These findings suggest potential correlations between serum E2 concentrations and the phases of oogonial proliferation and pre-vitellogenesis.(1) Fshβ and Lhβ showed stronger signals and higher transcript levels from 590 to 1050 dph than at earlier stages, implying their active involvement during primary oocyte development. (2) Fshβ and Lhβ at lower levels were detected during the phases of ovarian differentiation and oogonial proliferation. (3) E Epinephelus bruneus, is widely distributed throughout East Asia and is highly regarded in the marine fish industry for its superior quality and commercial value [E. bruneus typically reaches maturity within 4 years [The longtooth grouper, al value . This sp 4 years . However 4 years . Recent  4 years . Neverth2), and testosterone (T) exert both positive and negative feedback loops to regulate the brain and play a pivotal role in the neuroendocrine regulation of reproductive behaviour in fish [Gonadal sex differentiation, development, and maturation constitute pivotal physiological processes regulated by the endocrine system, primarily the brain–pituitary–gonadal (BPG) axis, in many fish species, including groupers ,5. Gonad in fish .Odontesthes bonariensis [fshβ and lhβ increased during sex differentiation in the red-spotted grouper, E. akaara [Sparus aurata [Monopterus albus [Conger myriaster [Anguilla japonica [Dicentrarchus labrax [2 and T in the BPG axis of the longtooth grouper. Consequently, technological advancements in the realms of artificial reproduction and seedling production for the longtooth grouper have been impeded. Therefore, in this study, we aimed to ascertain (1) the appearance and localisation of Fshβ- and Lhβ-producing cells in the pituitary gland, (2) the gene expression of gnrh1, fshβ, and lhβ in the brain and pituitary gland, and (3) E2 and T levels in the serum from 53 to 1435 days post-hatching (dph) in the longtooth grouper.Previous studies have identified the presence of Fshβ and Lhβ during various stages of the teleost life cycle. For example, immune signals of Fshβ and Lhβ were detected in the pejerrey, ariensis , and the. akaara . In addis aurata , and theus albus . Fshβ isus albus ,13,14, Jyriaster ; Japanesjaponica ; and Eurs labrax . HoweverE. bruneus were collected on 15 June 2015 and reared in indoor recirculating aquaculture tanks with slow-flowing and aerated sea water. The fish were exposed to a natural water temperature ranging from 2.18 ± 0.21 °C to 27.46 ± 0.37 °C and natural photoperiod conditions , and the dosages of anaesthesia for various teleosts  were usen = 20) via decapitation. However, obtaining both a single gonad and pituitary gland from fish at 53 dph proved challenging due to their small size. Consequently, for these younger specimens, the posterior section of the visceral cavity and the heads of 10 individuals were fixed in Bouin’s solution. The heads of the remaining 10 specimens were fixed using RNAlater reagent . The gonads of fish ranging from 174 to 1435 dph (n = 15) and brains with pituitary glands (n = 6) were fixed in Bouin’s solution for histological analysis. The pituitary glands and brains from another nine specimens were extracted and either fixed in RNAlater reagent or stored at −80 °C. Unfortunately, obtaining blood samples from fish at 53 dph was not feasible due to their small size. Blood from fish between 174 and 1435 dph (n = 15) was collected from the caudal vasculature using a heparinised syringe. Following centrifugation at 1500× g for 15 min, serum samples were collected and stored at −80 °C. All tissues preserved in Bouin’s solution were initially fixed for 24 h and subsequently stored in 70% ethanol at 4 °C.Head tissues were removed from specimens  ,20. In b20, 2022 Fundulus heteroclitus) FSHβ or LHβ, were generously provided by Dr. Shimizu [Concurrently, the sections of the entire brains with pituitary glands were dehydrated and subsequently immersed in a 10 mM citric acid solution  and maintained at 90 °C for 15 min. The primary antisera used, namely, anti-mummichog . The frozen samples were left to defrost in a 2 mL tube at 4 °C for 5 min. An amount of 500 µL of TRIzol® reagent was added to the tube, and the tissues were crushed using a homogeniser. Subsequently, 500 µL TRIzol® Reagent was added and the mixture was centrifuged at 12,000× g and 4 °C for 10 min. The supernatant was transferred to a new 1.5 mL tube. Thereafter, 200 µL of chloroform  was added to the tube, stirred for 5 min, and centrifuged at 12,000× g and 4 °C for 15 min. After centrifugation, 500 µL of isopropanol was added to the supernatant. In addition, 3 µL of ethachinmate and 9.9 µL of sodium acetate  were added, and the mixture was incubated at 4 °C for 10 min. Subsequently, the mixture was centrifuged at 12,000× g and 4 °C for 10 min. An amount of 1 mL of 75% ethanol was added to the resulting supernatant. The mixture was centrifuged at 12,000× g and 4 °C for 5 min, and 30 µL of diethylpyrocarbonate-treated (DEPC) water was added and stirred until the RNA was dissolved. RNA quantification was conducted using a NanoDrop 2000 spectrophotometer . RNA integrity was assessed using gel electrophoresis.For gene detection, total RNA was extracted from the head and the brain or pituitary gland of fish at 53 dph and 174 to 1435 dph, respectively. RNA was extracted using TRIzol18 primer (50 pmol/µL), 2 µL of random hexamer primer (600 pmol/µL), 4 μL of transcriptase reaction buffer (5×), 0.5 µL of RNase inhibitor (40 U/µL), 2 µL of dNTPs (10 mM each), and 0.5 µL of reverse transcriptase (20 U/μL) using a Transcriptor first-strand cDNA synthesis kit . The tube, containing 20 µL of the mixture, was placed in a Gene Amp PCR System 2400 thermal cycler  and reacted as follows: 25 °C for 10 min, 50 °C for 1 h and 85 °C for 5 min. Complementary DNA was used as a template for PCR amplification.In a PCR tube, 1 μg of RNA from the head and brain, or 150 ng from the pituitary gland, was diluted to 10 μL with PCR-grade water. This mixture was reacted with 1 µL of anchored-oligo(dT)PCR amplification products were subjected to electrophoresis using a 1.5% agarose gel. Initially, 0.75 g of Agarose S  was added to 50 mL of TBE buffer (1×) within a 100 mL beaker. This mixture was briefly heated in a microwave oven for 30 to 60 s. The resulting melted agarose mixture was then carefully poured into a Gel Maker Set , following which the comb was inserted and left for 1 h for solidification of the gel. The gel was removed and placed in a mini gel tank . TBE buffer (1×) was poured into the tank, and the gel was submerged within it. For the cDNA amplification, 10 µL reaction mixtures were prepared, each containing 1 µL of cDNA, 1 µL of forward and reverse primers for each gene, 1 µL of dNTPs (2.5 mM), 1 µL of buffer (10×), 5 µL of PCR-grade water, and 0.05 µL of Advantage 2 polymerase . Additionally, 5 µL of Gene Ladder 100  was added to serve as a size reference. Electrophoresis was conducted at 100 V for a duration of 40 min. The gel was then immersed in a 500 µg/mL solution of EtBr and gently shaken on an oscillating machine  for 30 min. Subsequently, the gel was briefly rinsed via immersion in distilled water for 10 s, and then, placed in a gel projector  to assess the desired products. The bands of gel containing the products of interest were cut, weighed, and stored in a 1.5 mL tube.g and 4 °C for 1 min. The substrate liquid was disposed of. An amount of 500 µL of new QG buffer was added to the spin column and centrifuged at 12,000× g and 4 °C for 1 min. Thereafter, the substrate liquid was discarded, and 750 µL of PE buffer was added and incubated at 4 °C for 5 min, and centrifuged at 12,000× g and 4 °C for 1 min. The substrate liquid in the collection tube was discarded. To remove the residual liquid in the spin column, this collection tube was centrifuged at 12,000× g and 4 °C for 1 min. The spin column was then placed on a new 1.5 mL tube. An amount of 50 µL of PCR-grade water was added to the spin column and incubated at 4 °C for 1 min, and then, centrifuged at 12,000× g and 4 °C for 1 min. The DNA concentration was measured using a NanoDrop 2000.The gel was extracted using a QIAquick Gel Extraction Kit . The target products were cut from the gel and weighed in the tube, and they were placed on ice for 30 min. Buffer QG with a quantity of approximately three times the gel weight was added to the tube and incubated at 50 °C for 10 min. Isopropyl alcohol  in the same quantity as the gel weight was added. This mixture was then transferred into a new 2 mL collection tube and centrifuged at 12,000× ® Kit (Invitrogen) and Competent Quick DH5α  were used for ligation and transformation. The product of gel extraction was placed on ice for thawing. Thereafter, 2 µL of product, 0.5 µL of salt solution, and 0.5 µL of TOPO vector were added to a 1.5 mL tube, mixed via pipetting, and incubated at 4 °C for 30 min. Competent Quick DH5α was placed on ice for thawing. Thereafter, 3 µL of competent cells was added to the tube and gently shaken up and down for mixing. The mixture was then incubated on ice for 5 min. The tube was placed in a water bath  and subjected to heat-shock treatment for 30 s at 42 °C without shaking. Immediately after this, the tube was placed on ice. In a Bechtop, 500 µL of Super Optimal broth with Catabolite repressor (SOC) medium (Invitrogen) was added to the tube. The tube was tightly capped, placed in an oscillating machine, and horizontally shaken for 1 h at 37 °C. Thereafter, 250 µL of SOC medium was added to the tube again. The tube was placed in the oscillating machine and horizontally shaken for 3 h at 37 °C. To prepare the plate, agar medium was produced with LB agar and 50 µg/mL kanamycin. This medium was poured into a plate and stored at 4 °C until further use. Once the plate was taken out, it was incubated at 37 °C for drying. Following this, 20 µL of X-gal (40 mg/mL) was spread onto the LB plate using a plastic rod. Thereafter, 50 µL of the mixture from the tube was added to the same LB plate and incubated at 37 °C overnight.A TOPO TA Cloning® PCR Master Mix Kit ; 5 µL of EmeraldAmp PCR Master Mix (2 × Premix), 1 µL of M13 forward primer , 1 µL of M13 reverse primer , and 3 µL of PCR-grade water