Pseudomonas aeruginosa through a systemic approach, considering that the metabolic pathway of PVD synthesis is regulated by the quorum-sensing (QS) phenomenon. The methodology comprised three main stages: (i) Construction, modeling, and validation of the QS gene regulatory network that controls PVD synthesis in P. aeruginosa strain PAO1; (ii) construction, curating, and modeling of the metabolic network of P. aeruginosa using the flux balance analysis (FBA) approach; (iii) integration and modeling of these two networks into an integrative model using the dynamic flux balance analysis (DFBA) approximation, followed, finally, by an in vitro validation of the integrated model for PVD synthesis in P. aeruginosa as a function of QS signaling. The QS gene network, constructed using the standard System Biology Markup Language, comprised 114 chemical species and 103 reactions and was modeled as a deterministic system following the kinetic based on mass action law. This model showed that the higher the bacterial growth, the higher the extracellular concentration of QS signal molecules, thus emulating the natural behavior of P. aeruginosa PAO1. The P. aeruginosa metabolic network model was constructed based on the iMO1056 model, the P. aeruginosa PAO1 strain genomic annotation, and the metabolic pathway of PVD synthesis. The metabolic network model included the PVD synthesis, transport, exchange reactions, and the QS signal molecules. This metabolic network model was curated and then modeled under the FBA approximation, using biomass maximization as the objective function . Next, chemical reactions shared by both network models were chosen to combine them into an integrative model. To this end, the fluxes of these reactions, obtained from the QS network model, were fixed in the metabolic network model as constraints of the optimization problem using the DFBA approximation. Finally, simulations of the integrative model  were run using the DFBA approximation to get (i) the flux profile for each reaction, (ii) the bacterial growth profile, (iii) the biomass profile, and (iv) the concentration profiles of metabolites of interest such as glucose, PVD, and QS signal molecules. The CCBM1146 model showed that the QS phenomenon directly influences the P. aeruginosa metabolism to PVD biosynthesis as a function of the change in QS signal intensity. The CCBM1146 model made it possible to characterize and explain the complex and emergent behavior generated by the interactions between the two networks, which would have been impossible to do by studying each system’s individual components or scales separately. This work is the first in silico report of an integrative model comprising the QS gene regulatory network and the metabolic network of P. aeruginosa.Computational modeling and simulation of biological systems have become valuable tools for understanding and predicting cellular performance and phenotype generation. This work aimed to construct, model, and dynamically simulate the virulence factor pyoverdine (PVD) biosynthesis in  In recent years, computational modeling of biological systems has acquired a significant role in understanding and predicting cellular performance. Computational modeling for explaining cell behavior has been accomplished by numerous discrete, continuous, and spatially explicit models describing subcellular systems and processes that biological systems need to survive . Indeed,Escherichia coli and biofilm formation by Pseudomonas aeruginosa, respectively [Following the development of next-generation sequencing techniques, a key objective has been to relate annotated genome sequences to cellular physiological functions. Therefore, systems biology has been designing new computational strategies to reconstruct biological networks (genomic and metabolic) and to model and dynamically simulate biological systems in order to study, from a holistic perspective, the regulation of mechanisms leading to the expression of diverse phenotypes. Thus, the extensive dataset of measurements of all biomolecules at the system level favors integration from the molecular to the whole organism level. In recent decades, several computational studies have made developing multiscale models of some biological systems possible. For example, Thiele et al. and Biggs and Papin have shown the advantages of multiscale models for understanding metabolic and macromolecular synthesis in ectively ,5.P. aeruginosa [pvd genes, which encode enzymes involved in PVD synthesis and which are regulated by the quorum-sensing (QS) phenomenon [-in this case, induced under conditions of iron deficiency, and responsible for synchronizing phenotype expression in a bacterial community—in this case, the factor virulence PVD expression) [Following a holistic approach, this work aimed to explain the synthesis of the siderophore pyoverdine (PVD), a virulence factor in ruginosa . The PVDenomenon  (a mecharession) . The secP. aeruginosa has two QS systems, LasI/LasR and RhlI/RhIR. The signaling molecule of the LasI/LasR system is N-(3-Oxododecanoyl)-L-homoserine lactone (3O-C12-HSL), which is synthesized by LasI and detected by the LasR receptor. The RhlI/RhIR system is regulated by N-butanoyl-L-homoserine lactone (C4-HSL), synthesized by RhII, and detected by the RhIR receptor. Both QS systems are hierarchically organized, such that the LasI/LasR system regulates the function of the RhlI/RhIR system, allowing further fine-tuning of the QS feedback on specific genes [Pseudomonas Quinolone Signal (PQS) system. This system encodes the synthesis of 2-heptyl-3-hydroxy-4-quinolone (2C7-3OH-4(1H)Q) that forms a complex with the available ferric ion (Fe3+) and plays an essential role in the pvd genes expression. At the cellular level, the quinolone complex positively controls the expression of the Pvd-type proteins, which is responsible for PVD maturation, synthesis, and exportation [ic genes . These Qortation .pvdL, pvdI, pvdJ, and pvdD genes [pvdA, pvdF, and pvdH genes. According to the model proposed by Visca et al. [3+ complex, which is transported back into the intracellular space by the FpvA carrier. Iron is then released from PVD to the periplasm by a mechanism involving iron reduction (Fe3+ to Fe2+), and PVD is then transported out of the cell via a specialized carrier of the siderophore recycling process [Most PVD biosynthesis is carried out in the bacterial cytosol by four non-ribosomal peptide synthase (NRPS) proteins encoded by the dD genes . Togethea et al. , the Pvda et al. ,11,12 th process ,14,15,16P. aeruginosa is proposed here for the first time to describe and understand the influence of the QS phenomenon on PVD biosynthesis in P. aeruginosa from a systemic perspective. That model involves the process of bacterial communication mediated by the QS phenomenon for synthesizing signal molecules that regulate the expression of pvd genes to generate enzymes that metabolically catalyze the production of the virulence factor. Furthermore, this integrative model combined two biological network models: (i) The QS gene regulatory network model, based on deterministic approaches and whose simulation results were the concentration of chemical species and the fluxes of chemical reactions; and (ii) the model of the P. aeruginosa metabolic network based on the flux balance analysis (FBA) approximation. This approximation addressed the biomass maximization problem constrained by mass balance and thermodynamic conditions to obtain the optimal distributions of reaction fluxes through the metabolic network [P. aeruginosa metabolic networks. Then, the fluxes of these shared reactions, obtained from the QS network, were used as a constraint system to model the P. aeruginosa metabolic network by dynamic flux balance analysis (DFBA) [P. aeruginosa strain PAO1 metabolism to PVD biosynthesis as a function of variations in QS signal intensity.An integrative  model that combines the QS gene regulatory network and the metabolic network of  network ,19,20,21s (DFBA) ,23. The P. aeruginosa QS network as a gene regulatory network for the expression of pvd genes encoding enzymes for PVD synthesis and maturation; (ii) construction of the P. aeruginosa genome-scale metabolic network, including PVD biosynthetic reactions using the FBA approximation; and (iii) integration of these two network models into a multiscale model using the DFBA approximation  construction of a deterministic model of the ximation .The following elements were used to construct and model the QS gene regulatory network, (i) bibliomic information about the genes involved in the QS process, (ii) a language/platform for the biological representation of the retrieved bibliomic data, and (iii) a language/platform for the mathematical modeling of the biological representation.Bibliomic information was retrieved from generic and specialized databases, such as the Scopus and Web of Science bibliographic indexes and specific biological databases. In addition, several search queries were used to find relevant documents and database entries that provided the maximum number of chemical species for modeling the biological process.https://www.genome.jp, accessed on 5 March 2016), KEGG [The QS network model was built using the standard System Biology Markup Language (SBML) format. The CellDesigner 4.4 software  was used6), KEGG ,26, BioC6), KEGG ,28, UniP6), KEGG  and PubC6), KEGG ,31 datab3+ were fixed . In addition, each reaction incorporated the kinetic constant (k) values  and the links of each interaction between related molecules. The corresponding ODEs for mRNAs and proteins in the transcription and translation processes are exemplified in Equations (1) and (2), respectively [For QS modeling, the initial concentrations of genes, QS regulatory proteins, and available cytosolic Fex(t) for a series of time points [To construct the deterministic QS model, elementary kinetics, based on the law of mass action for all chemical species, was assumed for all reactions in the system. QS model simulations were run using SOSlib  in the Ce points .P. aeruginosa. When the microbial population density increases in these cultures, the concentration of QS signal molecules that regulate the expression of bacterial phenotypes, such as PVD, also increases [P. aeruginosa metabolic network.After testing the stability of the QS network by the behavior of signal molecules, intracellular ferribactin production, and intracellular and extracellular PVD production, a single condition was set for the different simulation scenarios, namely, the initial concentration of the extracellular signal molecule PQS (E-PQS). This ranged from 0.01 μM to 0.1 μM with 0.01-unit intervals and from 0.01 μM to 0.1 μM with 0.1-unit intervals for a total of 20 simulation scenarios . These sncreases ,35,36. IP. aeruginosa metabolic network required a previously published generic cell model of the bacterium, the specific metabolic pathway of PVD synthesis, and software for modeling the hundreds of reaction equations representing the metabolic pathways. The in silico construction of the P. aeruginosa published in the literature; (ii) the P. aeruginosa genome annotation in the PseudoCAP database; and (iii) the metabolic pathway of PVD synthesis in the MetaCyc database (PseudoCyc), which was extended by bibliomics. As in the IMO1056 model, each reaction was manually mapped against data in biological databases .The computational modeling was based on three data sources: (i) The genome-scale metabolic network model iMO1056 , which w, BioCyc , ModelSE, BioCyc , Metanet, BioCyc , Transpo, BioCyc , TCDB [4, BioCyc ) for preCuring a metabolic process refers to making the network mathematically feasible by removing pathologies such as metabolites that are not produced or consumed by any reaction in the model, eliminating thermodynamically infeasible cycles, and validating the production of metabolites in the biomass reaction.P. aeruginosa metabolic network, the list of reactions in the network was represented as a stoichiometric matrix Before curating the ineering , (ii) adineering ,44, and ineering . In thisineering , Prigentineering , and Kimineering , in whicP. aeruginosa metabolic network modeling using a steady-state FBA approximation [https://www.gams.com/, accessed on 20 March 2023).The ximation  compriseh−1 (K3) .P. aeruginosa metabolic network represent two types of biological processes; both are part of a living cell but differ in time scale, spatial location, control mechanisms, and chemical species that usually work separately. However, both networks share