both ventricles, the uneven cTnI, cMyBP-C phosphorylation levels and pCa50 values implicate different responses for the systolic function.Heart failure with preserved ejection fraction (HFpEF) and right ventricular (RV) dysfunction are frequent complications of diabetic cardiomyopathy. Here we aimed to characterize RV and left ventricular (LV) remodeling and its prevention by vardenafil (a long-acting phosphodiesterase-5A (PDE-5A) inhibitor) administration in a diabetic HFpEF model. Zucker Diabetic Fatty (ZDF) and control, ZDF Lean (Lean) male rats received 10 mg/kg vardenafil  per os, on a daily basis for a period of 25 weeks. In vitro force measurements, biochemical and histochemical assays were employed to assess cardiomyocyte function and signaling. Vardenafil treatment increased cyclic guanosine monophosphate (cGMP) levels and decreased 3-nitrotyrosine (3-NT) levels in the left and right ventricles of ZDF animals, but not in Lean animals. Cardiomyocyte passive tension (F The prevalence of metabolic disorders such as obesity and type 2 diabetes mellitus (T2DM) and the burden of heart failure (HF) increase worldwide . T2DM isWorsening right ventricular (RV) performance is a common, and poor prognostic feature of HFpEF with a hypothetical RV cardiomyocyte dysfunction ,5. MoreoNitro-oxidative stress is thought to downregulate the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathway in metabolic models of HFpEF ,12. OxidHere we aimed to compare the functions and compositions of LV and RV cardiomyocytes in a diabetic rat model of HFpEF. We found mechanical and molecular differences between RV and LV cardiomyocytes, as well as distinct responses for long-term PDE-5A inhibitory therapy.All animal care and experimental procedures involved in this work conformed strictly to the EU Directive 2010/63/EU of the European Parliament and the Guide for the Care and Use of Laboratory Animals used by the US National Institutes of Health . The experimental protocol was approved by the institutional ethics committee of the Semmelweis University (permission number: PE/EA/418-4/2020).n = 8), vardenafil-treated controls , vehicle-treated diabetic , and vardenafil-treated diabetic rats  [ad libitum. Daily per os drug treatment [10 mg/kg body weight (BW) vardenafil dissolved in 0.01 M/L citrate buffer] or vehicle (0.01 M/L citrate buffer) administration via drinking water was initiated at the age of 7 weeks and continued until the end of the experimental period. Functional and biochemical measurements were performed. Hearts were excised, the left (LV) and right ventricle (RV) were dissected in cold saline solution on ice, placed in sterile cryovials, and immediately shock-frozen in liquid nitrogen at the age of 32 weeks. LV and RV tissue samples were thereafter stored at −80 °C.Male 7-week-old Zucker diabetic fatty (ZDF) rats were studied. ZDF diabetic (fa/fa) and control Lean (+/?) rats  were randomized into four groups: vehicle-treated controls  . Animals2, 100 mM KCl, 2 mM EGTA, 4 mM ATP, 10 mM imidazole; pH 7.0, 0.5 mM phenylmethylsulfonyl fluoride, 40 μM leupeptin and 10 μM E-64, all from Sigma-Aldrich, St. Louis, MO, USA) using a tissue homogenizer at 4 °C. Mechanically isolated cardiomyocytes were then subjected to incubation in ISO completed with 0.5% (v/v) Triton X-100 detergent  for 5 min, which eliminated all membrane structures. Isolated and permeabilized single cardiomyocytes were attached with silicone adhesive  between two stainless insect needles, linked to a very sensitive force transducer  and an electromagnetic motor  in ISO at 15 °C. After adjustment of the sarcomere length to 2.3 μm, force measurements were performed by transferring the cells from relaxing solution -2-aminoethanesulfonic acid, 37.11 mM KCl, 6.41 mM MgCl2, 7 mM EGTA, 6.94 mM ATP, 15 mM creatine phosphate; pH 7.2 all from Sigma-Aldrich, St. Louis, MO, USA) to activating solution  in the presence of protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, 40 μM leupeptin and 10 μM E-64. Ca2+ concentrations were expressed in pCa units calculated as follows: -lg[Ca2+]. The pCa of the activating and relaxing solutions was 4.75 and 9.0, respectively. Solutions with intermediate [Ca2+] were prepared by mixing relaxing and activating solutions (pCa 5.4–7.0). Ca2+-activated force (Factive) values at intermediate [Ca2+] were normalized to maximal Ca2+-activated force  and then fitted to a modified Hill-equation , generating a sigmoid curve to define the Ca2+-sensitivity of the contractile system (pCa50). Ca2+-independent passive tension (Fpassive) of isolated cardiomyocytes was measured by shortening the cells to 80% of their initial lengths for 8 s in relaxing solution after a quick release–restretch maneuver (30 ms). Original forces of every individual cell were normalized to cardiomyocyte cross sectional-area, calculated by the width and height of the corresponding cardiomyocyte. Force values are expressed in kN/m2.Isometric force measurements