were added to the tube and mixed. The white bacterial colony was chosen and dipped using a toothpick; the colony was then immersed into the tube mixture and stirred well. The tube was then placed on a thermal cycler, and the reaction was set as follows: 40 cycles of 98 °C for 10 s, 58 °C for 30 s, 72 °C for 1 min, and finally, at 4 °C forever. After the reaction, the product was checked using electrophoresis with 1.5% agarose gel as previously described.To check whether the plasmid contained the target gene, PCR cloning was carried out using an EmeraldAmpThe LB medium was produced using LB broth base (Invitrogen) and ampicillin (100 µg/mL). Three millilitres of LB medium was added to a 10 mL disposable tube. The toothpick was gently dipped into the white bacterial colony, including the purpose gene, and then immersed into the LB medium and stirred well. The disposable tube was placed in the oscillating machine and horizontally shaken at 37 °C overnight. Subsequently, the bacterial culture appeared turbid, and the tube was stored at 4 °C until plasmid extraction.Bacillus coli using a QIAprep® Spin Miniprep Kit . The bacterial culture solution was extracted and stirred well. This solution (1 mL) was then added to a 1.5 mL centrifuge tube and centrifuged at 12,000× g and 4 °C for 1 min. The supernatant was then discarded completely. The remaining culture solution (2 mL) was treated in the same manner. Then, 250 µL of P1 buffer, 250 µL of P2 buffer, and 350 µL of N3 buffer were added in that order, stirring well after each step. The tube was centrifuged at 12,000× g and 4 °C for 10 min. The spin column was then placed in a 2 mL collection tube, and the supernatant was added to the spin column and centrifuged for 1 min. After centrifugation, 750 µL of PE buffer was added to the spin column and centrifuged for 1 min. After centrifugation, the residual liquid in the spin column was discarded; centrifugation was conducted for 1 min and the liquid in the collection tube was discarded. The spin column was inserted into a new 1.5 mL tube and 20–50 µL of PCR-grade water was added to the membrane of the spin column, and then, incubated at 4 °C for 1 min. Then, the tube was centrifuged at 12,000× g and 4 °C for 1 min, and the plasmid concentration was measured using a NanoDrop 2000. Subsequently, the plasmid was stored at 4 °C for sequencing analysis. The plasmid was extracted from the In the tube, 600 ng of plasmid, 0.64 µL of M13 forward primer (10 pmol/µL), and moderate Ultra-Pure™ distilled water (Invitrogen) were added to a final volume of 14 mL. This plasmid mixture was sequenced by Fasmac Co., Ltd. .gnrh1 (GenBank Accession No. FJ380047) were designed according to the GenBank database using the Primer3Plus software available online (primer3plus.com/ accessed on 15 May 2020) and manufactured by Fasmac Co., Ltd.  as previously described by Xu et al.  [The primers for 0, 2022) ,20 Supplfshβ (GenBank Accession No. EF583919) and lhβ (GenBank Accession No. EF583920) were obtained from Ryu et al. (2013) [fshβ and lhβ were detected using a FastStart Essential DNA Probes Master , which was performed as previously described by Xu et al.  [The probe primers for . 2013)  and prod13  and p2 and T concentrations were quantified using an Estradiol EIA kit or a Testosterone EIA kit  according to the manufacturer’s instructions.Steroids were extracted from the serum samples using diethyl ether . Diethyl ether (including steroids) was evaporated using nitrogen. The dried extracts were subsequently dissolved in enzyme immunoassay (EIA) buffer. The Egnrh1, fshβ, lhβ, E2, and T contents at each age as the dependent variables. The degrees of freedom (dfs) and p-values were determined. Differences were considered statistically significant at p < 0.05.All data were analysed using SPSS software . The normality of the continuous data was tested using Shapiro–Wilk tests. All data are reported as the mean ± standard deviation (SD). Statistical differences between independent samples were assessed using one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. The one-way ANOVA model included age as the independent variable and the The gonadal development of the 53 dph grouper was in the undifferentiated phase. The gonad was located near the gut A and conHE-stained pituitary gland sections are shown in The sagittal sections showed good immunoreaction with anti-Fshβ and -Lhβ, and were used to visualise the appearance and distribution of Fshβ and Lhβ immune signals in the pituitary gland. Fshβ signals were detected in the PPD and PI areas at 53 and 1050 dph . The expression of lhβ was significantly higher at 590, 687, and 1050 dph than at the other stages.The expression of 2 concentrations were significantly higher at 174 and 333 dph than at 590 dph (p < 0.05) and were significantly higher at 1435 dph than those at any other stage .The EChanna punctata [Rutilus rutilus caspicus [In the present study, we examined the gonadal development of the longtooth grouper and observed distinct phases of development. At 53 dph, gonadal development was in the undifferentiated phase. Subsequently, at 174 and 333 dph, the oocytes were in the oogonial phase, and the primary oocyte phase occurred between 590 and 1435 dph. The oogonial and primary oocyte phases observed in this study were longer than those previously reported. For example, Sao et al. 2012)  document012  docupunctata , and Cascaspicus . ConsequE. malabaricus [E. akaara [2 levels were higher at the onset of ovarian differentiation and decreased towards the end of differentiation in both species [2 level at 53 dph could not be detected due to challenges in obtaining blood samples from small specimens. Therefore, the specific E2 changes during ovarian differentiation in longtooth groupers remain unclear.Devlin and Nagahama (2002)  reportedabaricus , and bet. akaara . Additio species ,28. In t2 and T levels at 174 and 333 dph, respectively, during the oogonial proliferation phase. Androgens can be converted to oestrogens by aromatase, serving as both androgen and oestrogen precursors [2 synthesis [Heteropneustes fossilis, T present in the plasma vitellogenin serves as a precursor for oestrogen synthesis during ovarian recrudescence [2 levels during the annual reproductive cycle of Pleuronectes platessa. While studies have predominantly explored the role of T as a precursor or inhibitor of E2 during the maturation phase in teleosts, limited information is available regarding the oogonial proliferation phase for comparison with our findings. The observed increase in T levels during oogonial proliferation in our study may be due to its precursor, triggering E2 synthesis. For example, Yaron and Levavi-Sivan (2011) [2 stimulation in teleosts, demonstrating that E2 directly influenced oogonial proliferation in the Japanese huchen, Hucho perryi [2 levels observed in our study may be associated with oogonial proliferation in longtooth groupers.Our study revealed significantly higher Eecursors . Moreoveynthesis ,31. In tdescence . In addin (2011)  reportedo perryi . Thus, t2 levels at 590 dph, followed by increases at 687 and 1050 dph and a final significant increase at 1435 dph. Previous studies have reported low E2 levels during pre-vitellogenesis, followed by a significant increase during vitellogenesis in various fish species, such as the Arctic charr, Salvelinus alpinus [Acipenser gueldenstaedtii; and the stellate sturgeon, A. stellatus [2 levels were insufficient to trigger vitellogenesis in captivity in the dusky grouper, E. marginatus [2 levels occur only when vitellogenin production necessitates significant E2 stimulation to initiate vitellogenesis in most teleosts. Based on previous reports, vitellogenesis in captively reared longtooth groupers occurs between April and May [2 level at 1435 dph suggests its potential association with pre-vitellogenesis and vitellogenesis preparation.During the primary oocyte development phase from 590 to 1435 dph, our study detected a significant initial decrease in E alpinus ; the Rustellatus . Additiorginatus . These f and May  and at afshβ and lhβ gene expression has been identified in the differentiating gonad of rainbow trout, Oncorhynchus mykiss [Oryzias latipes [Furthermore, Fshβ and Lhβ immune signals were detected in the PPD and PI areas at most stages. Notably, at 53 and 1050 dph, Fshβ signals were detected exclusively in the PPD and PI areas, whereas Lhβ signals were detected only in the PPD area. These results are consistent with those of other studies that have previously discussed the different distribution patterns of Fshβ and Lhβ cells in the PPD and PI areas (type-1) or the PPD area (type-2) . Additios mykiss . Howevers mykiss  and Japa latipes .fshβ and lhβ gene expressions were significantly higher during these stages. Consistent with our findings, Fshβ and Lhβ immune signals have been detected during primary oocyte development in the Malabar grouper [Scomber japonicus [Monopterus albus, Lhβ signals were first detected in the early primary growth oocytes; however, Fshβ signals were not detected until the secondary growth oocyte [The signal intensities of Fshβ and Lhβ increased greatly at 590, 687, and 1050 dph during primary oocyte development, and  grouper , and Fsh grouper . However grouper  and chubaponicus . In the h oocyte . In the 2 concentrations were observed at 174, 333, and 1435 dph, while T concentrations showed significant increments at 174 and 333 dph. These findings suggest potential correlations between E2 concentrations in the serum and the phases of oogonial proliferation and pre-vitellogenesis.Based on our findings, we can draw the following conclusions: (1) Fshβ and Lhβ signals were detected even before ovarian differentiation, with their intensity increasing notably during oogonial proliferation; (2) Fshβ and Lhβ exhibited more robust signals and higher transcription levels from 590 to 1050 dph than those observed at earlier stages, indicating that both hormones were active during primary oocyte development; (3) significant increases in E2 in early gonadal development in the longtooth grouper. Artificial propagation and seedling production techniques have not yet been fully successful in this species. To gain a comprehensive understanding of the endocrine regulation mechanism throughout the reproductive cycle, further research should focus on monitoring the changes in these reproductive hormones within the BPG axis during maturation and sex change phases in the longtooth grouper.This study has shed light on the roles of Fshβ, Lhβ, and E