chemical species, such as the enzymes encoded by the QS network genes that catalyze some biochemical reactions in the P. aeruginosa metabolic network. The challenge was to combine these two networks to function as an integrated multiscale model. The work of Mallmann and collaborators inspired the methodological proposal to solve the challenge [Pseudomonas aeruginosa metabolic network model, and (ii) using the fluxes of the reactions shared by both model as constraints in the multi-stage FBA .Under the multi-stage FBA approximation, both network models were combined by fixing in the metabolic model, as equality constraints, the fluxes of the shared reactions, obtained from the QS network simulations according to each simulation scenario where E-PQS concentration was changed . The unigDW−1h−1 . SimulatQS and P. aeruginosa metabolic networks, Subject to:scenario . FinallyP. aeruginosa metabolic network and the interaction between both networks over time was possible. Furthermore, the DFBA approximation proposed by Mahadevan et al. [The behavior of the integrated model over time was observed by dynamic simulation using the DFBA approximation. Thus, observing the influence of the QS network on the n et al.  was usedQS and P. aeruginosa metabolic networks. Simulations were run to obtain (i) the flux profile of each reaction, (ii) the growth rate profile, (iii) the biomass concentration profile, and (iv) the concentration profiles over time of the metabolites of interest, i.e., the QS signal molecules, glucose, and PVD. The optimization problem was solved as a nonlinear programming problem (NLP) using the CONOPT v3.15N solver in GAMS software . This model includes the equality and inequality constraints of the system (thermodynamics) and the fluxes of the QS network reactions fixed in the P. aeruginosa metabolic network (according to scenarios 4+ = 104.127 mM), phosphate (Pi = 3.88 mM), sulfate (SO42− = 0.286 mM), biomass (X = 0.1 g/L) and oxygen (O2 = 1 mM). The initial concentrations of glucose, nitrogen, phosphate, and sulfate were calculated according to the approximate composition of the Luria Broth (LB) culture medium reported in the technical specifications of the commercial culture medium.Subject to:P. aeruginosa strain PAO1  was grown in Luria Broth medium  in a 1-L BioStat®A bioreactor , equipped with a disc-type impeller, a ring-type diffused bubble aeration system, pH and pO2 probes, a chiller for temperature control, and an aeration system for continuous and automatic control of the laboratory airflow. Cultures were grown in triplicate batches under controlled pH = 7, Tº = 37 °C, agitation = 200 rpm, oxygen saturation = 20%, and air flow = 2 L*min−1 (2 vvm). Cultures were incubated for 24 h, and aliquots were collected at 1-h intervals.The integrated model, named CCBM1146, was evaluated qualitatively by comparing the behavior pattern of PVD production and biomass production between data obtained from in silico simulations and those obtained from in vitro cultures of P. aeruginosa cultures were taken to measure the absorbance at 600 nm by spectrophotometry. The mean values were analyzed by logistic regression using the StatSoft statistical software to fit the data according to Equation (6) and obtain the following specific growth equation for strain PAO1.Aliquots of 1 mL of the P. aeruginosa were taken and centrifuged at 14,000 rpm for 5 m at 4 °C. The pellets were discarded, and the supernatants were stored at −80 °C for subsequent determination of the PVD profile following the protocol of Meyer and Abdallah [After incubation was complete, three aliquots (1 mL) of the in vitro cultures of Abdallah ,54,55. Thttps://doi.org/10.17632/2xzzkmnpfx.1). In addition, this QS network model comprised 103 biochemical reactions, including DNA transcription, mRNA translation, protein complex formation, inhibition reactions, and molecule-to-molecule interactions with positive, negative, or unknown effects (k) values reported in the literature. The values of k not reported in the literature were assumed to be within the range of published k values for similar reactions . For model construction, a system of 114 ODE was generated using a total of 103 kinetic parameters and represented using the SBGN graphical notation of the SBML models.The QS network model described here consists of 114 subcellular chemical species, including proteins, small molecules, genes and their corresponding mRNAs, and nine complexes containing different molecules . In all simulation scenarios, the concentration of the E-PQS signal molecule was modified to emulate the behavior of in vitro cultures of P. aeruginosa, where population growth increases as a function of time (more cells per unit volume) (The QS network was modeled in the twenty simulation scenarios described in  volume) . The per volume) . https://doi.org/10.17632/2xzzkmnpfx.1). In this simulation, increasing concentrations of QS signal molecules were not considered because the purpose was to assess the basal capacity of the model to reproduce the QS circuits and produce QS signal molecules intracellularly. In silico intracellular production of the chemical species of interest corresponded to simulated concentrations of 0.0234 μM for 3O-C12-HSL, 0.0102 μM for C4-HSL, 0.00099 μM for PQS; 0.0158 μM for ferribactin, and 0.0635 μM for PVD . Subsequently, simulations run according to the scenarios in The low concentration of these chemical species showed that the system on its own produced a basal amount of signal molecules, ferribactin, and PVD in response to the concentration of Fewhich depends directly on the QS phenomenon [The highest PVD yield (0.122 μM) was achieved with 0.6 μM E-PQS  established by the BioCyc database (https://doi.org/10.17632/y9htx3fcjm.1).The curation process was complemented by (i) evaluation of metabolite production and consumption in the biomass reaction by reverse engineering; (ii) detection and resolution of pathologies in the 880 metabolites of the network: 29.51% had pathologies, 17.25% were resolved and 12.26% with unresolved pathologies remained in the model; (iii) detection and resolution of TICs: 23 TICs were identified and entirely resolved in the network. Finally, the proposed model, CCBM1146, comprised 1123 reactions, 880 metabolites, 136 protein complexes and transport proteins, and 1146 genes coding for 826 enzymes. Each reaction was manually assigned a metabolic system and its corresponding labeled subsystems according to the ontology pathway  obtained for the objective function of the model was 0.55 h−1 . After simulation, 352 reactions with active fluxes were obtained, including those for synthesis, transport, and exchange of the QS signal molecules  and PVD. This relatively small number of reactions is principally due to the structure of the biomass equation. Also, it could be related to the addition of PVD to the biomass reaction, which triggers the activation of a small set of essential reactions of the central metabolism (including biomass metabolites and PVD synthesis reactions). Furthermore, since PVD is a secondary metabolite, these reactions result in PVD production upon activating necessary intermediary metabolism and secondary metabolism reactions in the model.The definitive CCBM1146 model and the steady-state FBA approximation were used to obtain the flux distribution of reactions in the P. aeruginosa metabolic network model. Since these networks have different types of biological language, the flux values of nine reactions shared by both networks were considered to be useful for merging the behavior of chemical species in both types of network models. These nine shared reactions were involved in the synthesis and transport of QS signal molecules, ferribactin synthesis, and PVD synthesis and transport. After applying the multi-stage FBA approximation in six simulation scenarios of the integrated model  the QS signaling pathway, (ii) a regulatory pathway for pvd gene expression, and (iii) the effect of these two processes on the P. aeruginosa metabolic network for PVD biosynthesis. These three processes were encompassed here in a multiscale model to explain the influence of the QS phenomenon on the metabolic pathway of PVD biosynthesis in P. aeruginosa.A thorough understanding of complex biological systems’ behavior requires studying and comprehending the interplay of several processes that often occur at different spatial and temporal scales in the biological system as a whole ,2,61. ThP. aeruginosa proposed in this work is the first approximation that includes the three QS systems reported for this microorganism. It also incorporates the autoregulatory mechanisms of these three QS systems as well as the mechanisms responsible for the expression of pvd genes, the transport of PVD to the extracellular space, and the chelation and entry of iron into the bacterium through the PVD/Fe3+ complex. The data obtained from QS model simulations are supported by the fact that any system in dynamic equilibrium tends to reach a steady state, which translates into an equilibrium that resists external forces of change (perturbations). When the system is perturbed, the regulatory systems—in this case, the QS systems—respond to the signals emitted to establish a new equilibrium; this function is executed by the QS systems themselves and is known as feedback control. All biological processes that integrate and coordinate the functioning of living organisms are examples of the homeostatic regulation required by the system to ensure cell survival. Thus, the biological system is driven to the homeostatic state expected from its behavior, which is likely to maintain stability while adapting to the optimal conditions for survival. This argument is supported by the work of Gonzalez et al. [The deterministic in silico model of the QS gene regulatory network in z et al. , which sz et al. .P. aeruginosa in culture, i.e., as the bacterial population increased (more cells per unit volume), the extracellular concentration of QS signal molecules per unit volume also increased. It was also evident that the signal molecules of all three QS systems were synthesized intracellularly and that as their concentration decreased, PVD production increased; that is, the QS network model reproduced known biological behavior and worked according to the expected cellular dynamics concerning the QS systems  forms a complex with the Fur protein, Fur/Fe2+, that binds to the promoter regions of pvd genes (iron-repressible or iron-regulated genes), thus inhibiting their transcription. Under limiting intracellular iron concentrations, the Fur-mediated repression decreases, and a positive transcriptional regulation of pvd genes ensues [3+ complex at the extracellular level, its subsequent entry into the cell via an ABC-type transport system, and finally, to the release of Fe2+ into the cytosolic space.The proposed deterministic QS network model emulated in silico the natural behavior of  systems . It is is ensues ,64,65. TP. aeruginosa [On the other hand, it should be noted that the simulated extracellular PVD B behavedruginosa . Thus, tP. aeruginosa [In addition, the model acquired the emerging property of resilience, characteristic of adaptive biological systems. Although this property was not modeled, it became apparent when the model was disturbed as the QS regulatory system responded to outputs to establish a new dynamic equilibrium that reached a steady state. The generated equilibrium resisted disturbances, understood as external forces of change. The in silico results were similar to those reported on the natural behavior of QS mechanisms in ruginosa ,71.P. aeruginosa by combining two networks with different temporal and spatial scales, namely, the QS communication network responsible for regulating the expression of pvd genes and the bacterial metabolic network of PVD biosynthesis.This study proposed a systemic approach to design an integrative multiscale model to understand the influence of the QS phenomenon on the expression of the PVD metabolic phenotype of The CCBM1146 metabolic network model is an improved version of the genome-scale metabolic network iMO1056 developed by Oberhardt et al. . The CCBPseudomonas aeruginosa in cultures incubated under controlled conditions—intended to validate some of the results of the CCBM1146 model—showed that the stationary phase was reached between 14 h and 15 h of culture , which, like PVD, has a high iron affinity, and the newly discovered narrow-spectrum metallophore Pseudopaline (PSP), that acts as a virulent factor too and