were performed as previously described . Deep-frw/v) sodium dodecyl sulfate (SDS), 75 mM DTT, 50 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, bromophenol blue, 40 µM leupeptin and 10 µM E-64) for 45 min. Protein amounts of supernatants were determined after centrifugation , using a dot-blot technique with bovine serum albumin (BSA) standard and the concentration of samples was adjusted to a final concentration of 2 mg/mL. Agarose-strengthened 2% SDS-polyacrilamide gels were used to separate N2B titin. Total phosphorylation status of titin protein was assessed by Pro-Q® Diamond phosphoprotein staining  according to the manufacturer’s protocol, while total amount of protein was stained by Coomassie blue .Frozen LV and RV samples were thawed and permeabilized as described above. Myocardial samples were thereafter homogenized in sample buffer  Tween 20 (PBST) for 30 min. Next, cTnI phosphorylation-sensitive antibodies were used to determine the levels of protein phosphorylation at protein kinase A (PKA)- and protein kinase C (PKC)-specific cTnI (Ser-23/24 (1:1000), Ser-43 (1:500) and Thr-143 (1:500), Abcam, Cambridge, UK) and PKA-specific cMyBP-C Ser-282  phosphorylation sites. The signal was detected with a peroxidase-conjugated anti-rabbit IgG secondary antibody  on nitrocellulose membranes. Total amount of proteins was visualized with super sensitive membrane stain . Signals were detected with an MF-ChemiBIS 3.2 gel documentation system . Enhanced chemiluminescence (ECL) signals of site-specific phosphorylation of cTnI and cMyBP-C were normalized to corresponding Western immunoblot stains. Samples were typically loaded at least in duplicates (from n = 4–6 different rat hearts for each group) during at least 4 independent runs for a given protein assay. All original scans and the number of Western immunoblot observations  and cardiac myosin binding protein-C (cMyBP-C) was carried out in 4% and 12% polyacrilamide gels, respectively. After polyacrilamide gel electrophoresis and protein blotting procedure, membranes were blocked with 2% BSA diluted in PBS containing 0.1%  were deparaffinized and stained for 3-nitrotyrosine (3-NT) or cGMP in immunohistochemistry assays according to previous examinations ,20.Immunohistochemistry for 3-NT was performed to assess the presence of nitro-oxidative stress in our model groups. After deparaffinization and blocking, LV and RV sections were incubated in the presence of 3-NT primary antibody  at 4 °C overnight, and thereafter secondary antibodies were applied  for 30 min at room temperature. Black colored nickel-cobalt-enhanced 3,3’-diaminobenzidine  was used for the development.For the determination of myocardial cGMP levels, LV and RV sections were deparaffinized and following antigen retrieval (in citric acid buffer) and blocking, sections were probed with an anti-cGMP antibody . After washing, slides were incubated in the presence of secondary anti-rabbit antibody  and developed using Fast Red .Images of five identical areas of each section  were taken using a light microscope at 200× (3-NT) or 400× (cGMP) magnifications . 3-NT and cGMP positive areas were determined by color (dark grey and red) thresholding with the ImageJ software  by an independent investigator. The percentages of positively stained tissue areas to total areas  were calculated.2+-evoked contractures of myocyte-sized preparations were recorded with a custom-built LABVIEW Data Acquisition platform and analyzed with LabVIEW analyzing software . Signal intensities of protein bands were quantified by using the ImageJ image processing program  and Magic Plot  software. Statistical significance was tested by analysis of variance (ANOVA followed by Bonferroni’s post hoc test) by GraphPad Prism 6.0  software to evaluate the results. Values are given as mean ± SEM. Statistical significance was accepted at p < 0.05.CacGMP levels and the extent of nitro-oxidative stress were tested by immunohistochemistry. cGMP specific staining intensity was less in both LV and RV tissue samples of ZDF hearts than in those of the Lean or Lean + Vard groups A,B. Convpassive was significantly higher in both LV and RV cardiomyocytes of the untreated ZDF group than in those of the control groups. Additionally, the increase in Fpassive seen in the ZDF group was prevented by facilitating the cGMP-PKG pathway by PDE-5A inhibition, while vardenafil was without effects in both LV and RV cardiomyocytes in the Lean group  was tested in permeabilized LV and RV cardiomyocytes at different Ca2+ concentrations  was similar in all groups for both ventricles, independent of disease or vardenafil treatment (2+-sensitivity of isometric force production (pCa50) of LV cardiomyocytes was significantly higher in ZDF rats than in Lean animals, and this difference was reverted by vardenafil treatment  of left and right ventricular cardiomyocytes of ZDF animals. Distinct myofilament protein phosphorylation patterns of Lean and ZDF hearts in the absence