Furthermore, we hypothesized that both β-elemene and compound 5a could be therapeutically effective. Therefore, combining these drugs against mutated FLT3 cells could be promising. In this context, dose–matrix combination-based cellular inhibition analyses, cell morphology studies and profiling of 43 different apoptotic protein targets via combinatorial treatment were performed. Our studies provide strong evidence for the hypothesis that β-elemene and compound 5a combination considerably increased the therapeutic potential of both compounds by enhancing the activation of several key targets implicated in AML cell death.Acute myeloid leukemia (AML) is one of the cancers that grow most aggressively. The challenges in AML management are huge, despite many treatment options. Mutations in FLT3 tyrosine kinase receptors make the currently available therapies less responsive. Therefore, there is a need to find new lead molecules that can specifically target mutated FLT3 to block growth factor signaling and inhibit AML cell proliferation. Our previous studies on FLT3-mutated AML cells demonstrated that β-elemene and compound  Cancer is one of the deadliest diseases worldwide and has always been challenging. It is a very heterogeneous disease, with more than 200 species, and its causes are very complex. Hallmarks such as proliferation, invasion, and metastasis abilities; angiogenesis; and resistance to drugs are the main features of cancer. In addition, by influencing biogenesis, it can use all processes to its advantage, such as epithelial mesenchymal transition or microRNAs. A wide range of therapeutics including small inhibitory molecules, smart drugs, repurposed drugs, natural products, targeted agents such as siRNA, or monoclonal antibodies address this multi-mechanistic disease. Both diagnosis and treatment processes vary depending on the type of cancer and its origin ,5,6,7,8.It is reported that FLT3 inhibitors have demonstrated promising results in clinical settings with minimal side effects ,18. The 5a, an oxindole-based compound, was one of the most potent drugs and showed remarkable activity, specifically against FLT3 ITD-mutated AML cells. Oxindole-based compounds are classified as endogenous hetero-aromatic compounds with wide biological activities. They have been used as scaffolds for designing biological drug compounds [In earlier studies, drugs combined against cancer have led to positive results in cancer patients . Therefoompounds .5a demonstrated potent effects against FLT3/ITD+ AML cells [5a and β-elemene. Our results in this study show that this combination approach altered the cell death pathways in FLT3-ITD-mutated acute myeloid leukemia cells.Our previous results utilizing β-elemene and ML cells ,25. In t5a in combinational treatments. In our previous studies, it was determined that the IC50 value of β-elemene on MV4-11 cells was 25 µg/mL and compound 5a was 4.5 µM. Based on this, the dose matrix analysis was performed by combining the IC50, upper, and lower doses of the compounds. The doses were 10, 20, 25, and 50 µg/mL for β-elemene and 2.25, 4.5, 9, and 18 µM for compound 5a. Cells were treated with these doses and combinations for 72 hours. At the end of 72 h, cellular viability analyses were performed with the WST-1 test, and absorbance values were calculated relative to untreated control cells. The obtained viability values were analyzed with the SynergyFinder tool. β-elemene doses were entered into the software as a µM equivalent of µg/mL to perform analysis in the SynergyFinder tool. 5a and in 5a, which were 100% inhibitory, resulted in over 90% cell death in all combinational manners. The 9 µM compound 5a treatment showed a 78% inhibition efficiency in itself and other combinations. The 25 µg/mL β-elemene treatment, which is the IC50 dose for β-elemene, while its efficacy was 53.31%, showed inhibition in the range of 55–60% in combinations of 2.25 and 4.5 µM compound 5a. The situation was similar for the IC50 dosing of compound 5a. While it showed a 58.64% inhibition on its own, inhibition was around 58% in combinational treatments. No potentiating effect was observed in the combination of the IC50 values of the two compounds. The 2.25 µM compound 5a treatment showed a 19% inhibition by itself, while combinational doses with β-elemene showed increased efficacy. This increase was found to be 47% and above. Additionally, inhibition was 10.95% with the β-elemene 10 µg/mL dose, while with the 2.25 µM compound 5a, this value increased by 36.63% to reach 47.58%. In summary, all these results profiled the combinatorial cellular inhibition phenotype of β-elemene and compound 5a.A 4 × 4 dose matrix analysis was used to assess the cellular inhibitory efficacy of β-elemene and compound 5a by using models of four different hypotheses with the Synergyfinder tool. Thus, we aimed to determine both the interaction mechanism of these two drugs and the optimal dose combination for further studies. 5a according to four different synergy models: HSA (highest single agent), Bliss, ZIP (zero interaction potency), and Loewe. According to these models, the interaction between two drugs is evaluated mathematically as follows. It is classes as antagonism if the synergy score is below −10, additive if it is between −10 and 10, and synergism if it is higher than 10. In this study, the mean synergy scores of the models were calculated as 2.35 for HSA, −5.22 for Bliss, −5.31 for ZIP, and −8.31 for Loewe, respectively. Considering the obtained scores, we can say that there is no antagonism or synergy between β-elemene and compound 5a. Although the scores show that the relationship is additive, a closer look at the diagrams of the models reveals increased efficacy and synergy scores in combinations of 2.25 µM of compound 5a with different doses of β-elemene, especially in the HSA model. Our previous research has aimed to examine the detailed mechanistic properties of the two compounds in MV4-11 cells. After our studies centered on a 25-µg/mL β-elemene dose and a 9-µM compound 5a dose, we aimed to make further analyses in this study by combining 25 µg/mL of β-elemene with 2.25 µM of compound 5a. We thought that these two combinations would contribute to the modulation of potential signaling pathways, with a cellular inhibition efficiency of 59.48%. Using the potency of these two compounds, synergy analyses were performed to determine which combination would be used to modulate potential signaling pathways in MV4-11 cells. Many theories have been put forward from the past to the present in the applications carried out with the use of different compounds for certain targets. Although it is desired to determine a conclusion such as antagonism, non-interaction, and synergism for the interaction of drugs, this issue is still not fully settled. As Tang et al. asserted, the analysis of each combination should be evaluated against its pharmacology . Therefo5a or 25 µg/mL of β-elemene, or a combination of these two doses. An amount of 0.1% DMSO was used as a control. Cells were photographed after 72 h of treatments. As seen in Cell morphology analyses were performed to observe the phenotypic effects of selected doses on MV4-11 cells. Cells were treated 24 h after seeding with 2 µM of compound 5a for 72 h. Then, proteins from four individual groups  were isolated, and simultaneous immunoblotting analysis of 43 individual cell-death-associated proteins was performed using a Human Apoptosis Antibody Array kit . 5a. On the other hand, interestingly, Casp3 and Casp8 expression decreased significantly with the combination. This indicates that the combination altered equilibria in the apoptotic machinery compared to the singular treatment. One of the striking modulations occurred in CD40 and CD40L. Only the combination dose significantly increased CD40L, while CD40 was elevated in all three treatments. We can say that the combination contributes to the ligand activity, and accordingly, CD40 activity increases with the induction of β-elemene. Cyto-C was among the targets that increased only with the combination treatment. Although this increase does not seem to contribute to caspase cascade activation, it comes to mind that it may be increased as a result of the suppression of Bcl-2 family members by combination. DR6 increased after 72 h of treatment with both compounds, accompanied by a higher level of combination treatment. While Fas and its ligand (FasL) did not change in our previous studies, in this study, FasL increased with all three treatments, the highest in the combination. Fas was also similarly increased for β-elemene and combination but not 5a. All three of the heat shock proteins had increased for the two compounds in our previous studies. In this study, it was seen that lower dose 5a did not cause an increase in HSP27 and HSP60. It was also observed that the β-elemene-mediated increase in HSP27 decreased with the combination. Increases in HSP70 were noticeable in all three treatments. It was noted that 2 µM 5a tried to deal with these stress proteins. HTRA levels were characterized by a tremendous increase for all three treatments. Even though the levels of IGF-I and IGF-II, which have the ability to inhibit apoptosis, increased with the treatments, the combination attempted to bring them down. While no change was observed in 1, 3, and 4 of the IGFBP1-6 series, an increase was observed in 2.25 5a with β-elemene, as expected, and consistent with our previous study. But it was remarkable that this was brought down with the combination. Compound 5a underwent this reduction significantly at the 9 µM dose, where 2.25 µM supplementation reduced the induction of β-elemene. Although the story behind this is complex, we think it may be related to the reduction in IGF-II. No significant, combination-related changes were observed in Livin, p21, p27, and p53. While SMAC contributed positively