is involved in nickel and zinc uptake [The methodological strategy employed in this study and the results indicate that the proposed multiscale CCBM1146 model offers valuable insights into the influence of the QS phenomenon on the metabolism and subsequent behavior of c uptake ,76. ThisP. aeruginosa. In the future, this integrative model could help to infer the different metabolic phenotypes that may arise among other microorganisms when exposed to different concentrations of the signal molecules of their own QS circuits responsible for regulating the synthesis of their virulence factors. Furthermore, the DFBA approximation applied to the CCBM1146 model evidenced its descriptive capability for the profiles of biomass and PVD, showing a similar behavioral pattern of an in vitro culture of P. aeruginosa; this makes it more valuable and suitable for evaluating, in the future and from holistic and dynamic perspectives, how different conditions can affect the behavior of P. aeruginosa for the synthesis of different virulence factors regulated by QS. From a systemic perspective, this work presents a methodological strategy to design a novel multi-scale and multi-class model proposal by combining two classes of models with different scales. The CCBM1146 model helped to characterize and explain the complex and emerging behavior derived from the interactions between these two models, which would have been impossible by studying each model or scale separately. Moreover, the CCBM1146 model served to understand and infer the natural influence of PQS intensity on the metabolic PVD biosynthesis in P. aeruginosa metabolic network for PVD biosynthesis. Combining these two network models was possible by fixing the fluxes of reactions shared by both models as system constraints in the multi-stage FBA and DFBA approximation. Thus, it was possible to model the influence of the QS phenomenon on the P. aeruginosa metabolism to PVD biosynthesis as a function of QS signal intensity.Moreover, the model could be used to test experimental conditions in vitro. Finally, this work proposes, for the first time, the integration of the quorum-sensing gene regulatory network with the However, though in this work a sensitivity analysis was performed for the QS network model, for further modeling works under the steady-state and dynamic-state flux balance analysis approach of the CCBM1146 integrated model, it is recommended that a sensitivity analysis be performed.


CCD4-based marker CPRFC1, and LG3_13.146 marker for flesh colour in peaches, papayas, and cherries, respectively.Breeding fruit species is time-consuming and expensive. With few exceptions, trees are likely the worst species to work with in terms of genetics and breeding. Most are characterized by large trees, long juvenile periods, and intensive agricultural practice, and environmental variability plays an important role in the heritability evaluations of every single important trait. Although vegetative propagation allows for the production of a significant number of clonal replicates for the evaluation of environmental effects and genotype × environment interactions, the spaces required for plant cultivation and the intensity of work necessary for phenotypic surveys slow down the work of researchers. Fruit breeders are very often interested in fruit traits: size, weight, sugar and acid content, ripening time, fruit storability, and post-harvest practices, among other traits relevant to each individual species. The translation of trait loci and whole-genome sequences into diagnostic genetic markers that are effective and affordable for use by breeders, who must choose genetically superior parents and subsequently choose genetically superior individuals among their progeny, is one of the most difficult tasks still facing tree fruit geneticists. The availability of updated sequencing techniques and powerful software tools offered the opportunity to mine tens of fruit genomes to find out sequence variants potentially useful as molecular markers. This review is devoted to analysing what has been the role of molecular markers in assisting breeders in selection processes, with an emphasis on the fruit traits of the most important fruit crops for which examples of trustworthy molecular markers have been developed, such as the MDo.chr9.4 marker for red skin colour in apples, the  Fruits play an important role in the human diet. Some have sustained and still somehow sustain populations in parts of the world where other food sources are scarcely available. Staple fruits can provide a large part of starch, proteins, and fats from fresh or processed fruits. Staple fruits include some fruit trees that are of paramount importance in some regions of the world. Among others are breadfruit, plantains and bananas, olive oil, and coconut oil. Modern, more evolved societies see fruits and vegetables as sources of beneficial compounds: they are good for our health. They contain natural active principles, ranging from primary metabolites  to secondary metabolites known as phytochemicals .The total world fruit production for 2020 was 887,027,376 metric tons. In 1961, production was 200 million tonnes. The People’s Republic of China, India, and Brazil were found to be the largest producers ..® Apple Actinidia, a simple-to-use molecular marker has recently been developed which allows for this type of selection on seedlings  . V . VCpCYCbf (blood-flesh) that was first described by Warner et al.  wa waPrunusRosaceae, the understanding of the genetic changes that resulted in this fruit size increase between domesticates and their small-fruited wild relatives is most advanced in tomato . S. SCitrus progeny .Diospyros kaki are unisexual. The varieties bear seeds when the male flowers are present. Pollination constant astringent (PCA), pollination variation non-astringent (PVNA), and pollination variant astringent (PVA) types with seeds produced unmarketable fruits. DlSx-AF4  and amplified fragment length polymorphism (AFLP) marker, has been confirmed in a persimmon germplasm collection, and the results obtained were in agreement with the phenotype data. Because they stop accumulating tannins during the early stages of fruit development, PCNA (pollination constant non-astringent) cultivars are greatly desired because their mature fruits are not astringent. In Japanese varieties, the PCNA trait is recessive to the non-PCNA trait and is controlled by a single locus, AST. The area related to AST has been the focus of numerous efforts. The most encouraging findings came from a study that pinpointed an area that was closely connected to the AST gene [Flowers on DlSx-AF4  53] is  is DiospAST gene . On the AST gene . MicrosaAST gene  validateIn fleshy fruits, softening is a sign of ripening and can have both positive and negative effects on postharvest stability and texture. In order to increase fruit quality through breeding, it is important to focus on the timing and degree of pre-harvest ripening as well as the accompanying textural changes after harvest.Fruit ripening is a complicated process that involves the development of flavour, colour, cell wall changes and softening, starch breakdown, and aroma, all of which are significant contributors to the distinctive fruit qualities. The process is controlled at many levels by a complicated network of interactions between the plant hormones and essential transcription factors .Md-ACO1, a gene involved in ethylene biosynthesis in apple, is mapped. This PG–ethylene-related gene has been positioned in the apple genome on chromosome 10, which contains several QTLs controlling fruit firmness and softening . A. A141].  storage . By sequersicum) . Four SNng MdPG1 .The two QTLs of fruit firmness (FF)F were located on LG1 and LG2 of a maternal parent in a bi-parental cross of apricot. A total of 117 candidate genes for FF were identified in two intervals. These candidate genes were involved in processes such as cell metabolism, cell wall composition or degradation, vesicular transport, transcription factors, and signal transduction .PFWQTL-PM11, PFWQTL-G6, and MQTL-G4.1, were involved in the control of auxin and peroxidase activity (1 Mb). Moreover, PFWQTL-G11 candidate genes for the ethylene response (ETR) were found (1.5 Mb). In QTLs associated with numerous quality traits, including PF, genes involved in cell wall metabolism were discovered. Three of these nine QTLs—pHWQTL-PM1 7.1, DMCWQTL-PM9.2, and PFWQTL-G6—corresponded to large consensus QTLs with R2 values of more than 25.0%. Before considering MAS selection, they need now be confirmed in other genetic situations, and their confidence intervals would need to be improved . T. T205]. o et al.  recently NAC TFs ,220,221.On LG11 in a 4-year average, one QTL for harvest date in grape was found. Despite the fact that the position of its summit changed over the years, it was situated in the upper to the middle section of LG11 .Nishio et al.  identifiPyrus pyrifolia Nakai). Two ACC synthase genes, MdACS1 [MdACS3 [MdACS1 was strongly associated with preharvest fruit drop in the apple. Both Japanese pear and apple ACC synthase genes  have various functions regulating harvest time, preharvest fruit drop, and fruit storage capability [A QTL with a significant correlation to harvest time was discovered at the top of LG15, next to the PPACS2 locus in Japanese pear  on G4 in some crossings [Sr/sr) gene is located in this same region, and it has been proposed that the sr allele, which determines the SR character, is one allele of MD [MD gene. Additionally, Nuez-Lillo et al. [Prupe.4G186800 NAC transcription factor gene (MD [sr/sr) of this gene, which has been localised to linkage group 4 of the peach genome, confers the SR phenotype and is responsible for determining this trait. It has been suggested that the causative mutation is a massive 26.6 kb deletion that includes the sequence of a transcription factor for the NAC  has been repeatedly reported by other authors in peach ,220,225 rossings ,220. Thele of MD . In a pele of MD  also disle of MD  to a pleo et al.  discovertor gene  that Pirgene  oIn Japanese plum, a QTL was strongly localised in LG4 between 6 and 12 Mbp. The occurrence of this QTL is highly conserved within Prunus species, being related the MD locus with NAC family transcription factors .H, which was linked to a slower rate of ripening across all phases from open flowers to the green/red stage, the presence of the AFLP marker P12M121-194 on chromosome 2 is connected with a shorter time to red fruit (RiMADS 01, a MADS box gene that significantly influenced the rate of ripening. According to Danielevskaya et al. [QTLs controlling the ripening phases in raspberry were found across four chromosomes, namely, 2, 3, 5, and 6. Each of the groups had markers that had a significant influence at various ripening stages. In contrast to the Hh genotype of Gene ed fruit . A wide a et al. , these ga et al. .There is little doubt that genomic technology will hasten the creation of new cultivars of a variety of fruit crops, influencing the process of choosing genetically elite progeny for development as prospective new commercial cultivars as well as for use as parents in the following stage of breeding. With the use of next-generation sequencing, GWS can now be used to screen individuals who carry a significant number of SNP markers that are densely scattered throughout the genome. The same methods can also be used to find allele-specific markers for foreground MAS application before GWS. Since reliable phenotyping is a precondition for both the training of a GWS model and for genome-wide association studies, such activities call for strong collaboration between researchers from several fields. Although the search for molecular markers associated with agronomic traits has assumed an important role in breeding programs of fruit species, it frequently seems necessary to validate the results obtained in a greater number of varieties and genotypes. There are 44 molecular markers immediately available to the community of breeders, for 9 characters, and they are usable in 15 different fruit species or genera. However, it is evident that there is the need to deepen the studies relating to the many QTLs identified in order to be able to obtain useful markers for an efficient assisted selection. For many important traits, with complex genetic control, for example, fruit-soluble solid content or dry matter, important for fruit shelf life, we still do not have reliable markers, despite the considerable work done in many species.