and presence of chronic preventive PDE-5A inhibition helped to explain these differences.Results of this study demonstrate uniform increases in FThe RV is thought to be more vulnerable to hemodynamic and nitro-oxidative stress than the LV during the progression of heart failure . Neverthpassive of endomyocardial biopsies of HFpEF patients or ZDF rats provides a plausible link between cardiomyocyte mechanics and LV diastolic dysfunction [A characteristic elevation in cardiomyocyte Ffunction ,23. Thisfunction ,24. In vfunction  or PKG [function  increasefunction . Howeverfunction . Alternafunction .max was not affected in LV or RV cardiomyocytes of ZDF rats, although T2DM has been formerly associated with RV systolic dysfunction in HFpEF [50 was increased in LV cardiomyocytes, but it remained unchanged in RV cardiomyocytes in ZDF animals. Nevertheless, unlike in Lean controls, pCa50 was effectively decreased in cardiomyocytes of both ventricles by vardenafil treatments in ZDF rats. Similarly, LV and RV cGMP levels were elevated by long-term vardenafil treatment in ZDF animals, but not in Lean controls. These data suggest a more significant role for PDE-5A in the presence of diabetic cardiomyopathy than in its absence, and thus they are consistent with an elevated myocardial expression of PDE-5E in both ventricles during oxidative stress and heart failure [Fin HFpEF ,29. pCa5 failure .50 is known to be regulated by myofilament protein  phosphorylation at multiple phosphosites. For example, hypophosphorylation of cTnI at the Ser-22/23 sites results in an increase in pCa50 in human systolic HF [50, which was associated with hypophosphorylation of cTnI at Ser-22/23. The Ser-22/23 residues of cTnI can be targeted by PKA or PKG [max. Former studies demonstrated that PKCα-mediated hyperphosphorylation of cTnI at Ser-43/45 leads to a reduction in Fmax and pCa50 of LV cardiomyocytes from failing hearts of rodents [max or pCa50 [2O2 as a mimic of oxidative stress also affected PKC-mediated myofilament phosphorylation and Ca2+-sensitivity of force production in the rat [pCatolic HF . LikewisA or PKG . In thisA or PKG . As a maA or PKG . In cont rodents ,35 and h rodents . In contor pCa50 . An inteor pCa50 . Further the rat . While t2+/calmodulin-activated kinase II (CaMKII) [Phosphorylation of cMyBP-C was shown to be cardioprotective against ischemic injury , and pho(CaMKII) , being d(CaMKII) . Here we(CaMKII) . Interes50 and myofilament phosphorylation data implicated distinct pharmacological responses and potentially divergent consequences on the systolic functions of the RV and the LV by long-lasting PDE-5A inhibition. Conversely, our data also suggested that boosting the cGMP-PKG signaling pathway might prevent LV and RV diastolic dysfunction in a uniform fashion during diabetic cardiomyopathy and/or HFpEF.In previous studies, our group reported cell-to-cell , regiona


Ice formation on the aerodynamic surfaces of an aircraft is regarded as a major problem in the aerospace industry. Ice accumulation may damage parts, sensors and controllers and alter the aerodynamics of the airplane, leading to a range of undesired consequences, including flight delays, emergency landings, damaged parts and increased energy consumption. There are various approaches to reducing ice accretion, one of them being the application of icephobic coatings. In this work, commercially available polyurethane-based coatings were modified and deposited on NACA 0012 aircraft airfoils. A hybrid modification of polyurethane (PUR) topcoats was adopted by the addition of nanosilica and three-functional spherosilicates (a variety of silsesqioxane compound), which owe their unique properties to the presence of three different groups. The ice accretion on the manufactured nanocomposites was determined in an icing wind tunnel. The tests were performed under three different icing conditions: glaze ice, rime ice and mixed ice. Furthermore, the surface topography and wetting behavior (static contact angle and contact angle hysteresis) were investigated. It was found that the anti-icing properties of polyurethane nanocomposite coatings strongly depend on the icing conditions under which they are tested. Moreover, the addition of nanosilica and spherosilicates enabled the reduction of accreted ice by 65% in comparison to the neat topcoat. In various industries, such as aerospace, telecommunication, automotive or wind power plants, implemented materials are subjected to not only mechanical loads but also adverse weather conditions. One of the most important problems in aviation is ice formation on the aerodynamic surfaces, which may cause major issues, such as the alternation of the aerodynamics of an aircraft, jamming, damage of parts and sensors and even, in the case of thick ice layers, premature stall ,2,3. TheThere are various methods of dealing with the icing problem, including mechanical or chemical ice removal as well as thermal and electrical deicing systems ,8,9,10. 