to the apoptosis machinery with an increase in all three treatment types, Survivin also increased and negatively influenced it. The substantial rise observed in the combined treatment for Survivin demonstrated that the two drugs exhibited this target-specific synergy. Soluble tumor necrosis factor receptors 1 (sTNF-R1) and 2 (sTNF-R2) were significantly increased with combination treatment, especially for sTNF-R1. On the other hand, while there was no change in TNF-β, there was a decrease in TNF-α only in the combination. All the TRAIL death receptor series (TRAILR 1–4) showed significant increases with 72 h of 25 µg/mL β-elemene treatment. The fact that it increased both apoptosis mediators, TRAILR1 and TRAILR2, and decoy receptors, TRAILR3 and TRAILR4, is somewhat difficult to explain. Finally, XIAP protein increased significantly with all three treatments and showed an inhibitory effect on the apoptosis process. Considering all these massive change dynamics, 25 µg/mL β-elemene combined with 2.25 µM 5a caused regulations in various cell death targets. After the analyses we performed with the combinatorial approach, a protein-level-based array analysis was performed to investigate how the selected combination affected the cell death pathways. As a result of treatment of the cells with oxindole compound at a dose of 9 µM for 48 h in our previous research; upregulated proteins were identified as Bad, HSP27, HSP60, HSP70, HTRA, Livin, p27, and TNFβ, and downregulated proteins as Caspase8, IGFBP-2, IGFBP-4, and Survivin . On the 5a targeted FLT3-ITD in AML cells and demonstrated strong inhibition of proliferation [5a could provide a strong synergistic effect against FLT3-ITD-mutated AML cells. FLT3 mutations comprise the current therapy under the banner of drug resistance. Blocking FLT3-mutated oncoproteins will disrupt growth factor-mediated signaling cascades. The pharmacological synergism of anticancer drugs has been utilized in clinical settings and has produced promising results [5a and β-elemene, we used models based on many hypotheses with the synergy finder tool [50 of β-elemene and compound 5a was not changed by the combination. These observations suggested that the combination of compound 5a and β-elemene demonstrated no potentiating cellular inhibition. We found that the effects of β elemene and 5a were additive without any sign of antagonism indolin-2-One) (InChI=1S/C16H13NO3/c1-20-15-9-10(6-7-14(15)18)8-12-11-4-2-3-5-13(11)17-16(12)19/h2-9,18H,1H3,) was synthesized as we have previously described [15H24 and molecular weight of 204.35. Alternatively, stock solutions, compound 5a and β-Elemene  exclusion method [The FLT3-expressing and ITD-mutant acute myeloid leukemia cell line MV4-11 (Cat. no. CRL-9591) was purchased from the American Type Culture Collection . MV4-11 cells were cultured in Iscove’s Modified Dulbecco’s Medium  supplemented with 10% heat-inactivated fetal bovine serum , 100 U/mL penicillin, and 100 µg/mL streptomycin  and 2.5 µg/mL plasmocin . Cells were routinely cultured in cell culture flasks at 37 °C in a humidified standard incubator with 5% COn method .5a combinations on MV4-11 cells. Cells were treated with different doses of β-elemene and compound 5a with a 4 × 4 dose matrix above and below their IC50s which were selected in accordance with our previous studies [4 cells/well and cultured for 24 h. Doses were created with serial dilution from the main stocks of β-elemene or compound 5a. For matrix combinations, β-elemene was studied at 50 µg/mL, 25 µg/mL, 20 µg/mL, 10 µg/mL and, compound 5a was studied at 18 µM, 9 µM, 4.5 µM and 2.25 µM. 24 h after seeding, compound sets were applied to the cells triplicate, and incubated for 72 h at 37 °C with 5% CO2. At the end of the incubation period, WST-1 reagent  was added to each well, and plates were incubated for 3 h under the conditions mentioned above. Then, the absorbance of each well was measured with a microplate reader (Infinite® 200 PRO)  at a wavelength of 450 nm and 600 nm (as the reference). After that, the recorded absorbance values were used to calculate the percentages of cell viability and inhibition. The data were analyzed with the SynergyFinder tool [A drug combination assay was performed to evaluate the synergistic potential of β-elemene and compound  studies ,25. Firsder tool , and synder tool ,57.5 cells/well. After 24 h of incubation, cells were treated with β-elemene (25 µg/mL), compound 5a (2.25 µM), and a combination dose of 25 µg/mL of β-elemene and 2.25 µM of compound 5a. As a negative control, a medium containing 0.1% DMSO was used. Cells were incubated with compounds for 72 hours at 37 °C and 5% CO2. Subsequently, cells were photographed for morphological change assessment under an inverted microscope, a Leica DM IL LED with a DFC-290 camera  [Analysis of cellular morphology after treatments was performed as previously described . In brieGermany) .5 cells/well in a 6-well plate for simultaneous detection of 43 human apoptotic marker concentrations in cell lysates. At the end of 24 h of incubation, cells were exposed to β-elemene (25 µg/mL), compound 5a (2.25 µM), and a combination of compound 5a (2.25 µM) and β-elemene (20 µg/mL) for 72 h. As a negative control, 0.1% DMSO-containing growth medium was used. At the end of treatment, cell pellets were lysed in 400 µL 1× lysis buffer and incubated at 4 °C for 30 min with gentle shaking. The lysates were then spun down for 10 min at 4 °C at 14,000 rpm and supernatant was collected. A Pierce BCA Protein Assay Kit  was used to quantitatively measure protein concentrations [® XF Imaging System . The intensity score of each array spot was measured with the ImageJ software program , and the following formula was used to normalize the expression of individual proteins in each membrane: X(Ny) = X(y) ∗ P1/P(y), P1 = mean signal density of positive control spots on the reference Array, P(y) = mean signal density of positive control spots on Array “y”, X(y) = mean signal density for spot “X” on array for sample “y”, X(Ny) = normalized signal intensity for spot “X” on Array “y”.A Human Apoptosis Antibody Array kit  was used to detect related genes in the human-apoptosis-signaling pathway, as we have previously described ,25. MV4-trations . Antibodt-test. p-values below 0.05 were considered statistically significant.The statistical significance between experimental groups were calculated using Student’s 5a. We demonstrated that both drugs showed strong inhibition in the proliferation of FLT3-mutated AML cells. The current study demonstrated that the combination of β-elemene and compound 5a induces pharmacological inhibition of FLT3-mutated signalling and inhibits AML cell proliferation without antagonizing each other. Several important proteins were modulated via combination treatment, signifying the importance of using a combination as an approach against aggressive AML cancers. We propose that these results be validated in in vivo models so that this combination can be used with AML patients in the future.Current therapy against AML faces many challenges, like drug resistance, because of the FLT3 tyrosine kinase receptor mutations. Identifying new molecules targeting the mutated FLT3 AML cancers is crucial based our previous studies on β-elemene and compound


Cell cycle synchronization method could be used to improve the diagnostic sensitivity of oncologic FDG positron emission tomography.18F] fluoro-2-deoxy-d-glucose (FDG) uptake in vitro and in vivo. Methods: Human prostate cancer cells (PC-3) were treated with 10 μM mibefradil for 24, 48, and 72 h to induce G1 arrest. Cell cycle distribution was analyzed at 0, 4, 8, 12, 15, 18, and 24 h after mibefradil withdrawal. Cellular uptake was measured after incubating cells with [3H] Deoxy-d-Glucose (DDG) for 1 h at the same time points used in the cell cycle analysis. The correlation between [3H] DDG uptake and each cell cycle phase was evaluated in the early (0–12 h) and late phases (15–24 h) of synchronization. In vivo FDG PET imaging was performed in PC-3-bearing mice at baseline, 24 h, and 48 h after mibefradil treatment. Results: The G0/G1 fraction of PC-3 cells was significantly increased from 33.1% ± 0.2% to 60.9% ± 0.8% after 24 h mibefradil treatment, whereas the S and G2/M fractions were decreased from 36.3% ± 1.4% to 23.2% ± 1.1% and from 29.7% ± 1.3% to 14.9% ± 0.9%, respectively, which were similar to the results by serum starvation. Mibefradil treatment for 24, 48, and 72 h increased the number of cells in S phase at 18–24 h after withdrawal; however, only the 72 h treatment increased [3H] DDG uptake . [3H] DDG uptake was positively correlated with the size of the S phase fraction and negatively correlated with the size of the G0/G1 fraction in the late phase of synchronization. DDG uptake was significantly increased by mibefradil-induced cell cycle synchronization and correlated with the sizes of cell cycle fractions. In vivo FDG PET imaging also demonstrated a significant increase in tumor uptake after mibefradil treatment. Quantified tumor FDG uptake (%ID/g) increased from 4.13 ± 2.10 to 4.7 ± 2.16 at 24 h, and 5.95 ± 2.57 at 48 h (p < 0.05). Conclusion: Cell cycle synchronization could be used to increase the diagnostic sensitivity of clinical FDG positron emission tomography.Background: We investigated whether cell cycle synchronization induced by the T-type calcium channel inhibitor mibefradil could increase tumoral 2-[ Then, cells were washed twice with 2 mL of ice-cold PBS and incubated with 250 μL of 0.1 N NaOH for 10 min at room temperature. Cell lysates (150 μL) were incunated with 10 mL of liquid scintillation fluid  for 15 min; then, radioactivity was measured using a liquid scintillation counter . For the protein assay, 10 μL of cell lysate was incubated with 150 μL of Pierce 660 nm protein assay reagent  for 20 min in 96-well plates (in duplicate). Absorbance was measured using a microplate reader . Cellular [3H] DDG uptake (%uptake/μg) was calculated as the total radioactivity in the cell lysate divided by the radioactivity of 0.2 μCi [3H] DDG and the total amount of protein in the cell lysate (μg). This experiment was performed in triplicate.Cells were seeded and treated as