Schizophrenia is a devastating disease characterized by frequent relapses, cognitive decline, and emotional and functional disability, with unknown causes. The phenomenology and clinical course of schizophrenic disorders are different between the two genders, which is thought to be related mainly to the effects of steroid sex hormones on the nervous system. Regarding inconsistencies in the studies, we aimed to compare the levels of estradiol and progesterone between schizophrenia patients and healthy individuals.This cross-sectional study was conducted on 66 patients referred to the specialized clinical psychiatric ward of a teaching hospital in the north of Iran, for 5 months in 2021. Thirty-three schizophrenia patients confirmed by a psychiatrist based on DSM5 criteria were included in the case group, and 33 individuals without a psychiatric disease were included in the control group. We completed a demographic information checklist for each patient, along with the Simpson-Angus extrapyramidal side effect scale (SAS) for drug side effects and the positive and negative syndrome scale (PANSS) for the severity of the disease symptoms. Then, a 3-ml blood sample was taken from each participant to determine the serum levels of estradiol and progesterone. The data were analyzed by SPSS16 software.P = 0.4). However, the mean serum level of progesterone was significantly lower in schizophrenia patients (0.37 ± 1.39 pm/dl) than in control subjects (3.15 ± 5.73 pm/dl) (P < 0.001). The PANSS and SAS scores were not significantly correlated with the level of sex hormones (P > 0.05). Serum estradiol and progesterone levels based on sex significantly differed between the two groups .Thirty-four (51.5%) and 32 (48.5%) participants in this study were male and female, respectively. The mean serum level of estradiol was 22.33 ± 13.65 pm/dl in schizophrenia patients and 29.36 ± 21.32 pm/dl in the control group, showing no significant difference between the two groups (Considering the hormonal differences between schizophrenia patients and control subjects, determining hormonal levels in these patients and using complementary hormonal therapies with estradiol or similar compounds can be beneficial as the starting point of schizophrenia treatment, where therapeutic responses can draw the future developmental framework. Schizophrenia is a devastating disease characterized by frequent relapses, cognitive decline, and emotional and functional disability. This disorder encompasses positive  and negative  symptoms and is recognized as a cognitive disorder highly resistant to pharmaceutical treatment –3. SchizEndogenous progesterone (P4) is humans’ most potent and abundant hormonal subgroup. This hormone is synthesized both centrally and peripherally from pregnenolone, a cholesterol-derived steroid precursor. Progesterone (a precursor of estrogens), testosterone , and various types of glucocorticoids and mineralocorticoids play an essential role in the health and growth of men and women. It has been reported that serum progesterone levels range between 1 and 3 nM in men, with slight fluctuations with age. On the other hand, premenopausal women experience a variable serum level of progesterone according to the menstrual cycle. Accordingly, the lowest concentration of progesterone has been reported in the early follicular phase (0.3–2 nM) and the highest in the middle of the luteal phase. The serum level of progesterone drops considerably after menopause .Estradiol is the most potent endogenous estrogen in humans and the main focus of this study. Estrogens are synthesized both centrally and peripherally. Estradiol is a hormone that exists in both men and women; however, its serum levels are lower in adult men than in women, showing the least age-related fluctuations. The lowest and highest estradiol levels are seen in the early follicular stage (an average level of 150 pM) and on the day of ovulation (an average level of 670 pM), respectively. In the middle of the luteal phase, estradiol shows an average level of 500 pM . In geneWhile studies consider a role for the fluctuations of sex gonadal hormones in the phenomenology of schizophrenia in women and men, there is little research on the profiles of gonad hormones and neurosteroids in this disorder . Sun et Besides, estrogen, estradiol, and progesterone levels differ between men and women, and a few studies have been conducted on this issue in Iran. Therefore, we aimed to compare estradiol and progesterone levels between patients with schizophrenia and healthy individuals.This cross-sectional study was performed on patients referred to specialized psychiatric clinics and ward of the Martyrs of 5th Azar Teaching and Medical Center in Gorgan (north of Iran) for 5 months in 2021.The participants were included in this study by convenience method sampling. A total of 72 people entered the study. Three persons from the case group were excluded. After some participants were withdrawn from the study, in total, this study was performed on 66 participants. Thirty-three people in the case group were selected based on DSM5 criteria and with the approval of a psychiatrist, and 33 people in the control group were selected from patients referred to the psychiatric wards of a teaching hospital (Martyrs of 5th Azar) in Gorgan who did not have a psychiatric illness. This hospital is a specialty hospital with a psychiatric ward.The inclusion criteria were the age of 18 to 50, schizophrenia diagnosis based on DSM5 criteria by a specialist, and admission during the luteal phase (for women). The inclusion criteria in the control group encompassed people visiting the laboratory without a psychiatric illness. The exclusion criteria included a history of taking Oral Contraceptive Pills (OCP) or hormonal contraceptives in the last 3 months, a history of other neurological disorders requiring treatment, the presence of polycystic ovaries or other diseases with hormonal changes such as hyperprolactinemia, Edison, or tumor, postmenopausal and pregnant women, lactating mothers 3 months after delivery, and those not satisfied with the study.A sample size of 70 people was calculated based on the Yazici et al. study  with a tA checklist was used for each patient to record demographic characteristics and serum levels of estradiol and progesterone. This checklist included multi-digit codes, age, sex, education, height, weight, medical history, history of psychiatric disorders and frequency of episodes, history of extrapyramidal complications, history of Electro convulsive Therapy (ECT), history of drug use, and history of supplement use. The positive and negative syndrome scale (PANSS) was used to assess the severity of positive and negative symptoms in schizophrenic patients, with three subscales: positive symptoms with seven questions, negative symptoms with seven questions, and general symptoms with 16 questions. This questionnaire’s validity and reliability have been confirmed by Ghamari Givi . The SimAfter obtaining informed consent from each person , 3 ml of blood was taken and transferred to the laboratory in ice boxes as soon as possible. The hormone levels were measured with micrometer accuracy by immunoassay  or Enzymt-test (for two groups) or ANOVA test (for more than two groups) were performed to compare estradiol and progesterone data with normal distribution; otherwise, Mann–Whitney or Kruskal–Wallis test was used. The effect size (Cohen’s D) was also performed in two groups, interpreted as trivial (0–0.19), small (0.5–0.79), moderate (0.5–0.79), and large (> 0.8)  . The PeaWe selected 35 people for the case group based on DSM5 criteria confirmed by a psychiatrist and 37 people for the control group. Three persons from the case group were excluded due to dissatisfaction with the study, sampling error, and coronary heart disease. In total, this study was performed on 66 participants.The demographic characteristics, including sex, race, marital status, history of ECT, and opium use, were statistically different between the two groups. Although serum estradiol levels were lower in the schizophrenic group than in the control group, it was insignificant due to a small effect size. However, the progesterone level was statistically different between the two groups with a medium effect size .A power analysis was conducted using G*Power version 3.1.9.7 for relation between estradiol level in two groups , based on data from study. With a significance criterion of α = 0.05 and effect size = 0.39 and sample size = 33 in each group, the obtained power was been 34%.The questionnaire scores were interrelated, but no significant correlation (Pearson or Spearman) was found between the estradiol and progesterone levels and any of the questionnaire scores .We did not find a statistically significant association between categorized estradiol and progesterone levels in men and women and questionnaire scores. Because of the non-normal distribution of estradiol and progesterone data, we performed the Kruskal–Wallis test .After including age and sex in the linear regression model, only the age for progesterone in the control group was statistically significant .Schizophrenia is a debilitating psychiatric disease. Many recent studies have reported differences in the severity and duration of the disease between the two genders, which have been attributed to the effect of sex hormones . Therefovia inducing anti-apoptotic mechanisms and promoting cell survival. Furthermore, this hormone has an inhibitory effect on glutamatergic synapses. Allopregnanolone, a progesterone metabolite, was shown to inhibit the dopamine-induced release of glutamate in the prefrontal cortex , Negative Positive and Negative Syndrome Scale (NPANSS), and General Positive and Negative Syndrome Scale (GPANSS). Consistently, some studies have stated that the serum level of progesterone has a negative correlation with the total PANSS score and positive PANSS scores, suggesting a relationship between low hormone levels and the severity of the disease in schizophrenic patients .In contrast with our study, in Weiser et al. study (2019) , which eConsidering the hormonal changes in schizophrenic patients compared to control subjects and their significant relationship with the severity of positive symptoms, determining hormonal levels in these patients and implementing complementary hormonal therapies such as estradiol and similar compounds can be beneficial as the starting point for schizophrenia treatment, where therapeutic responses can draw the future developmental framework.The study limitations included the small sample size and the restrictions caused by the COVID-19 pandemic, demanding compliance with health protocols and, therefore, a reduction in visits to health centers, including psychiatric clinics, which prolonged the patient recruitment process and obtaining samples. Also, considering the effects of some drugs, such as risperidone and aripiprazole, on the level of the hormones assessed, it is necessary to adjust for the effects of such drugs in future studies. In addition, not considering sex differences intra-individual and ethnic biological variations were other limitation in the present study. A clinical trial study is suggested to investigate the difference hormone levels on schizophrenic patients.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Golestan University of Medical Sciences (IR.GOUMS.REC.1400.346). The patients/participants provided their written informed consent to participate in this study.NS and ZS: conceptualization and methodology. MZ and DK: data curation, software, and writing – original draft preparation. AR: visualization and investigation. MZ and NS: supervision. DK and AR: validation. NS: resources. All authors reviewed and edited the manuscript and approved the submitted version.