2, TiO2, ZrO2) together with a thin layer of highly hydrophobic components , etc.) rendered the surface superhydrophobic n, where R may be a hydrogen atom, an alkyl group, an aryl group and their derivatives. The character of this group is interesting due to the possibility of obtaining new, unique properties through various modification procedures and reactive or non-reactive substituents at the silicon atom located in the corner of the cage. Silsesquioxanes are hybrid compounds that combine the features of inorganic (siloxane core of silsesquioxanes) and organic  compounds. These reagents owe their popularity to a growing number of applications in various industries, mainly due to good thermal and mechanical properties, dispersion properties as well as the ease of their further functionalization towards desirable rheological or solubility properties octasiloxane (POSS14).−5 eq. of Karstedt catalyst. Reaction mixture was heated in reflux and stirred until the full conversion of Si–H was detected by FT-IR and 1H NMR.Vinyltrimethoxysilane (0.049 mol), hexene (0.074 mol) and allyl-OFP (0.074 mol) in molar ratio 2:3:3 were added to the solution of octaspherosilicate  in toluene. The mixture was constantly stirred and heated to 60–70 °C, then added to the system in an amount that varied from 8 × 10® Pilot XIII air spray gun  and left to dry for 3 h. Meanwhile, PUR topcoats with 3 wt % content of nano-silica, POSS14 (5 wt %) and DC88 (5 wt %) were prepared by mixing the liquid additive with PUR using a magnetic stirrer. Before spraying, nano-silica suspensions on the modified PUR coating were prepared by means of an ultrasonic gun  for 30 min. The suspensions were then sprayed on top of the already applied primer and left to dry for 24 h. The different compositions prepared are shown in The 2024 aluminum alloy NACA0012 profiles were first cleaned in acetone. Initially, both primer components were mixed according to the manufacturer’s indications, then sprayed on Al profiles by means of a Walther1H and 13C{1H}, NMR spectra were recorded on a Bruker Ultrashield 300 MHz operating at 300 and 75 MHz, respectively. 29Si{1H} NMR spectra were recorded on a Bruker Ascend 400 MHz Nanobay operating at 79 MHz. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet iS50 Fourier transform spectrophotometer  equipped with a diamond ATR unit with a resolution of 0.09 cm−1. The spectra were collected in the 400–4000 cm−1 range; 16 scans were collected for each spectrum.For the characterization of in-laboratory synthesized POSS-based compounds ® CG204  coating thickness tester at least six times across the sample using the eddy current measurement mode.The coating thickness was measured by an ExtechWettability properties (static contact angle (SCA), advancing contact angle (ACA), receding contact angle (RCA), sliding angle (SA)) were measured by a contact angle measurement system . All angles of each sample were measured at least three times across the sample surface using the sessile drop method by dispensing 3 µL (SCA), 5 µL  and 10 µL (SA) of deionized water on the sample’s surface. The contact angle hysteresis (CAH) was calculated as the difference between ACA and RCA.a and Sz, both of which are defined in detail by Zhang et al. [Surface roughness was measured by means of a laser scanning confocal microscope . The test standard was ISO-25178. All the measurements were acquired on a 1550 µm × 1550 µm area with 0.67 µm spacing. Three different randomly selected areas were measured on each sample. The obtained parameters were Sg et al. .3.Ice accretion tests were performed by the use of icing wind tunnel . The iciIce reduction was calculated from the obtained ice accretion values using formula:−1, respectively, due to the stretching and bending of the Si-H group). For all of the compounds, the hydrosilylation proceeded with the completion of the reactions (~99%). The structure and purity of the modifiers were confirmed by NMR analysis. It was observed that the hydrosilylation of olefns proceeded selectively to the β-isomer, except for trimethoxysililethyl, where the formation of α-isormes was also observed in a proportion of ~21% in all of the cases. Due to the applied synthesis procedure, the distribution of the functional groups in the obtained compound was statistically complex. We obtained a mixture of compounds from the group distribution maximum for the structure in POSS-based compounds were prepared according to the synthesis procedure described in α and β group in a 21:79 ratio.The selectivity of the reaction, determined by NMR spectroscopy, showed that the product contained a mixture of trimethoxysililethyl 1H NMR : δ (ppm) = 6.19 − 5.93 , 3.90 , 3.58–3.53 , 1.68–1.61 , 1.30–1.27 , 1.13 , 0.89–0.86 , 0.60 , 0.15, 0.14, 0.12 ;13C NMR : δ (ppm) = 107.78 , 75.77 (O-2CH-CH2-CH2-Si), 67.64 , 50.68 (OMe), 33.16, 31.73 (hexyl), 23.16 (O-CH2-2CH-CH2-Si), 23.02, 22.74, 17.77, 14.25 (hexyl), 13.52 (O-CH2-CH2-C2H-Si), 8.60 (Si-2CH2CH-Si), 7.37, 5.27 (Si3)CH (CHSi), 0.42 (Si-2CH2CH-Si), 0.22 (hexyl SiMe2), −0.38 (fluorinated group SiMe2), −1.05 (trimethoxysilylethyl group SiMe2);29Si NMR : δ (ppm) = 13.37 − 12.73 (SiMe2), −41.61 (Si (OMe)3), −108.90–(−109.07) (core);19F NMR : δ (ppm) = −119.90, −125.90, −130.57, −137.46, −137.46 .The coating thickness was measured as described in a previous paper . The resIt can be seen that chemical modification increases the roughness of the material. The difference is quite small for Sample 5, whereas it is the most apparent for Sample 2. Sample 4 exhibits low values of Looking at the standard deviation of the measurements, it seems that the average height was similar in different areas of the samples; nevertheless, there are quite significant differences in the values of maximum height. To visualize the presented results, selected profiles for each sample are shown in It should be noted that, in the profiles shown in The wettability parameters of the samples are presented in The contact angle of the reference sample is a bit lower than All of the chemically modified samples are characterized by lower contact angle hysteresis values than the reference sample, which means they are more hydrophobic. Once again, the hybrid modifications resulted in the best outcomes. Furthermore, the values of the sliding angle are also the lowest for Samples 2 and 6, In 2 + DC88 (Sample 2), as the ice reduction was equal to 65%. However, it should be noted that all of the chemical modifications led to a significant reduction in ice accretion, over 55%.In the highest temperature, 2 + DC88 brought in effects similar to each other, around 30% ice reduction.In the middle temperature, 2 and POSS14 were added (Sample 6) as there was a drop of over 40%. The other modifications resulted in a decrease of approximately 30%, with the worst result for Sample 3.In the lowest temperature, Comparing ice reduction in different temperatures, chemically modified coatings exhibit the best anti-icing properties when glaze ice is accreted. It is the most challenging to design a coating that would prevent the formation of mixed ice as three out of five samples showed the lowest ice reduction in In 2 + DC88 and SiO2 + POSS14, are the most hydrophobic ones. They are quite rough. However, the Sa value of Sample 3, modified with only SiO2, is similar to that of Sample 6, yet its contact angle is similar to Samples 4 and 5, which are characterized by smooth surfaces. The possible explanation is that hierarchical structures were achieved for Samples 2 and 6, which resulted in better hydrophobic characteristics.The samples with hybrid modifications, SiOIt could be observed in the Results part of this paper that the coatings that achieved the best results in preventing ice accretion of one kind of ice did not necessarily achieve the best outcomes for the other types. Therefore, the rest of this section is divided into three parts in order to discuss in detail the relationship between the tested parameters for glaze, rime and mixed ice.In The highest ice reduction was obtained for Sample 2, which is the roughest one. It also exhibits the second highest contact angle. There seems to be no clear pattern in the aspect of increasing ice reduction by the means of roughness as the values above 62% are achieved by Samples 2, 3 and 4, which are characterized by the highest, medium and lowest values of The influence of roughness and wettability on mixed ice accretion is presented in Sample 6 is represented by the highest ice reduction, medium roughness and highest contact angle. Once again, the influence of roughness on ice accretion is not clear. Nonetheless, there is a clear correlation between the contact angle and ice reduction values. The conclusion may be drawn that the more hydrophobic the coating is, the more icephobic behavior it exhibits. The dependence is not linear as there is a significant decrease in ice reduction for Sample 3 compared to Sample 4, yet the difference in their contact angle is quite small. Having that said, there is clearly a trend.The anti-icing behavior of coatings in In the case of rime ice accretion, the most hydrophobic Sample 6 once again exhibits the best anti-icing properties. Moreover, the least hydrophobic Sample 3 is the one with the lowest ice reduction. The wettability of Samples 4 and 5 is similar, and so is their ice reduction value. Both characteristics of Samples 4 and 5 are lower than those of Sample 3. However, the relationship between contact angle and anti-icing cannot be stated as in the case of mixed ice due to the fact that Sample 2 is the second most hydrophobic one yet the second least icephobic. There is no clear correlation between ice accretion and the roughness of the samples.The fact that there was no correlation found between the roughness of the surfaces and ice accretion in any temperature may lead to a conclusion, presented in Part 4.1. of this paper, that some of the samples (namely 2 and 