described for cell cycle analysis. At each time point, cells were washed twice with 1 mL of PBS and incubated with 0.2 μCi  DDG uptake for 24 h after removing mibefradil   D.3H] DDG uptake after 48 h with 10 μM mibefradil treatment   D.3H] DDG uptake at 15–24 h after mibefradil withdrawal (3H] DDG uptake by treated cells was 118.1% at 15 h (p = 0.030), 128.5% at 18 h (p = 0.021), and 140.1% at 24 h (p = 0.001). In the cell cycle analysis, the S fraction of treated cells was larger than that of control cells at 18 (35.3% ± 1.1% vs. 25.2% ± 0.7%) and 24 h (34.5% ± 0.9% vs. 25.7% ± 1.3%), whereas the G0/G1 fraction was smaller at 18 (45.7% ± 0.5% vs. 55.2% ± 0.7%) and 24 h (42.2% ± 1.0% vs. 57.1% ± 0.7%)  C.3H] DDG uptake significantly.Taken together, although 24, 48, and 72 h mibefradil treatments induced G1 arrest, only the 72 h treatment increased [3H] DDG uptake of treated cells ranged from 91.3 ± 12.0% to 145.8 ± 5.8% of controls. At passage 5, [3H] DDG uptake was significantly higher at 18  and 24 h  after drug removal. Similarly, at passage 8, [3H] DDG uptake was significantly higher at 18  and 24 h  after drug removal. By contrast, at passage 6, [3H] DDG uptake was significantly higher only at 24 h  after drug removal. Thus, [3H] DDG uptake was consistently higher in treated cells than in control cells only at 24 h after mibefradil withdrawal.As the passage of cells may influence the results, we repeated this experiment at three different passages  and late phases (15–24 h) of cell cycle synchronization. In the early phase, [3H] DDG uptake displayed no correlation with any of the cell cycle fractions  and a negative correlation with the G0/G1 fraction   was 4.0 ± 0.5, and measured to be 4.6 ± 1.14 at 24 h, and 4.45 ± 1.24 at 48 h ( > 0.05) A. No sigp < 0.05)  increased from 4.13 ± 2.10 to 4.7 ± 2.16 at 24 h, and 5.95 ± 2.57 at 48 h. The difference between baseline to 48 h and 24 h to 48 h achieved statistical significance ( < 0.05) .2+ channel inhibitor mibefradil for that purpose. As a result, we observed that mibefradil treatment synchronized tumor cells in the G1 phase, and FDG uptake was increased after drug removal both in vitro and in vivo. Additionally, FDG uptake was correlated positively with the S fraction and negatively with the G0/G1 fraction in the late phase of synchronization. To our knowledge, this is the first report of a successful increase in tumoral FDG uptake using cell cycle modulation by T-type Ca2+ channel inhibitor.The sensitivity of FDG PET is limited in some tumors for reasons related to low FDG accumulation in tumor cells. The best method of overcoming this weakness is to increase FDG uptake in those tumors. In the present study, we investigated cell cycle synchronization induced by the T-type Ca2+ channel inhibitor that blocks both T-type and L-type Ca2+ channels. Mibefradil was effectively used in the management of hypertension and chronic stable angina; nevertheless, it was withdrawn from the market because of serious drug–drug interactions with β-blockers, digoxin, verapamil, and diltiazem [2+ channel inhibitors to control cell cycle progression and inhibit tumor cell proliferation [2+ currents and induced necrosis in those cells [Mibefradil is a Cailtiazem . Howeverferation . As a sise cells . Similarse cells . In addise cells . This sy3H] DDG uptake significantly only after 72 h of treatment. Although the pattern of change in the cell cycle distribution and the respective sizes of the cell cycle fractions over time were similar with different treatment durations, increased [3H] DDG uptake was not achieved by 24 h or 48 h treatment. When we repeated the same experiment for validation, the result corroborated that 72 h mibefradil treatment significantly increased [3H] DDG uptake by PC-3 cells. Although the degree of increase varied with the cell passage number, [3H] DDG uptake at 24 h after mibefradil removal was consistently higher than that by control cells. This suggests that FDG uptake in clinical PET imaging could be increased by cell cycle synchronization, which would improve the sensitivity of PET in tumors with low FDG uptake.We found that mibefradil-induced cell cycle synchronization increased [p = 0.002) [3H] DDG uptake was positively correlated with the size of the S fraction in late-phase synchronization. The cell cycle distribution of tumor cells may be changed by chemotherapy. One of the popular chemotherapeutic drugs, 5-fluorouracil, increased the size of the G1 fraction in breast cancer cells in a time-dependent manner; however, it decreased FDG uptake by 50% after 72 h of treatment [3H] DDG uptake was negatively correlated with the G1 fraction and positively correlated with the S fraction. Meanwhile, FDG uptake was also increased by 48 h doxorubicin treatment (33%); however, only the size of the G2/M fraction was increased. In contrast to this result, we did not observe any correlation between FDG uptake and the size of the G2/M fraction.As has been mentioned, the correlation between FDG uptake and the proliferative capacity of tumor cells has been evaluated in a variety of manners, although the variation in FDG uptake according to the cell cycle phase has rarely been studied. In untreated non-Hodgkin’s lymphoma tissues, there was a significant association between high FDG accumulation  and a large S fraction  , which ireatment . Convers3H] DDG uptake at 0 and 4 h after 72 h treatment was variable compared to that at the other time points. The standard errors of [3H] DDG uptake at 0 and 4 h were 12.0% and 15.8%, respectively. Therefore, we separated cells into early and late phases based on the time after synchronization, and according to our expectation, [3H] DDG uptake by PC-3 cells displayed a significant correlation with the S and G0/G1 fractions only in the later phase. This result suggests that PC-3 cells may be unstable early after long-term cell cycle synchronization, and some time may be required to increase FDG uptake in cancer patients in the clinic.As shown in 3H] DDG uptake at later time points. In the same manner, although G1 arrest eventually produces a large S fraction in cancer cells, it would not potentiate tumor proliferation.A previous study investigating the effect of flavonoids on the proliferation and cell cycle distribution of cancer cells revealed that flavonoid-treated human breast cancer cells (MDA-MB-435 and MCF-7) and human colon cancer cells (HT-29) returned to their normal cycling within a day of flavonoid removal . This isIn vivo PET studies showed a gradual increase in FDG uptake 48 h after mibefradil treatment. In contrast, the control group did not show an interval change in FDG uptake. Although various conditions like tumor growth pattern and anti-tumoral effect can influence FDG uptake in this study, the results illustrated the cell-cycle-modulating drugs can be helpful to enhance the FDG uptake of the tumor, which is especially expected with tumors with low FDG uptake like prostate cancer. To overcome the potential confounding factors that influence tumor FDG uptake including tumor heterogeneity, tumor growth rate, and blood glucose level, further studies will be warranted.3H] DDG uptake and membranous glucose transporter-1 expression. In addition, 2-DDG treatment in combination with thiazolidinediones showed significant anticancer effects in colon cancer in vitro and in vivo, while 2-DDG alone showed no significant effect. Although the correlation between the cell cycle phase and 3H-DG uptake has not been investigated, these results support the use of mibefradil for enhancing tumoral FDG uptake in cancers.Recently, we have reported that cell cycle synchronization using thiazolidinediones enhanced anticancer effects of 2-DDG . Like mi18F-FDG PET is a well-known effective imaging technique to increase tumoral uptake [18F-FDG accumulates more in the tumor at the delayed time points because, unlike inflammatory cells, malignant tumors have low glucose-6-phosphatase activity. Dual-time-point imaging has shown superior or similar diagnostic performances compared with the standard imaging method in various cancers such as lung and breast cancers. However, it also has limitations in that 18F-FDG accumulation on the delayed imaging is inconsistent, and some inflammation also has a similar pattern of FDG accumulation to cancers. Therefore, dual-time-point imaging needs to be validated further.Dual-time-point (or delayed-time-point) imaging of l uptake . Dual-ti2+ channel inhibitor mibefradil resulted in the increased accumulation of cancer cells in S phase and subsequently increased FDG uptake. Animal studies to verify this outcome showed enhanced FDG uptake after mibefradil treatment. Cell cycle synchronization is expected to enhance the diagnostic sensitivity of FDG PET.In conclusion, cell cycle synchronization induced by the T-type Ca