Cocaine being a stressor, it may alter brain homeostatic function. This dysregulation may entrench cocaine use disorder.: This is a human laboratory study of the effects of intranasal desmopressin (a Vasopressin 1b receptor agonist) and oxytocin on ACTH secretion in cocaine use disorder patients versus a control group. It consisted of two endocrine challenges performed on consecutive days. On day 1, the effect of intranasal desmopressin (80 IU) on ACTH secretion was measured. On day 2, a pre-treatment with intranasal oxytocin (24 IU) preceded intranasal desmopressin to monitor its effect on desmopressin-induced ACTH secretion. We hypothesized that the effect of intranasal oxytocin in controls would differ from the effect in cocaine use disorder patients.: Forty-three patients were included in this study: 14 controls and 29 cocaine use disorder patients. Significant differences were noted in the direction of change of ACTH secretion between the two groups. In cocaine use disorder patients, overall ACTH secretion was on average 2.7 pg/ml/min higher after intranasal desmopressin than after intranasal oxytocin/desmopressin . The opposite was observed in controls: overall ACTH secretion averaged 3.3 pg/ml/min less after intranasal desmopressin than after intranasal oxytocin/desmopressin .: Intranasal oxytocin and desmopressin revealed a pattern of ACTH secretion in cocaine use disorder patients that is distinct from a non-addicted control group.  Drugs oQuotidian stressors, with or without drug cues, can cause relapse to cocaine and other drugs . Sinha aRecurrent alcohol or cocaine use was reported to produce chronic stress effects, depleting oxytocin stores in the hypothalamus and amygdala , and incWe present in this paper the results of a human laboratory study investigating the patterns of ACTH secretion in response to a challenge with intranasal desmopressin and to another challenge in which intranasal desmopressin insufflation is preceded by a pre-treatment with intranasal oxytocin. In accordance with the findings that chronic stress increases the effect of vasopressin on ACTH secretion, we hypothesized: 1) Intranasal desmopressin would increase ACTH secretion in cocaine use disorder patients more than in a non-addicted control group; 2) Pre-treatment with intranasal oxytocin would blunt the ACTH secretion induced by intranasal desmopressin more so in the cocaine use disorder group than in a non-addicted control group. The primary outcome was participant ACTH levels. This study was conducted in parallel with a double-blind, randomized, placebo-controlled 6-week clinical trial of intranasal oxytocin (24 IU). Results from this study are already published . It reve22.1Participants were recruited by local advertising. The study was conducted at the STARS clinic, a research clinic in the Division on Substance Use at the New York State Psychiatric Institute (NYSPI) and Columbia University. It was approved by the NYSPI Institutional Review Board (IRB 6093) and all participants gave a written informed consent. Medical screening included a history and physical examination, urine toxicology to verify cocaine use in cocaine use disorder patients, pregnancy test for women, an electrocardiogram, a comprehensive metabolic panel, and complete blood count. We used the MINI International Neuropsychiatric Interview to confirm or refute the diagnosis of cocaine use disorder or of comorbid psychiatric disorder in cocaine patients and controls . To veri2.2This study consisted of two intranasal endocrine challenges administered on 2 separate but consecutive days . For cocOn Day 2, patients and controls were given first a pre-treatment with 24 IU of intranasal oxytocin before receiving the same intranasal desmopressin challenge. Measurements of ACTH levels in serum samples were collected at 10, 20, 30, 45, and 90 min after the desmopressin challenge, as well as 10, 20, 30 min after the oxytocin pre-treatment.After collection, serum samples were frozen at −20 °Celsius, packed on dry ice, and shipped to Nathan Kline Laboratory in Orangeburg NY for analysis by ELISA methodology . Intra- 2.3All cocaine use disorder patients and controls were trained by staff to self-administer intranasal solutions for the laboratory sessions . Solutio2.4For their participation in the study, each control and cocaine use disorder patients received $150. We covered transportation costs ($5).2.5ACTH secretion outcomes were analyzed using a longitudinal mixed effects model with a random intercept to account for between-subject variability and a random effect of time from baseline to account for within-subject repeated measures. The fixed effects of baseline ACTH, patient group, time, and endocrine challenge was included in the model. The 3-way interaction of group*time*challenge was tested to examine the primary hypothesis of whether ACTH secretion provoked by intranasal desmopressin or intranasal oxytocin/desmopressin differs between cocaine and control patients, while adjusting for their baseline ACTH secretion. If the 3-way interaction was not significant, it was removed from the model, as well as any non-significant 2-way interactions between variables group, time, and challenge in the model.33.1p = 0.012).3.2n = 25), mean (SD), baseline ACTH levels was 32.4 (15.7) and 30.9 (18.9) for controls (n = 11), which were not significantly different between groups . To simply describe the outcomes, mean and standard deviation baseline levels of ACTH were computed. For cocaine patients  suggesting no differences in ACTH secretion over time between endocrine challenge type and group. When the non-significant 3-way interaction was removed, there was a significant 2-way group*challenge interaction . 292 = 2.91, p = 0.004). The opposite was observed among controls: ACTH secretion averaged 3.3pg/ml/min lower after intranasal desmopressin than after intranasal oxytocin/vasopressin , while controlling for baseline ACTH levels. However, there was no significant difference in ACTH levels overall between patients and controls within either challenge. There were no gender differences between mean ACTH level changes related to the endocrine challenges.The 3-way interaction of group*time*challenge was not significant  would represent the usual circumstances of most cocaine patients in that repeated cocaine use occurs in absence of any therapeutic intervention.As stated in the introduction, the laboratory study sought to understand how intranasal oxytocin could raise the odds of abstinence by testing the theory of chronic stress for its relevance to the treatment of cocaine use disorder. While each study unveiled significant effects, we could not find associations that tangibly linked the results of the laboratory and clinical trial studies. Hence, questions remain:: 1) Does either the desmopressin-induced increase in ACTH, or the decrease in ACTH due to intranasal oxytocin pretreatment relate to the increase in odds of abstinence from cocaine seen in the clinical trial? The lack of association may be because: 1) despite the voluminous literature on stress and addiction see , this asSome clinical trials that have yielded positive outcomes point to promising mechanisms. Suggestive of dysregulation in the dopaminergic pathways  amphetamHowever preliminary, the results of this study and of our previously published clinical trial  imply thThis study was supported by a R21 award from the National Institute of Drug Abuse (NIDA) DA035461 and Supplement DA035461–02S1. NIDA did not have a role in the study design, collection, analysis, and interpretation of the data, nor in the writing of this manuscript or the decision by the authors to submit this report for publication.Dr. Wilfrid Noël Raby, PhD MD: Principal Investigator of this study. Dr. Raby conceptualized the study, applied for the R21 that funded the study, conducted data collection, supervised the integrity of the study and of the data collected, and wrote the manuscript. Matthew Heller, BSc MSc: Worked as a research assistant in the conduction of the study and reviewed all the collected data for accuracy. Demetrios Milliaressis, BSc, MSc, PhD (Honorary): Worked as a research assistant in the conduction, reviewed all the collected data for accuracy, and participated in the editing of the manuscript. Jean Choi MSc: Performed the data analysis for the study and participated in the final editing of the manuscript. Cale Basaraba, MPH: Participated significantly in the data analysis. Martina Pavlicova PhD: Oversaw the initial design of the study and the data analysis. Frances R. Levin MD: Provided mentorship and guidance to the PI, and participated in the editing of the final manuscript. Sarah Church PhD: Provided material support to the study. Edward V. Nunes MD: Provided mentorship and support during all phases of this study and participated in the editing of the final manuscript.NIDA: Nothing to Declare. Dr. Wilfrid Noël Raby, PhD MD: Principal Investigator of this study. The PI has no conflict of interest to declare. Matthew Heller, BSc MSc: No conflict of interest to declare. Demetrios Milliaressis, BSc, MSc, PhD (Honorary): No conflict of interest to declare. Jean Choi MSc: No conflict of interest to declare. Cale Basaraba, MPH: No conflict of interest to declare. Martina Pavlicova PhD: No conflict of interest to declare. Frances R. Levin MD: No conflict of interest to declare. Sarah Church PhD: No conflict of interest to declare. Edward V. Nunes MD: No conflict of interest to declare.NIDA: Nothing to Declare. Dr. Wilfrid Noël Raby, PhD MD: Principal Investigator of this study. Dr. Raby conceptualized the study, applied for the R21 that funded the study, conducted data collection, supervised the integrity of the study and of the data collected, and wrote the manuscript. The PI has no conflict of interest to declare. Matthew Heller, BSc MSc: Worked as a research assistant in the conduction of the study and reviewed all the collected data for accuracy. No conflict of interest to declare. Demetrios Milliaressis, BSc, MSc, PhD (Honorary): Worked as a research assistant in the conduction, reviewed all the collected data for accuracy, and participated in the editing of the manuscript. No conflict of interest to declare. Jean Choi MSc: Performed the data analysis for the study and participated in the final editing of the manuscript. No conflict of interest to declare. Cale Basaraba, MPH: Participated in the data analysis. No conflict of interest to declare. Martina Pavlicova PhD: Oversaw the initial design of the study and the data analysis. No conflict of interest to declare. Frances R. Levin MD: Provided mentorship and guidance to the PI, and participated in the editing of the final manuscript. No conflict of interest to declare. Sarah Church PhD: Provided material support to the study. No conflict of interest to declare. Edward V. Nunes MD: Provided mentorship and support during all phases of this study, and participated in the editing of the final manuscript.None of the authors hold any financial interests or partnerships with people or organizations that could bias the results or the writing of this manuscript.