6) may have hierarchical structures formed, while the others do not have such structures.2 + DC88 and SiO2 + POSS14 were proven to perform better in terms of ice reduction than coatings with only one modifier. In higher temperatures  and the effects associated with the introduction of perfluorinated functional groups. The POSS14 additive used in the publication differs from the commercial DOWSIL 88 due to the fact that, in one molecule, we have both functional groups that bind chemically to the SiO2 surface (methoxide groups) as well as surface energy modifiers in the form of alkyl and perfluorinated groups. The variation of wettability in terms of the roughness of the samples may lead to the conclusion that a hierarchical structure was formed in the case of the hybrid modifications.Ice reduction of up to 65% was achieved by the means of the chemical modification of polyurethane coatings. Coatings with the hybrid modifications SiO


Drosophila. The ancestor of Bcd (AncBcd) emerged after a duplication of a Zerknullt (Zen)-like ancestral protein (AncZB) in a suborder of flies. AncBcd diverged from AncZB, gaining novel transcriptional and translational activities. We focus on the evolution of the HD of AncBcd, which binds to DNA and RNA, and is comprised of four subdomains: an N-terminal arm (NT) and three helices; H1, H2, and Recognition Helix (RH). Using chimeras of subdomains and gene rescue assays in Drosophila, we show that robust patterning activity of the Bcd HD (high frequency rescue to adulthood) is achieved only when amino acid substitutions in three separate subdomains  are combined. Other combinations of subdomains also yield full rescue, but with lower penetrance, suggesting alternative suboptimal activities. Our results suggest a multistep pathway for the evolution of the Bcd HD that involved intermediate HD sequences with suboptimal activities, which constrained and enabled further evolutionary changes. They also demonstrate critical epistatic forces that contribute to the robust function of a DNA-binding domain.Changes in regulatory networks generate materials for evolution to create phenotypic diversity. For transcription networks, multiple studies have shown that alterations in binding sites of cis-regulatory elements correlate well with the gain or loss of specific features of the body plan. Less is known about alterations in the amino acid sequences of the transcription factors (TFs) that bind these elements. Here we study the evolution of Bicoid (Bcd), a homeodomain (HD) protein that is critical for anterior embryo patterning in  The main components of transcription networks are transcription factors (TFs) and the cis-regulatory elements of target genes that contain TF-binding sites. During evolution, DNA sequence changes in either component can alter network topology, affect gene expression patterns, and ultimately induce functional changes that are selected by evolutionary pressures . A sequeOne issue with the TF evolution hypothesis is that changes in TF function that generate new functions might interfere with critical roles normally played by the TF. However, this issue can be mitigated by gene duplication events, which provide extra genetic material for the evolution of novel or modified functions . For exaDrosophila embryo. The ancestor of Drosophila bcd gene (ancbcd) emerged ~150 Ma in Cyclorrhaphan flies (a suborder of the Diptera [two-winged flies]) after a duplication of an ancestral gene (anczb), which also gave rise to the ancestor of bcd’s sister gene zerknullt (zen) (anczen) , and prevents its translation in anterior embryonic regions , a homeodomain (HD)-containing transcription factor that is critical for patterning anterior regions of the (anczen) . In Drosf AncBcd  and allof AncBcd . In the f AncBcd  and is ef AncBcd . Most Bcf AncBcd . Bcd als regions , 2002. E regions .Drosophila have completely different functions in vivo. Specifically, when expressed in a Bcd-like gradient in embryos lacking Bcd, Zen has no Bcd-like activity (The Zen and Bcd proteins in activity . Howeveractivity . These rK and m54>R) because these had previously been shown to be critical for Bcd’s DNA-binding specificity . In contrast, combining substitutions in the RH with those in H1 or NT alone generates suboptimal HDs with lower survival rates. The distributions of larval phenotypes between these genotypes suggest different mechanisms for generating similar morphologies. Taken together, these results also suggest a multistep pathway to explain the evolutionary transition from a nonfunctional AncZB HD to an AncBcd HD with robust in vivo function through alternative pathways.A and B, K and m54>R, are present in the RH subdomain, which directly contacts base pairs in the major groove of DNA  as a percentage of embryo length . In embryos carrying the AncZB_RHdiag embryos, the average position was at 82% EL   M–P. The  and btd P, which  : 1) WTL (wild-type like): larvae with easily detectable head structures , three thoracic segments, and eight abdominal segments. 