Most fabricationtechniques require post-annealing to convert the amorphous VO2 thin film into the VO2 (M1) phase. In this study,we provide a temperature-dependent XRD analysis that confirms thechange in lattice parameters responsible for the metal-to-insulatortransition as the structure undergoes a monoclinic to the tetragonalphase transition. In our study, we deposited VO2 and W-dopedVO2 thin films onto silica substrates using a high repetitionrate (10 kHz) fs-PLD deposition without post-annealing. The XRD patternsmeasured at room temperature revealed stabilization of the monoclinicM2 phase by W6+ doping VO2. We developed analternative approach to determine the phase transition temperaturesusing temperature-dependent X-ray diffraction measurements to evaluatethe a and b lattice parameters forthe monoclinic and rutile phases. The a and b lattice parameters versus temperature revealed phase transitiontemperature reduction from ∼66 to 38 °C when the W6+ concentration increases. This study provides a novel unorthodoxtechnique to characterize and evaluate the structural phase transitionsseen on VO2 thin films.Various methods have been used to fabricate vanadiumdioxide (VO Onthe other hand, the monoclinic VO2(M1) phase has been studiedextensively during the last few decades because it undergoes an abruptmetal–insulator transition (MIT) at ∼68 °C,3 which is reversible by altering the temperature, the electricalfield, incident illumination, and pressure strain properties. Suchreversible phase transition temperatures are associated with structuralmodification from a low-temperature monoclinic M1-phase to a high-temperaturerutile R-phase.3 An intermediate VO2(M2) polymorph phase with a β-angle of 91.88° couldbe stabilized at room temperature or exist during the MIT from M1→M2 → R. This can be achieved by doping with a low concentrationof various transition elements, such as W, Al, etc., and introducingstrain in the film.5Over the past few decades,numerous polymorph phases of vanadiumdioxide (VO2(M1) has a phase transition temperatureslightly higher than room temperature, arguably limiting its practicalapplications. As a result, several fabrication techniques have beenadopted to reduce the VO2(M1) phase transition temperatureto near room temperature by doping with high-valent transition metalssuch as Al3+, Ta5+, Mo6+, Nb5+, and W6+.11 Alternatively, to doping VO2 with transition metals;the phase transition temperature can be controlled by changing particlesizes, surface morphologies, and crystalline phases during the competitivenucleation growth mechanism.9 On the otherhand, doping with high-valent transition metals of the VO2 thin films leads to lattice distortion and thus induces local stressand strain, which have been shown to reduce the phase transition temperature.14 Thus, lowering the phase transition temperature of VO2 thin films is attractive for numerous applications such as IR uncooledbolometers, thermochromic coatings, optical switching devices, ultrafastswitching, smart radiator devices for spacecraft, and Mott transitionfield effect transistor.7 Recently, there have been a few studies on pure VO2(B)and VO2(M1) materials, focusing on structural unit cell a, b, and c lattice parametersto determine the MIT behavior and transition temperature.16Meanwhile, VO2 and transition metal-doped VO2 thin films, aimingto lower their transition temperatures, which include sputtering,hydrothermal, nanosecond laser (ns) PLD,11 RF-magnetron sputtering,and femtosecond (fs) PLD.17 Chen et al.11 synthesized Al3+-doped VO2 thin film onto silicon and soda-lime substrates using Al-doped V2O5 target and ns- PLD with a KrF excimer laserat a wavelength of 248 nm. A transition temperature of 40 °Cwas reported for VO2 doped with an Al3+ thinfilm compared to 67 °C for the pure VO2 thin films.Similarly, VO2 and WxVx1–O2 thin films depositedwere fabricated with a reactive pulsed laser deposition and a XeClexcimer ns-laser at a wavelength of 308 nm by Soltani et al.12 They observed a transition temperature of about36 and 68 °C for W-doped VO2 and VO2 thinfilms, respectively.Different techniques have been implemented to fabricate VO2 and WxVx1–O2 thin films onto a silica substratewithout post-annealing usingfemtosecond pulsed laser deposition at a repetition rate of 10 kHz.We systematically investigated the crystal structure of the VO2 and WxVx1–O2 films by using TEM and XRD patterns.In addition, the FullProf Software was utilized to analyze the temperature-dependentXRD pattern data to evaluate the a and b lattice parameters and to predict the phase transition temperaturesof these samples.In this study, we fabricated the VO22.12O5) and W6+-doped vanadium pentoxide (V2O5) targets withthe molar composition of (100 – x) V2O5–xWO3– were prepared. High-purity V2O5 (≥99.99%) and WO3 (99.99%) materials were purchasedfrom Alfa Aesar. About 25 g batch of pure V2O5 power and the appropriate amount of WO3 and V2O5 powers were weighed to prepare W-doped V2O5 powder material. The WO3 and V2O5 powders were thoroughly mixed using a mortar and apestle until a homogeneous mixture was obtained. Each powder samplewas pressed into a pallet (PLD target) with dimensions of 30 mm ×40 mm × 2 mm using a Spec press with a 1 t load for 5 min. Anultrasonic bath was used to clean the 20 mm, 30 mm × 1 mm silicasubstrates at 50 °C followed by an acetone and isopropyl alcoholrinse and dried with a high-purity nitrogen gas-gun. The substrateand the target were mounted into respective holders within the PLDchamber. The PLD chamber was then evacuated to a base pressure of10–7 Torr before backfilling to a working pressureof 70 mTorr using high-purity process oxygen (99.99%). The separationdistance from the substrate to the target was kept at 60 mm, and thesubstrate temperature was maintained at 700 °C. Pure V2O5 and W-doped V2O5 targets wereablated to deposit thin films with a KMLabs Wyvern 1000–10solid-state Ti:sapphire laser/amplifier and a laser fluence of 0.27J/cm2 at a 75 kHz repetition rate. The total depositiontime was in the region of 2 h.Vanadium pentoxide (V2.21 radiation = 1.54056 Å) at 45 kV and40 mA. The XRD patterns were measured from 10 to 60° with a stepsize of 0.02 for angle 2θ. Subsequently, the temperature-dependentstudies of XRD patterns were collected using a Malvern P’AnalyticalEmpyrean Diffractometer (CuKα1 radiation= 1.54056 Å) system equipped with an Anton Parr HTK1200 heatingstage unit. The temperature dependent XRD data was recorded in thetemperature ranging from 10 to 80 °C with an increment of 5 and10 °C. Each sample was mounted on an Anton Parr HTK1200 heatingstage with housing, then heated to the appropriate temperature andkept for 5 min to stabilize before XRD data was collected. The XRDmeasurements of the VO2 and WxVx1–O2 thin films wereanalyzed using the FullProf Suite software 3.00, and the pseudo-Voigtprofile function for Profile matching and Rietveld refinement wereperformed. The a and b lattice parametersof monoclinic and rutile VO2 phases were tracked and evaluatedat different temperatures using Le Bail analysis to determine thephase transition temperature. The X-ray photoelectron spectra (XPS)were recorded on an Omicron energy analyzer (EA-125) with an Al Kα(1486.6 eV) X-ray source. Temperature-dependent resistivity measurementsdata were performed from 25 to 100 °C for heating and coolingusing the Ossila Four-Point Probe .The surface topographywas examined and recorded using a Carl Zeiss EVO MA15 scanning electronmicroscopy (SEM). Following the SEM imaging, ImageJ software was utilizedto determine isolated particle distribution deposited on the substrate.A focused ion beam (FIB)  machine was employedto prepare an in situ TEM cross section of each thin film. The FEITecnai TF20 transmission electron microscope fitted with a HAADF detectorwas utilized to acquire cross-sectional images, together with highreflectance TEM images and selected area electron diffraction (SAED)patterns. The room temperature X-ray diffraction patterns of the as-preparedsamples were recorded using a P’Analytical X’Pert diffractometerphase without impurity. The SAED pattern also reveals the characteristicsof long-range ordered polycrystalline structures. Furthermore, themagnified HRTEM image has interplanar spacing correlating to an out-of-planeand in-plane spacings of 0.342 and 0.171 nm, which correspond to (110)and (−211) planes of VO2 (M1) as illustrated in 2 along with interplanar spacings of 0.321 and 0.453nm, which correspond to (110) and (−111) zone axis. To validatethe crystal structure of the thin films prepared, electron diffractionpatterns of samples VW-0 and VW-2 SAED were employed to determinelattice parameters using SingleCrystal software for comparison. 2 polycrystalline lattice. Likewise, 2 lattice along the[110] and [−111] plane axes for the M1 and M2 mixed phasesindicated in red and blue.Bright-field TEM cross-sectional imagesof all the fabricated samples were prepared using a focused ion beam. 3.32 and WxVx1–O2 thin filmswere collected using a θ–2θ scan to investigatetheir crystalline phases, as depicted in 2 (sample VW-0) illustrate about seven prominent polycrystallinepeaks centered at 2θ = 27.82°, ∼33.37°, ∼37.04°,∼39.04°, ∼42.14°, ∼ 55.43°, and∼57.8°. These peaks corresponded to the following crystallographicplanes (hkl) of (011), (−102), (200), (−112),(210), (220), and (022), with the reflection of VO2 (M1)phase and crystal group of P21/c (JCPDS Card No. 72- 0514).19 The XRD patterns obtained for undoped VO2 thin film structures are comparable to polycrystalline structuresreported in the literature.1 Furthermore,the XRD patterns of various concentrations of W6+-dopedVO2 thin film samples exhibit additional orientation peaksat 12.22°, 15.01°, 17.86°, 30.13°, 35.80°,and 45.44° with increasing intensity as the W6+ contentincreases. These different diffraction peaks seen in 2(M2) and VO2(B) with a space group C2/m, which correlate with the JCPDS 70–31312 and JCPDS Card No. 65–7960.20 Theseresults confirm the formation of mixed phases consisting of VO2(M1), VO2(M2), and VO2(B) phases ofchemical formula W0.6V2.4O7 underthe current experimental condition. 