The roles of presenilins in mitophagy, autophagy, mitochondrial dynamics, and many other functions, non-γ-secretase related, also require close attention in future research. Resolution of contradictions in understanding of presenilins cellular functions are needed for new effective therapeutic strategies for AD.This review uncovers the intricate relationship between presenilins, calcium, and mitochondria in the context of Alzheimer’s disease (AD), with a particular focus on the involvement of presenilin mutations in mitochondrial dysfunction. So far, it is unclear whether the impairment of mitochondrial function arises primarily from damage inflicted by  There are over 300 mutations in the presenilins genes, many of which result in excessive production of the most hydrophobic/aggregate form of Aβ42, leading to neurodegeneration and dementia in familial Alzheimer’s disease (FAD). Other mutations, as PS1 L286V or M146V and PS2 N141I, may increase the likelihood of apoptosis independently of amyloid via mitochondrial deficiency or calcium imbalance  are a family of highly conserved transmembrane proteins located in endoplasmic reticulum (ER), endosomes, and lysosomes, at ER-mitochondria contact sites, nuclear envelope, Golgi apparatus, but primarily in compartments near the cell surface . The tworocesses . Howevermbalance . In addimbalance . Ca2+ sitheories .γ-secretase activity, not only in relation to amyloidogenic processing, but also with regard to non-amyloid substrates and γ-secretase-independent functions of presenilins. These diverse functions encompass the modulation of calcium homeostasis, mitochondrial function, autophagy, and mitophagy. The hypothesis suggests a relative primacy of mitochondrial dysfunction over amyloid accumulation in the pathogenesis of AD and is currently subject to debate , which plays a key role in the transmission of regulatory and proliferative signals in cells. Increased phosphorylation of kinesin light chain (KLC) by GSK3β promotes the dissociation of kinesin-I from membrane-bound organelles (MBOs), leading to diminished motility of mitochondria and synaptic vesicles. In fact, cultured hippocampal neurons expressing the PS1 M146V mutation exhibited reduced density of both mitochondria and synaptic vesicles , which actively transports Ca2+ from the cytosol to the ER, thereby helping to maintain low resting levels of cytosolic Ca2+. PS2-mediated calcium sequestration by SERCA pumps is vital for refilling ER calcium stores  production remain poorly understood . Studieseability . Number eability . At the  channel . Howeveroverload . The optoverload . A reducoverload . Furtherm stores .2+ ER overload, both overexpression of normal presenilins and some FAD-related mutations in them have been associated with increased Ca2+ release from the ER, depletion of Ca2+ stores, and an increase in cytosolic Ca2+  membranes, with a particular emphasis in PS2. PS2 functions as a positive regulator of the calcium connection between ER and mitochondria . The comoduction . Strikinmulation . These emulation .2+ for improving Ca2+ transfer from ER to mitochondria , will help to resolve the existing contradictions and identify the root causes of mitochondrial dysfunction in neurodegeneration. Accurate determination of the role played by presenilin in the mitochondrial cascade would provide valuable guidance to researchers in their quest for therapeutic targets and the development of treatment and prevention strategies.While the role of mutant presenilins is unquestionable, it remains uncertain whether their presence alone is sufficient and which factors contribute to the implementation of FAD. Disturbances in calcium homeostasis and associated mitochondrial dysfunction are anticipated to have significant implications in aging and degeneration, making it an area of paramount importance for research. Additionally, further elucidation is necessary regarding the multiple functions of presenilins in various brain structures and cell types that may contribute to particular mitochondrial dynamics and bioenergetics. Given the apparent failure of drug therapies targeting at amyloid peptide clearance, it is imperative to focus on alternative hypotheses regarding the etiology of AD to identify effective therapeutic interventions.EK: conceptualization and writing-review and editing. MM and LK: literature research and analysis and writing. MM: visualization. EK and MP: supervision. All authors contributed to the article and approved the submitted version.This work was supported by #NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006)—A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Obesity, and especially visceral obesity, contribute to various health issues, including type 2 diabetes and cardiovascular diseases (CVD). While BMI is commonly used to diagnose obesity, it has limitations and does not consider factors like fat distribution or muscle mass. This study aims to assess the relationship between BMI, percent body fat, waist circumference (WC), waist-to-height ratio (WHtR), and cardiovascular risk factors in Polish military flying personnel. Methods: This study involved 200 men from the Polish Air Force aged 38.8 ± 8.5 years. Anthropometric tests, body composition tests, and tests of biochemical markers of CVD were conducted. (2) Results: The prevalence of overweight and obesity varied based on the evaluation criterion; they were present in 63.5% of soldiers by BMI and in 52.5% by percent body fat; abdominal obesity was present in almost half (47%) of the surveyed soldiers according to WC and in 62.5% according to WHtR. All markers of obesity correlated positively with various biochemical markers of CVD, and 8.5% of subjects met the criteria for metabolic syndrome. (3) Conclusions: The prevalence of obesity in Polish military flying personnel, regardless of the evaluation criterion, is associated with significant metabolic complications in the form of lipid disorders and insulin resistance. Obesity is recognized by the World Health Organization (WHO) as an epidemic of the 21st century. It is considered a major public health problem and the fifth most important cause of death worldwide , 12% in Obesity, especially visceral obesity, contributes to the development of type 2 diabetes, insulin resistance, cancer, osteoarthritis, sleep apnea, and cardiovascular disease (CVD), such as hypertension, coronary heart disease, heart failure, supraventricular ventricular arrhythmias, and dyslipidemia ,11. It aAdipokine interactions, insulin resistance (IR), chronic inflammation, and increased blood clotting are known to contribute to the development of cardiovascular disease in obese individuals ,16. IR c2), according to WHO criteria [2 , and obesity is diagnosed with a BMI ≥ 30 kg/m2. While this index is widely used around the world because of its simplicity of use, it has many limitations. The interpretation of BMI values in adults does not depend on age or gender, and it does not reflect the content or distribution of body fat , the ratio of body fat to muscle tissue, or hydration status. It is well known that the effect of increased BMI may be related to expanded muscle mass , which is relatively common among soldiers [In diagnosing obesity, the most commonly used marker is the body mass index (BMI), which is calculated by dividing body weight (kg) by height squared  is an alternative measurement of visceral fat. A systematic review published in 2010 concluded that WHtR may be advantageous because it avoids the need for age-, sex-, and ethnicity-specific values . Higher Hence, the aim of the presented study was to evaluate the relationship between BMI, body fat content, waist circumference, and waist-to-height ratio and parameters of carbohydrate, lipid, and hormone metabolism, as well as insulin resistance, which are cardiovascular risk factors in Polish military flying personnel. Additionally, we evaluated to what degree the method used to determine overweight and obesity affects the values of the cardiovascular risk factors.This study included 200 men (mean age 37.8 ± 8.5 years) who are members of the active military flying personnel of the Polish Air Force who took part in the National Health Programme 2016–2020 Project. They were individuals who consecutively reported for mandatory annual anthropometric examinations to the Laboratory of Dietetics and Obesity Treatment as part of routine adjudicatory and medical examinations at the Military Institute of Aviation Medicine in Warsaw, Poland. They expressed a desire to participate in additional examinations carried out as part of the National Health Program 2016–2020. All of them had current fitness to fly certificates given by the Aeromedical Board . All procedures were conducted in September–November 2018. All procedures were approved by the Institutional Review Board of the Military Institute of Aviation Medicine, Warsaw, Poland (decision No. 01/2018 of 9 March 2018), and this study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All participants signed informed consent forms.The degree of overweight and obesity was assessed according to the body mass index (BMI) according to the World Health Organization (WHO) criteria   was also calculated. WHtR is defined as waist circumference divided by height; body height was measured with a Harpenden Anthropometer  to the nearest 1 mm in a standing, upright position without shoes. Physical measurements, including weight, standing height, and the circumferences of the waist and hips, were carried out according to standard protocol by trained assistants. Smoking habits were determined by self-questionnaires.-Total cholesterol (TC) . Values < 190 mg/dl were taken as the norm.-Triglycerides (TG) . Values of ≤150 mg/dl were taken as the norm.-Low-density lipoproteins (LDL-C) . Values < 115 mg/dl were taken as the norm.-High-density lipoproteins (HDL-C) .-Glucose . Values < 99 mg/dl were taken as the norm.-Insulin . Values of 2.6–24.9 µIU/mL were taken as the norm.-Ghrelin .All examinations were performed on soldiers after an overnight fast. Blood samples were taken in the morning around 7 o’clock. Concentrations of the following biochemical markers were determined using standard methods: Insulin resistance was diagnosed based on the insulin resistance index Homa-IR (Homeostatic Model Assessment) using the following formula :HOMA = [Blood pressure was checked by an internal medicine physician. All of the studied soldiers had blood pressures within the normal range. Nevertheless, it should be noted that we cannot exclude cases of treating hypertension outside the military health service and not informing the air medical board of this fact for fear of not receiving a positive opinion from the medical examiner. 