2) ΔHead: larvae missing any of the five head structures mentioned above. 3) ΔHead+Abdomen: larvae with head defects and additional defects in abdominal segments. 4) ΔAbdomen: larvae with normal head structures, but with defects in abdominal segments. As positive controls, we assayed the rescue activities of transgenes containing the wild-type Alldiag), and tested the construct for rescue activity. These substitutions in multiple subdomains showed partial patterning activity, with more than 90% of larvae forming all three thoracic segments and at least one of the head structures mentioned above , CH(nt-H1-h2-RH), and CH(nt-h1-H2-RH)]. Transgenes containing two of these chimeric HDs strongly increased rescue activity compared with the transgene containing all changes in the RH alone (AncZB_RH)  transgene were classified as WTL  transgene, there was also a strong increase in rescue activity, but less than 40% were classified as WTL, with the rest evenly distributed among the other three phenotypic categories  rescue transgene  in the RH with those in each of the other subdomains [CH and CH(nt-H1-h2-RH)] directed the survival of 3% and 7% of those larvae to adulthood, respectively  to monitor the frequency of survival past larval stages for the experiments that produced WTL larvae J. As a bectively J. Althoubcd females containing the CH(NT-H1-h2-RH) were classified as WTL  in embryos carrying the AncZB_Alldiag construct was located at 70% EL, relatively far from the boundary position in embryos rescued by the AncBcd HD (54% EL)  that range from 68% to 63% EL  chimera, which completely failed to fully rescue  P. Thus, y rescue J, activat 67% EL D. Therefotd, btd, gt, and the pair-rule gene eve  chimera (B). We also observed a strong correlation between fully and partially rescuing chimeric lines and the positioning of eve stripe 1. Embryos carrying the three transgenes that fully rescue formed eve stripe 1 at 75–76% EL (nt-h1-H2-RH) and AncZB_Alldiag transgenes (both 0% full rescue) consistently formed this stripe more anteriorly  among the three constructs that fully rescue. Notably, the CH(NT-H1-h2-RH) transgene, which combines forward substitutions in three subdomains and rescues 40% of bcd mutant embryos to adulthood activated hb expression with a pbp at 63% (NT-h1-h2-RH) or CH(nt-H1-h2-RH) transgenes  and the AncBcd (full function) HDs, we showed that the substitutions in at least three separate subdomains  must be combined for full patterning activity.In this article, we used an in vivo RH). However, by combining the RH substitutions with several different sets of substitution in the NT and/or H1 or substituting all diagnostic residues across all subdomains, variable levels of suppression were achieved  increased that number to five . In this stripes P. Moreovhb, which encodes a critical cofactor for activation of all Bcd-dependent target genes  or farther posterior. Interestingly, the CH(NT-H1-h2-RH) construct, which rescues to adulthood with a frequency similar to that observed for the AncBcd HD control, forms a hb pbp at 63%. We propose that the position of ∼65% EL establishes the minimal amount of embryonic space required for the correct placement of gap and pair-rule stripes, robust formation of cephalic structures, and ultimately survival to adulthood. The pbp at 65% EL is significantly more anterior than those directed by the control AncBcd construct , which survive with high penetrance , another K50 HD protein. Unlike Bcd, Otd is highly conserved, and because it binds in vitro to DNA sequences similar to those bound by Bcd, it has been proposed that Bcd evolved to take over regulation of an ancestral Otd-dependent network  have been proposed as important maternal factors involved in anterior embryo patterning . It seems impossible that critical amino acid substitutions in all three subdomains occurred simultaneously at some point in the evolution of the AncBcd HD. However, we propose that critical substitutions in each of the three might have occurred in a specific temporal order, each of which endowed the protein with a novel property that could be positively selected in evolving flies .RHdiag) reduced HD activity compared with the K50R54 double substitution. This result suggests that interactions between amino acids constrained the historical order of substitutions in the RH subdomain.Assuming that the ancestral network was controlled by a K50 HD protein such as Otd , we propAlthough robust HD activity requires substitutions in three subdomains, forward substitutions in either NT or H1 substantially augment the rescue activity generated by changes in the RH alone. Specifically, AncZB HDs containing either combination (RH+NT or RH+H1) rescue a small percentage of embryos that survive to adulthood J. We proDrosophila Bcd protein. As such, these experiments do not take into account the evolution of other parts of the protein, which show even