2(M2) and VO2(M1) peaks centered at ∼26.80°(−111) and ∼27.82° (011) with the intensity ofthe M2 phase increasing as W6+ content increases. Theseresults indicate that the M2 phase becomes more stable and dominantover the M1 phase as the W6+ content increases. This isattributed to the induced microstrain caused by substituting W6+ ions into the VO2 lattice structure. The XRDpatterns agree with the TEM SAED patterns depicted in Following the SEM analysis, XRD patterns ofthe fabricated undoped VOd,of the four different VO2 films fabricated was determinedemploying the full-width-half-maximum (fwhm) values obtained fromdiffraction peaks at ∼26.80° and ∼27.82° andDebye–Scherrer equation.21Furthermore, the average crystalline size, The variation in thecrystallinity size at the two diffractionpeaks for each sample was approximately the same. Nevertheless, theaverage crystalline size obtained from the Debye–Scherrer equationwas <200 nm compared to the average particle size calculated fromthe SEM images depicted in 6+ ions in VO2 thin filmswas determined. The microstrain or strain effect plays an importantrole in the electrical and optical properties and transition temperaturesof the VO2 thin films. Therefore, the diffraction peakscentered at 2θ = ∼26.80° (M2) and ∼27.82°(M1) were used to investigate the microstrain or strain effect byfollowing the relationship.22Following the Debye–Scherrer equationanalysis, the microstraindistortion induced by W6+ content-doped VO2 thin films. It was observed that themicrostrain increased slightly with W6+ content, whichmay be attributed to the defect induced by W6+ in the VO2 lattice structure. Thus, such an increase in microstrainmay be ascribed to local structure modification of electron–electroninteractions in the VO2 thin film crystal structure, resultingin stabilization of the M2 phase.25 Furthermore, the dominatingof the M2 phase over the M1 stage at higher W6+ contentmay be ascribed to the differences in visible grain orientation andbreaks up of the V4+–V4+ bonds to formnew bonds such as V4+–W6+, V3+–W6+, and V3+–V4+.233.42 thin films. It is well-knownthat the valence states of V and W can significantly affect the VO2 thin film transition temperature.282 and Vx1–WxO2 thin film samples , which were deconvoluted with peak-fitting of XPS spectral ofhydroxyl (OH), oxygen (O s1), and V-2p to determine the prominentcharacteristics binding energies. The oxidation states of V-2p presentin the thin film sample surface are composed of typical two-peak patternsof V-2p1/2 and V-2p3/2, which are attributedto the spin–orbital splitting features. The binding energieswith peak positions due to spin splitting feature V-2p3/2, which occurred at ∼515 and ∼517 eV, are ascribedto V3+ and V4+ oxidation states of V speciesin pure and doped thin films,29, respectively. Similarly,the spectral feature V-2p1/2 has corresponding bindingenergy peaks at ∼523 and ∼524 eV, belonging to V3+ and V4+ oxidation states.29 According to Kurmaev et al.,30 the presence of V3+ valence states in all the thin filmsamples prepared may be attributed to the high-temperature environmentused during sample fabrication and oxygen vacancies, leading to thinfilm charge localization and surface segregation.X-ray photoelectron spectroscopy (XPS) analysis was performed toascertain the correct electronic states of vanadium(V) and tungsten(W) in the undoped and W-doped VO2– in the V–Obinding, while the OH peak occurred at ∼531 eV. Liu et al.27 reported that the presence of oxygen vacanciesin the crystal lattice had a great influence on the VO2 thin film transition temperature, electrical and optical properties.The spectral feature that emerged at 531.4 eV corresponds to the OHconcentration, which decreases with an increase in tungsten dopingconcentration. The presence of the OH content on the surface of theVO2 thin film may be ascribed to the environment and surface wateradsorption. XPS spectra depicted in 7/2 and W 4f5/2 induced by W6+ ions. The binding energy peak at 41.5eV is ascribed to V 3p.Meanwhile, the XPS spectral peak of O 1s appearedat ∼529eV, which can be assigned to O3+ and V4+. 3+ and V4+ valence state content percentageratios as a function of W doping concentration. The proportion ofV3+ decreases, and V4+ increases with increasingW concentration, which confirms the stabilization of the V4+ state. The chemical composition of each sample prepared was determinedto be VO1.69, VO1.47, VO1.21, andVO1.14 for samples VW-0, VW-1, VW-2, and VW-3. This demonstratesthat oxygen deficiency increases by increasing the W content underthe same fabrication condition.The influence of the W content on theV valence states was investigatedby fitting the area under the curves of V3.52 and W6+ doped VO2 thin film microstructuresby performing temperature-dependent XRD measurements. This providesa clearer quantitative understanding of how the W6+ contentaffects the VO2 thin film crystal structure and latticeparameters during the MIT mechanism from the M1 → M2 →R and M1 → R transition. 2 and W6+-doped VO2 films, with 2θ between 27.4° and28.4° and at temperatures ranging from 25 to 85 °C coveringthe range over which the physical properties are changing. The diffractionpeak of the VO2(M1) (011) phase of the thin films at alow-temperature range undergoes a change to the R(110) phase at ahigh temperature (JCPDS file 01–079–1655). In 31 Similarly, sample VW-1 reveals three diffractionpeaks identical to those of sample VW-0. The M2(201) intermediatestructure may be ascribed to different mechanisms, such as strainand stress at the thin film interface, doping with W6+ anddefects on the thin film.24 In the caseof sample VW-3, two peaks at 2θ of 27.65° and 27.84°can be attributed to the monoclinic M1 phase at a lower temperaturein the presence of the metallic R phase at a higher temperature.Following the observed temperature-related changesto the physical and optical properties, we investigated the VO2 and W6+-dopedVO2 thin film samples. The VO2 diffraction peaksoccur at ∼55.43° (220) and ∼57.8° (022) andare also shifted to the higher angle at the low-temperature range,corresponding to the monoclinic M1 phase. At the elevated temperature,the XRD patterns move to lower angles, indicating a phase transitionfrom monoclinic M1 to the metallic R phase as a result of the heatingprocess. Meanwhile, shifting the M1 structural phase to a higher 2θangle during heating results from the strain induced at the thin filmand silica substrate interface, leading to a mesoscopic phase separation.These results demonstrate that the various diffraction peaks seenin the VO2 and W6+-doped VO2 thinfilms fabricated by fs-PLD can be used to predict VO2 (M1)phase transition temperature accurately.Furthermore, a and b werecalculated using FullProf software to help shed lighton the subsequent measured behavior. The trend of a and b lattice parameters as a function of temperaturewas obtained by using temperature dependent XRD patterns in the rangeof 2θ from 26° to 60°. a and b withtemperature for samples VW-0, VW-1, VW-2, and VW-3 exhibiting hysteresisproperties, respectively. A-lattice (b-lattice) graduallyshifted to a lower (higher) value as the temperature increased, witha clear distinction between the insulator state (M1) and metallicstate (R). This trend indicates that a-lattice and b-lattice parameters are associated with contraction andexpansion in the VO2 thin film samples during heating fromroom temperature up to 85°. The range of the a and b lattice parameters seen in samples VW-0,VW-1, VW-2, and VW-3 are in good agreement with the observations byLiu et al.,15 who deposited VO2 thin films on (0001)-Al2O3 single-crystalsubstrates using RF magnetron sputtering. Similarly, the trends ofthe a and b lattice parameters asa function of temperatures are comparable to our results.The local crystallinelattice parameters 2 andW6+-doped VO2 thin films were evaluated by employingthe first derivative logarithms of lattice parameters a and b for the temperature {i.e. d[log(a&b)]/dT}. a&b)]/dT versus temperature, which was fitted with the Lorentz equation usingOriginPro software. The phase transition temperatures (Tt) of the thin films were determined using the expression a and b parameters. It is observed thatthe average transition temperature of samples VW series decreasesfrom ∼66 to 38 °C as the W6+ concentrationincreases from 0.0 to 1.5 wt %. Thus, such a decrease in phase transitionis mostly attributed to an increase in W6+ doping concentration,induced microstrain and particle sizes, as illustrated in 2 thin film induces compressive strain along the a axis, which can lower the transition temperature to nearroom temperature, as shown in 5 where they synthesizedW6+-doped VO2 thin film samples with W6+ concentrations of 0%, 0.5%, 1%, 1.5%, and 2% using a cosputteringmethod and followed by post-annealing. They measured temperature-dependenttransmission in the near-infrared region and reported tuning the phasetransition temperatures from 64.3 to 36.5 °C. Similarly, Rajeswaranet al.24 fabricated polycrystalline WxVx1–O2 thin films using ultrasonic nebulized spray pyrolysis ofaqueous combustion mixtures, with W6+ content varying between x = 0.2 and 2.0 at. %. The authors reported that transitiontemperatures decreased from 68 to 25 °C by doping the VO2 with 2.0 at% of W6+ and measuring the temperature-dependentresistance of the thin films. According to these literature results,the variation in the transition temperature is affected by the natureof the VO2 thin film phases, such as M1, M2, T, and R,together with surface morphology and orientation of the grains andtheir grain boundaries.32 According toTang et al.23 and He et al.33 the loss of direct bonding between the V4+–V4+ homopolar and V3+–V4+ heteropolar bonds by doping W6+ with VO2 destabilizes the VO2 semiconducting phase to lower thephase transition temperature. In addition, a high doping concentrationof the W6+ valence state may lead to a boost of free-electronconcentration and then lead to a transition temperature drop.34 It is also important to note that the transitiontemperatures obtained from our study are comparable to temperature-dependentresistivity transition temperatures of similar doping concentrationsreported elsewhere.36The MIT transition temperatures of the pure VO3.6T]. The resulting curves are shown in Th, and cooling, Tc phase transition temperature. Similar temperature-dependentresistivity measurements were performed for samples VW-1 and VW-2to determine the transition temperature, which is not shown (to bepublished later). a and b lattice parameterspresented in The temperature-dependentresistivities of the thin films prepared were investigated by usinga four-point probe purchased from Ossila Ltd. The electrical resistivitywas recorded from room temperature to 100 °C for comparison withthe temperature-dependent XRD results illustrated in 42 and W6+ doped VO2 on silica substrates. The thin films’ surface morphology,particle size, and crystal orientation were confirmed using SEM androom temperature XRD measurements. The XRD measurements revealed mixedphases of the highly dense polycrystalline monoclinic crystallinestructures of VO2(M1) and (M2) for W6+-dopedVO2 thin film samples. With increasing W6+ concentration,the VO2(M2) phase becomes dominant and stable and existstogether with VO2(M1) and VO2(B) phases; however,it suppresses the XRD peak intensity of the VO2(M1) phasedue to the W6+ content. Thus, this is ascribed to the straininduced by doping the VO2 with the W6+ ionsand the uniformly distributed W6+ in the VO2 matrix, favoring the VO2(M2) phase formation insteadof the VO2(M1) phase. The temperature-dependent measurementsshowed a remarkably sharp change in the a and b lattice parameters from room temperature to a high temperatureof about 85 °C. These lattice parameter changes result in a sharpdecrease at the MIT temperature, corresponding to the structural phasetransformation from monoclinic M1 to the metallic R phase. The phasetransition temperature decreases from ∼66 to 38 °C whenincreasing the W6+ concentration. This study demonstratesthe nature of the changes in the temperature-dependent lattice parameters,offering the potential to understand and more accurately predict thestructural phase transitions of VO2 and W6+-dopedVO2 thin films, which affect the resistivity and opticaltransmission behavior as a function of temperature.A high repetition ratefemtosecond-PLD approach has been used todeposit thicker VO