2 and, at the same time, fasting glucose ≥ 100 mg/dl and non-HDL cholesterol ≥ 130 mg/dl, where non-HDL cholesterol = total cholesterol − HDL cholesterol) [Based on the results of these tests, the percentage of soldiers meeting the criteria for metabolic syndrome (waist circumference ≥ 102 cm or BMI ≥ 30 kg/mesterol)  was calcThe results of the above tests were compared according to the eligibility criteria for overweight and obesity, both by (1) BMI, (2) body fat percentage, and (3) waist circumference. In addition, the results of laboratory tests were compared with reference ranges and presented as the percentage of subjects with out-of-normal parameters, according to BMI and percent body fat. The latter is due to the fact that the effect of increased BMI may be related to expanded muscle mass , which is relatively common among soldiers ,25,26.2 and Kruskal–Wallis tests were used. If the Kruskal–Wallis test confirmed differences between groups, multiple comparisons of mean ranks were then used for all groups. Relationships between variables were checked using the Spearman correlation. A statistical significance value of p < 0.05 was assumed, and the normality of distributions was checked using the Shapiro–Wilk test. All analyses were performed using STATISTICA version 13.0 PL software.The results for quantitative variables were presented in the form of descriptive statistics  and, for qualitative variables, in the form of frequencies and graphs. For comparative analyses between groups , chiThe main demographic variables are presented in As expected, the prevalence of overweight and obesity in the surveyed soldiers differed according to the evaluation criterion see . As an ip < 0.001) compared to overweight and normal weight subjects. This relationship was not observed between groups defined according to percent body fat (p > 0.05)  of the surveyed soldiers according to WC, and in 62.5% according to WHtR . However > 0.05) .p < 0.001). In the obese soldiers, compared to the remaining soldiers, both average waist circumference (107 cm vs. 84 cm) (p < 0.001) and the WHtR were higher .Soldiers with diagnosed obesity according to BMI had significantly higher scores on all body composition and anthropometric parameters  than thop > 0.05; see p < 0.001). The comparative analysis of total body water content, lean body mass, and skeletal muscle mass in the groups of soldiers divided according to body fat content did not show any significant differences (p > 0.05). The results of laboratory blood tests of military flying personnel divided into groups according to BMI, body fat content, and waist circumference are shown in p < 0.001).The statistical analysis showed no significant differences between the ghrelin and blood glucose concentrations of the soldiers studied in groups divided by BMI and body fat content . Signifip < 0.05). Nutritional status was not related to ghrelin concentrations in the blood of the subjects , is not suitable for assessing percent body fat. The main limitation of BMI, as mentioned in the introduction, is that it is impossible to distinguish BF from lean mass and central fat from peripheral fat . This isSimilar observations were made by De Lorenzo et al.  in compaThe results of this study also indicate high prevalence of lipid disorders among Polish military flying personnel , which is consistent with unfavorable trends in the general population. Depending on the study sample selection, the prevalence of dyslipidemia in Poland is estimated at 60–80% of individuals in a population over 18 years of age . Lipid mOur study found a significant association between soldiers’ obesity and carbohydrate–lipid disorders. The subjects with diagnosed obesity had significantly higher levels of biochemical parameters, such as total cholesterol, triglycerides, LDL-C, insulin levels, and HOMA-IR index, and lower HDL-C levels than participants at normal weight. In the US military, similar trends are observed . These dThe health situation is much more favorable in the French army, where among the 1589 soldiers surveyed, less than 5% of the population reported a diagnosis of comorbidities: diabetes 2%, high blood pressure 1%, and dyslipidemia 1.5% . The large number of overweight soldiers with unfavorable body fat distribution observed in our study group is worrisome, because in light of current data, obesity of the visceral type is an independent and significant risk factor for atherosclerosis and its complications, hypertension, and type II diabetes, among others. One of the direct consequences of visceral obesity is insulin resistance. The result of reduced tissue sensitivity to insulin is hyperglycemia and lipid disorders (mainly hypertriglyceridemia). Increased insulin secretion by pancreatic islet beta cells gradually leads to their exhaustion and the development of pre-diabetic states and diabetes. Hyperglycemia, lipid disorders, and hyperinsulinemia causing endothelial dysfunction and vascular wall remodeling accelerate the development of atherosclerosis and the onset of its clinical consequences . In the Military flying personnel, due to the tasks they perform, should be characterized by impeccable health, which, unfortunately, our study did not confirm. A prescription for existing metabolic disorders in the military environment could be the dissemination of the Mediterranean diet as a recommended dietary model. This is because this diet has many health benefits . StudiesAs this is a cross-sectional observational study, is was only possible to elucidate associations between various measures of obesity and risks for CVD (including insulin resistance). Future follow-up studies will allow us to evaluate causal relationships between these measures in this cohort. Additionally, we did not measure the levels of spontaneous physical activity or diet of the studied soldiers, which are factors known to counteract the development of metabolic disorders. This will also be taken into account in further studies.The findings suggest that the prevalence of obesity in Polish military flying personnel, regardless of the evaluation criterion, is associated with significant metabolic complications in the form of hyperlipidemia and insulin resistance, all of which are risk factors for cardiovascular disease. Significant differences in blood glucose concentrations were noted only between groups divided according to waist circumference. Yet BMI alone should not define obesity, which is particularly important in the case of military personnel, who are often found to have greater skeletal muscle mass than in the general population. Hence, it is recommended to include in the assessment of the nutritional status of soldiers routine body composition measurement tests in order to avoid overestimation or underestimation of obese individuals in this professional group. Prevention programs aimed at early assessment and modification of cardiovascular risk factors in the military flying personnel population are sorely needed, especially those aimed at weight reduction. In addition, the outcome of this study suggests that measurement of blood insulin levels should be included in mandatory medical certification examinations, along with glucose levels, to counteract the development of diabetes.


Staphylococcus aureus and gram-negative Escherichia coli by 85% and 75%, respectively. This may be attributed to the more complex cell membrane structure of E. coli compared to S. aureus. This demonstrates the potential of nanoemulsion-based PDT as an effective alternative to traditional antibiotics for treating multidrug-resistant microorganisms.Multidrug-resistant microorganisms have become a significant public health threat, and traditional antibiotics are becoming ineffective. Photodynamic therapy (PDT) is a promising alternative that utilizes photosensitizers and light to produce Reactive Oxygen Species (ROS) that can kill microorganisms. Zinc phthalocyanine (ZnPc) is a promising photosensitizer due to its strong affinity for encapsulation in nanoemulsions and its antimicrobial properties. In this study, nanoemulsion was prepared using Miglyol 812N, a surfactant, and distilled water to dissolve hydrophobic drugs such as ZnPc. The nanoemulsion was characterized by its particle size, polydispersity index, Transmission Electron Microscope and Zeta potential, and the results showed that it was an efficient nanocarrier system that facilitated the solubilization of hydrophobic drugs in water. The use of ZnPc encapsulated in the nanoemulsion produced through the spontaneous emulsification method resulted in a significant reduction in cell survival percentages of gram-positive  Staphylococcus aureus and Escherichia coli, which can lead to severe local and systemic complications. S. aureus is responsible for a range of illnesses from minor skin infections to life-threatening conditions such as meningitis, pneumonia, endocarditis and sepsis [E. coli causes both intestinal and extraintestinal infections, including bloodstream infections, pneumonia, skin abscesses and meningitis [Common bacterial infections, including cellulitis, abscesses and postsurgical infections, are typically caused by pathogens such as d sepsis . E. coliningitis .However, over the years, many bacteria have developed resistance to antibiotics, which poses a significant threat to public health ,4. As a Photodynamic therapy was identified as a promising technique against bacteria, fungi and viruses, as well as an anticancer therapy, as early as 1990 . AntimicaPDT uses a non-toxic dye called a photosensitizer (PS) that is activated by light in the presence of molecular oxygen (O2) of foreign cells to produce Reactive Oxygen Species (ROS), which can induce cytotoxic effects . PSs tenS. aureus and gram-negative bacteria such as E. coli is highly influenced by the structure of the photosensitizer (PS). The difference in inactivation between the two is associated with the cell wall structure. Gram-positive bacteria have single cell walls composed of peptidoglycan with teichuronic acids and lipoteichoic, which induce permeability. Conversely, the cell wall of gram-negative bacteria is more complex, comprising the peptidoglycan layer, lipopolysaccharides (which are strongly negatively charged), phospholipids, lipoproteins and proteins [The effectiveness of photoinactivation on gram-positive bacteria such as proteins ,17.Several strategies have been developed to increase the penetration of light-sensitive agents into bacterial cells and improve the effectiveness of aPDT. The first step is the selection of appropriate photosensitizers such as phthalocyanines, which are used as dyes and pigments. They belong to the second PS generation and have an intense blue-green color with strong absorption in the red visible region (approximately 600–760 nm), enabling deeper light penetration, efficient ROS generation, high photostability, low toxicity in the dark and easy chemical modification compared to other photosensitizers ,19. The S. aureus and gram-negative E. coli.Therefore, the aim of this study is to assess the effect of antimicrobial photodynamic therapy using Zn phthalocyanine encapsulated in nanoemulsion on gram-positive Photosensitizer: Zn phthalocyanine was purchased from Sigma Aldrich Co., Ltd. ;Solvents:High-performance liquid chromatography (HPLC) Acetone was purchased from ;Tetrahydrofuran (THF) was purchased from ;Essential Oil: Miglyol 812N was gifted from Cremer oleo division, Germany;Biopolymer surfactants: Poloxamer 188 was purchased from (Sigma Aldrich Co.);Lipophilic surfactant: Span 80 was purchased from (Sigma Aldrich Co.);Hydrophilic surfactant: Tween 80 was purchased from .S. aureus ATTCC6538 and E. coli ATTCC8739) were obtained from the Department of Botany and Microbiology, Faculty of Science, Cairo University.The bacterial strains used in this study  at the NAWAH scientific center, Egypt, was used to measure the particle size distribution (mean size), polydispersity index (PdI), zeta potential and viscosity;The Transmission Electron Microscope (TEM)  at the National Research Centre (NRC), Cairo, Egypt, was used to characterize the structure and morphology of the