greater levels of amino acid sequence divergence. Further, all our experiments were performed in modern-day Drosophila embryos, and do not take into account changes in the cis-regulatory elements of target genes that coevolved with the AncBcd protein. However, as the genome sequences of more insects become available, it should be possible to use reconstruction strategies to define the ancestral sequences of the complete AncBcd protein and the regulatory regions it interacts with. Furthermore, the ever-increasing use of CRISPR/Cas9 techniques for gene editing in nonmodel organisms should allow for testing ancestral protein and regulatory sequences in multiple insect species. Although these methods cannot create the ancestral systems themselves, they should make it possible to discover general features that permit a transcription factor and its target regulatory sequences to coevolve.Our results shed light on the mechanisms involved in the evolution of the AncBcd HD, but are limited by the fact that all chimeric HDs were inserted into the modern-day Cyo bcd+/Sco; E1bcd/E1bcd, yw; TM3B, Sb, Ser/D and ΦC31 (y+); 38F1 (w+). We cloned an injection plasmid (piattB40-Bcd) containing two inverted ΦC31-specific recombination sequences, a Gmr-GFP reporter, and a polylinker flanked by 1.9-kb bcd promoter and 0.8-kb 3′-UTR. The bcd coding region was amplified by PCR from pBS-SK+ cDNA clones, digested with RsrII and AscI and ligated into piattB40-Bcd in between Bcd promoter and 3′-UTR. This main plasmid was used to generate Dm Bcd protein with different ancestral HDs, which are predicted as described and published in , ished in . We usedished in . Each trhttp://weblogo.berkeley.edu/logo.cgi) (The logo generation platform (ogo.cgi)  was usedogo.cgi)  format aogo.cgi) . The codogo.cgi) .In situ hybridizations were performed as previously described (escribed . BrieflyGuinea pig anti-Cad  (1:400) Cuticle preparations were performed on embryos aged 24–30 h at 25 °C as previously described . BrieflyDark-field views of whole larvae were imaged at 200× magnification; DIC images of cephalic regions are imaged at 400×. Each image was sorted into one of four categories that encompassed the variation in phenotype of first instar larvae both within each transgenic fly line, and across all fly lines analyzed. The different phenotypic categories are; WTL: larvae containing all head segments , ΔHead: larvae missing one or more head segments with normal abdominal and thoracic segments, ΔAbdomen: larvae with variable defects in abdominal segments but normal head and thorax. If an embryo showed both abdominal defects and head defects, it was classified as ΔHead+Abd. The number of WTL, ΔHead, ΔHead+Abd and ΔAbdomen larvae were counted for each transgenic line, tabulated, and graphed as a percentage of the total number of embryos analyzed for that transgenic line.For lines producing WTL larvae, we set up hatching assays to assess survival of these larvae to pupa stage and then adulthood. For hatching assays, females containing rescue constructs were allowed to lay embryos on fruit juice plates for 1 h and then over 100 eggs were picked and incubated at room temperature until pupae formed and adults eclosed. Pupae and adults were counted, and compared with the number of embryos tested for each experiment.hb anterior expression, stained embryos of appropriate ages were imaged at 200× on a Zeiss Axioskop. Briefly, coordinates were established for each embryo so that the x- and y-axes were tangential to the ventral and anterior sides, respectively. A–P positions were displayed as percent of embryo length (EL%) with the anterior pole as 100%. pbps were determined by visual estimation as the distance from the anterior tip to the most posterior position of hb anterior expression and these results were confirmed by ImageJ analyses  (To measure pbps of _003070) .Embryo images were loaded into ImageJ, and a Region of Interest (ROI) that was approximately 35% width of DV length from 95% to 60%  to analyze expression patterns was generated. The width of the ROI was kept constant when imaging all embryos, but the length was varied such that the length of the ROI spanned the length of the whole embryo. For each embryo, an intensity profile plot  was generated for the ROI. The midpoint of the curve that represents the edge of the boundary of expression of target gene was selected as the position of the boundary of gene expression. This numerical position was divided by the total embryo length to normalize the pbps by % EL, and pbps from individual embryos were averaged. 100% EL denotes the anterior tip, and 0% represents the posterior tip of the embryo.eve stripe patterns, stained embryos at nc14 were positioned as described above and imaged at 200×. For each embryo, an intensity profile plot  was generated using ImageJ   (To measure _003070) , and ana3, 2021) .Molecular Biology and Evolution online.msab051_Supplementary_DataClick here for additional data file.