Propionibacterium bacteriophage PAC1 was characterized. The enzyme (PaAmi1) was cloned into a T7 expression vector and expressed in E. coli BL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity of PaAmi1 was confirmed using isolated peptidoglycan from P. acnes. The antibacterial activity of PaAmi1 was investigated using live P. acnes cells growing on agar plates. Two engineered variants of PaAmi1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes of Propionibacterium bacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towards P. acnes and the enterococci species Enterococcus faecalis and Enterococcus faecium. The results of the present study suggest that PaAmi1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins.The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase  from  Antimicrobial resistance (AMR) is one of the most serious global threats of this century. The continuous increase in the emergence of multidrug-resistant (MDR) bacteria is estimated to raise the number of deaths to 10 million annually by 2050 ,4,5,6,7.The cell wall of the bacteria consists of the polysaccharide peptidoglycan (PG), which is composed of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues linked by β-1→4 bonds. The glycan strands are cross-linked via short peptides linked to each MurNAc residue ,19,20. TEndolysins are classified into three major groups: amidases, peptidases, and glucosidases , based oPropionibacterium acnes is an aerotolerant, anaerobic, Gram-positive bacterium that normally colonizes the sebaceous glands on human skin. It is characterized as a dominant human skin commensal. Over 80% of adolescents and an increasing number of adults are affected by acne vulgaris, a skin condition caused by Propionibacterium acnes. The intense use of antibiotics has led to the emergence of antibiotic-resistant P. acnes strains, which could transfer their antibiotic resistance genes to other commensal bacteria of the human skin microbiome through horizontal gene transfer  for selecting a strain capable of producing PaAmi1 at high levels. The last two strains showed no growth after the transformation, so seven strains were selected for further studies. The enzyme was expressed as an insoluble protein in inclusion bodies  at 37 °C and 47 °C   on lytic activity was evaluated (2+ appears to enhance significant (approx. 50%) lytic activity, confirming that PaAmi1 displays zinc-dependent catalytic activity, similar to other amidases  for potential AMP sequences was achieved using the MACREL algorithm  in order to determine the best expression strain for PaAmi1.The WI, USA) , which pE. coli BL21 Shuffle® T7 Express cells in LB medium containing ampicillin (100 μg/mL) was used to inoculate fresh 2xYT medium (ampicillin 100 μg/mL) and was incubated at 37 °C under shaking at 180rpm until OD at 600 nm reached 0.5 to 0.6  and two times with double-distilled water. Each time, the pellet was vortexed and centrifuged at 13,000× g for 15 min. Next, the pellet was solubilized with the following buffer: 2 M urea-0.1 M Tris-NaOH buffer, pH 12, containing 1 mM DDT. Protein refolding was carried out with overnight dialysis against 20 mM Tris, pH 9. The enzyme solution was stored at 4 °C until further use [Overnight culture of 5 to 0.6 . Furtherther use ,76. Prot600nm of 0.5–0.6, and the reduction of the turbidity of the cell suspension was monitored for 90 min at 25 °C in the presence of PaAmi1 . The course of the reaction was monitored by taking the absorbance at specific time intervals. Likewise, in the control cuvette, the same components were added, but instead of PaAmi1, dialysis buffer was added. The lytic activity (%) was calculated after a 90 min reaction using Equation (1):Bacterial cells were suspended in appropriate buffer to a final ODPaAmi1 was assessed by measuring the turbidity reduction of different bacterial cell suspensions  at 600 nm using 50 mM KH2PO4/K2HPO4, pH 6.24. The lytic activity of PaAmi1 was evaluated using Equation (1). Lyophilized Micrococcus lysodeikticus cells were obtained from Sigma-Aldrich . DH5a E. coli was obtained by Thermo Fisher Scientific, Waltham, MA, USA. The other bacterial strains were much a appreciated gift from the Department of Microbiology, “Aghia Sophia” Children’s Hospital, Athens (Dr. A. Stathi) and described elsewhere [2PO4/K2HPO4, pH 6.24) before being used in the turbidity reduction assay.In that case, the reduction of the control assay (without enzyme) is subtracted, and the effect of the enzyme on the cell suspension is evident. The antimicrobial spectrum of lsewhere . The bacPaAmi1 was assessed using the turbidity reduction assay and employing Micrococcus lysodeikticus or Propionibacterium acnes cells as substrate. The following buffers (50 mM) were used: Glycine/HCl pH 3.0; CH3COOH/CH3COONa pH 4.0; CH3COOH/CH3COONa pH 4.5; CH3COOH/CH3COONa pH 5.0; MES/NaOH pH 5.5; MES/NaOH pH 6.0; MES/NaOH pH 6.5; HEPES, pH 7.0; HEPES pH 7.5; HEPES pH 8.0; and Na2B4O7/H3BO3 pH 9. The lytic activity of PaAmi1 was also evaluated in the presence of different divalent metal ions (1 mM): ZnCl2, NiCl2, MnCl2, MgCl2, CoCl2, CaCl2, and CuCl2 or 5 mM ZnCl2. The activity of PaAmi1 was performed in three temperatures  in order to determine the optimal temperature for lytic activity.The effect of pH (pH 3–9) on the activity of P. acnes cells was isolated according to Fukushima and Sekiguchi, (2016) [Peptidoglycan from , (2016) . FollowiPropionibacterium acnes cells were dyed with Remazol Brilliant Blue R (RBB-dyed cells) according to Zhou et al., 1988 [Autoclaved l., 1988 . The RBBPropionibacterium acnes cells were cultivated, and then a 0.5 McFarland standard was prepared. Appropriate dilutions were made until approximately 104 cells were suspended in each reaction tube containing 50 mM MES/NaOH buffer, pH 6.0, and ZnCl2 (5 mM). Furthermore, different amounts of purified PaAmi1 were added (0.05–1.0 mg), and the reactions were left at room temperature and under stirring for 3.5 h. A control reaction was also prepared under the same conditions, but instead of PaAmi1, dialysis buffer  was added. Following incubation, a sample (10 μL) of each reaction was transferred to an agar plate (COL S +) and incubated at 37 °C for 8 days under anaerobic conditions.PaAmi1 to give the engineered variants DS1PaAmi1 and PA1PaAmi1 was achieved by PCR. The amplification of the whole expression vector pETite C-HisKan vector  was performed by PCR using the KAPA Taq PCR Kit  and the following forward primers:ds1F(5′-ATGCGCATTCGCTTATTACAGCGCTTCAACAAGCGCCGCTTCATTCCGGCT-3′) andpa1F(5′-ATGCGCGTATTCCGCCGCGCAGCACGCATTGCACAGCGCTTCATTCCGGCT-3′).Fusion of the AMPs DS1 (RIRLLQRFNKR) and PA1 (RVFRRAARIAQ) at the N-terminus of 2O. The conditions used in the thermocycler were an initial denaturation at 98 °C for 4 min. The PCR protocol comprised 30 cycles of 10 s at 98 °C, 15 s at 62 °C , and 16 s at 72 °C (extension temperature). A final extension time of 10 min at 72 °C was performed after the 30th cycle. The amplified PCR products were incubated with T4 DNA ligase  at 16 °C for 16 h. Following ligation, the plasmids were treated with the restriction enzyme Dpn I , and the recombinant constructs were used to transform competent E. coli stellar cells for sequencing, and E. coli BL21 OverExpress™ C41(DE3) cells for heterologous expression.As a reverse primer in both PCRs, the pETite reverse primer was used (5′-CTCAAGACCCGTTTAGAGGC- 3′). The PCR reaction was carried out in a total volume of 15 μL, containing the following: 7.5 μL Clone Amp HiFi PCR Premix, 0.2 μM forward and reverse primers, 2 ng template DNA, and 6.6 μL HE. coli BL21 OverExpress™ C41(DE3) cells in an LB medium containing ampicillin (100 μg/mL) was used to inoculate fresh 2 × YT medium (ampicillin 100 μg/mL) and was incubated at 37 °C under shaking at 180 rpm until the OD at 600 nm reached 0.5 to 0.6. The expression of DS1PaAmi1 and PA1PaAmi1 was induced with 0.5 mM IPTG, followed by incubation at 20 °C for 20 h at 160 rpm. Purification of DS1PaAmi1 and PA1PaAmi1 was performed as described for the wild-type enzyme PaAmi1. The bacteriolytic activity of DS1PaAmi1 and PA1PaAmi1 was achieved as described for the wild-type enzyme PaAmi1.Overnight culture of PaAmi1 and PA1PaAmi1) was evaluated by incubating the enzymes (for 10 min) at different temperatures (4–90 °C) in a 50 mM MES/NaOH buffer at pH 6. Following incubation, the remaining enzyme activity was measured using turbidity reduction assays . The activity of PaAmi1 and PA1PaAmi1 was measured using M. lysodeikticus cells as substrates, while the activity of DS1PaAmi1 was measured using P. acnes cells. This is because DS1PaAmi1 displayed low lytic activity on M. lysodeikticus cells.The thermostability of the wild-type and the engineered variants -like regions exist in the genome of bacteriophages that can be exploited to design novel artilysins. One AMP sequence that was in silico predicted from the genome of the PAC1 phage, when fused at the N-terminus of PaAmi1, gave an engineered variant with enhanced operational stability and lytic activity towards P. acnes and the enterococci species Enterococcus faecalis and Enterococcus faecium. The engineered enzyme can be further studied as a potential antimicrobial agent with applications in the cosmetic and pharmaceutical industries.Enzyme therapy utilizing endolysins is a promising solution to the rising problem of antibiotic-resistant bacteria. In the present work, a new NALAA-2 from