nanoemulsion (NE) and Zn Phthalocyanine encapsulated in NE (NE + ZnPc). The total magnification was 8.00 kx, and the accelerating voltage was 200 kV;A light system composed of a red diode light  at the National Institute of Laser Enhanced Science (NILES), Cairo University, Egypt, was used to irradiate the photosensitizer;The water-miscible solvents (acetone and Tetra Hydro Furan) were evaporated in a rotary evaporator device at the National Research Centre, Cairo, Egypt, after complete homogenization of the nanoemulsion and the nanoemulsion with phthalocyanine;The Vortex Heidolph reax top  at the National Research Centre, Cairo, Egypt, was used to mix every mixture thoroughly.The nanoemulsion was prepared using a modified combination of two methods ,24 of spTo prepare the Zn phthalocyanine encapsulated NE, an organic solution (1) was initially prepared following the same method described in All formulations (NE and NE + ZnPc) underwent characterization based on their particle size distribution (mean size), polydispersity index (PDI) , zeta potential and viscosity. The mean size and PDI of colloidal dispersions were determined at 22 °C using the Backscattered laser-amplified scattering reference method at an angle of 180°. The zeta potential was measured based on the electrophoretic mobility of nanoparticles between electrodes in an electric field . All assThe structure and morphology of the NE and NE + ZnPc were characterized by diluting concentrated nanoemulsion in distilled water (1:10) by placing a small drop of the diluted sample onto a holey film grid and allowing it to dry. The samples were then observed under a transmission electron microscope (TEM) to examine their structures and morphologies.S. aureus was cultured on Tryptic Soy Agar-TSA, while E. coli was cultured on Luria-Bertani (LB) agar. Overnight cultures of S. aureus and E. coli were prepared by inoculating 10 mL of TSB and LB broth, respectively, followed by incubation at 37 °C. For the experiments, each culture was collected after serial dilution; S. aureus was diluted 1/10 (150 CFU/mL), while E. coli was diluted 1/100 (500 CFU/mL) due to its faster growth rate compared to S. aureus [. aureus .S. aureus and E. coli were transferred separately to cell culture plates containing 6 wells each and then exposed to red diode light with an irradiance of 38.1 mW/cm2 [For each bacterial strain, 50 µL of 1 mW/cm2  for varyS. aureus and E. coli were transferred to a cell culture plate according to A total of 50 µL aliquots of As described in S. aureus and E. coli were transferred to separate wells of a cell culture plate and then treated with different concentrations  of the safety concentrations of Zn phthalocyanine encapsulated in the nanoemulsion and distilled water. After half an hour of incubation in the dark, the samples were exposed to red diode light for varying times, as indicated in S. aureus and E. coli. The samples were then mixed thoroughly, and serial dilutions were plated onto Tryptic Soy Agar. Colonies were counted as previously described.Aliquots of 50 µL of S. aureus and E. coli were individually transferred to a cell culture plate and then added to the best concentration of NE + ZnPc of 1000 µL and distilled water. One group was directly exposed to red diode light without any incubation time (control), while the other groups were incubated for 30 min, 1 h, or 2 h with the prepared Zn phthalocyanine encapsulated in the nanoemulsion and each type of bacteria with distilled water before exposure to the red diode light, as shown in This experiment aimed to demonstrate the importance of incubation time between Zn phthalocyanine encapsulated in a nanoemulsion and each type of bacteria with distilled water prior to photodynamic therapy. For this, aliquots of 50 µL of The shape and color of the nanoemulsion produced and the nanoemulsion with phthalocyanine are shown in The morphology of NE and NE + ZnPc was characterized using transmission electron microscopy (TEM), as illustrated in The results demonstrated that both NE and NE + ZnPc samples exhibited desirable nanometric size distributions, as shown in S. aureus at 1 min. However, after 5 min, the bacterial survival percentage started to significantly decrease (p < 0.05), though, at 20 min, there was an increase in the bacterial survival percentage. In the case of E. coli, no significant light effect was observed on the bacterial survival percentage at 1 min or 5 min, but a significant decrease (p < 0.05) in the bacterial survival percentage was noted after 10 min, 15 min and 20 min  in an oil-in-water (O/W) nanoemulsion as a drug carrier using Miglyol 812N oil (triglyceride ester of saturated coconut) and use this prepared nanoemulsion with Zn phthalocyanine as an antimicrobial photodynamic therapeutic agent to control multidrug-resistant bacteria. The efficiency of antimicrobial photodynamic therapy can be greatly enhanced based on the photosensitizer type and the delivery system’s presence.Phthalocyanine was chosen as the preferred photosensitizer, a second-generation photosensitizer, due to its higher oxidative power and its ability to produce singlet oxygen effectively ,34. PhthIn this study, Zn phthalocyanines were chosen as the photosensitizer for aPDT applications due to their efficient encapsulation in nanoemulsions, making them better than other photosensitizers such as Aluminum chloride phthalocyanine entrapped in nanoemulsion, as reported in previous studies ,38,39. HPrevious studies utilized photosensitizers dissolved in aqueous media such as saline or DMSO, but these media were reported to be unstable and could cause damage to tissues and an increase in host cell permeability . Hence, The particle size of the nanoemulsion is critical for intravascular delivery, as the diameter of the smallest blood capillaries in the human body is in the 4–7 µm range, and larger particles may cause capillary occlusion. TEM measurements were performed to determine the size, morphology and size distribution of the prepared nanoemulsion. The results showed a nanometric size range of 30–500 nm for both the NE and NE + ZnPc formulations, as shown in The Zeta potential is a crucial factor for predicting the dispersion stability of nanoemulsions and measuring the surface charge of particles when suspended in a liquid. The value of Zeta potential depends on the physicochemical properties of the system. A positive zeta potential value of ±30 mV or higher is generally considered sufficient for ensuring the physical stability and long lifetime of the nanoemulsion . In our Viscosity is another crucial parameter for characterizing the physicochemical properties of nanoemulsions. It helps determine whether the system is an oil-in-water (O/W) or water-in-oil (W/O) nanoemulsion. Low viscosity indicates that it is an O/W type, while high viscosity indicates a W/O type nanoemulsion. Viscosity measurements for our prepared nanoemulsions were less than 21 cP or 30 cP, with a minimum viscosity of 10.68 cP reported in previous studies ,46,47. ITo ensure that the nanoemulsion and phthalocyanine encapsulated in the nanoemulsion, as well as red diode light, had no bactericidal effects on bacteria, repeatable dark toxicity experiments were conducted. Specific concentrations of the non-toxic effects of the nanoemulsion and Zn phthalocyanine encapsulated in the nanoemulsion alone were determined to be 500, 700 and 1000 µL, and the minimal emission time with red diode light alone that had no bactericidal effect was determined. The results showed that toxic products (ROS) of antimicrobial photodynamic therapy (aPDT) were only generated when the photosensitizer was excited by a light source of a specific wavelength of 625 nm. This restricted oxidative damage to the site of the photosensitizer .S. aureus was 10 min, while in E. coli, it was 15 min. In previous studies, it was reported that the minimal safety time for red diode light alone that kept bacteria growth in S. aureus was also 15 min, whereas in E. coli, it was 30 min [E. coli required more time to be killed due to the complexity of its cell wall structure, which is more complex than that of S. aureus, as shown in S. aureus after 20 min. This may be caused by a mutation, which may have increased the bacterial resistance [S. aureus and E. coli with red diode light produced reactive oxygen species that caused oxidative damage sufficient to kill multidrug-resistant bacteria.Our dark toxicity experiments showed that the minimal safety time for red diode light alone that kept bacteria growth in s 30 min . This susistance . The bacS. aureus and E. coli, respectively, as shown in S. aureus compared to E. coli. The bactericidal effect against S. aureus was more effective than against E. coli due to differences in their cell membrane structures, as discussed previously and demonstrated in E. coli and S. aureus [S. aureus, E. coli and Candida albicans [The antimicrobial photodynamic therapy resulted in a photokilling percentage of 85% and 75% for . aureus . Additioalbicans .The incubation time of leaving the prepared Zn phthalocyanine encapsulated in nanoemulsion with bacteria in the dark before the antimicrobial photodynamic therapy is critical, and about half an hour is required as the optimum incubation period. Without this incubation time, there was no inhibition in the cell number. This incubation time allowed Zn phthalocyanines encapsulated in the nanoemulsion to easily enter strange cells such as multidrug-resistant bacteria, which then underwent antimicrobial photodynamic effects after exposure to red diode light. However, in case of longer incubation than 30 min, the cells’ mortality is caused by the toxicity of Zn phthalocyanines encapsulated in the nanoemulsion and not due to applying photodynamic therapy. The uptake of phthalocyanine (Pc) was observed on the cell wall as well as in the cytoplasm of the HA-MRSA after three hours of incubation, as reported in . AnotherThis study showed an interesting alternative method that maintains the hydrophobicity of the photosensitizer and allows it to function effectively by encapsulating it in the nanoemulsion in the nanometric range. This presents a synergistic approach for future trials in aPDT. Photodynamic therapy (PDT) has been recently demonstrated to be a powerful strategy for tumor destruction, and nanotechnology is also suggested to reduce treatment dosage and frequency by enabling the delivery of drugs within confined tissues using nanoemulsions . SeveralS. aureus and E. coli, respectively. This successful formation of Zn phthalocyanine encapsulated in nanoemulsion opens the door for the use of hydrophobic photosensitizers and drugs in various biological and medical applications. Future research will focus on investigating the positive effects of Zn phthalocyanine encapsulated in nanoemulsion as an antimicrobial photodynamic therapy agent for oral, skin, dental and cancer treatment.This study introduces a novel approach for using hydrophobic photosensitizers in aqueous media. The process involves encapsulating Zn phthalocyanine in a nanoemulsion using solvents, essential oils, biopolymers, lipophilic surfactants and hydrophilic surfactants. The resulting nanoemulsion is well-characterized, with a size range of 30–500 nm, and is stable at room temperature for an extended period. This approach has been shown to be effective in antimicrobial photodynamic therapy, with a photokilling percentage of 85% and 75% against