Oocyte in vitro maturation has massive potential for the generation of great numbers of embryos for research and for the application of assisted reproductive technologies, such as in vitro embryo production. However, the developmental ability of in vitro matured oocytes is lower than those matured in vivo. Here, incubating the oocytes with cAMP modulating agents for two hours before in vitro maturation decreased oocyte DNA damage and increased the number of embryos generated after in vitro fertilization. The present findings could help to develop new methods to improve the quality and developmental potential of in vitro matured oocytes.p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control . Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased ( Despite being one of the most promising assisted reproductive technologies, in vitro maturation (IVM) has become a major limiting factor for the in vitro production (IVP) of viable embryos in different species . Even wiBefore ovulation, oocytes arrest at the prophase I of the meiotic cycle . During Oocytes can also undergo nuclear and cytoplasmic maturation in vitro. Still, the two processes are greatly unsynchronized since the physical removal of cumulus-oocyte complexes (COCs) from antral follicles spontaneously resumes meiosis . This unFor this reason, alternative IVM systems that temporarily maintain high intra-oocyte concentrations of cAMP, and thus meiotic arrest, have been proposed . TheHence, the aim was to evaluate cAMP modulators forskolin and IBMX effects on completion of meiosis, cytoplasmic reorganization, and cumulus cells’ activity in sheep COCs, and their contribution to subsequent embryonic development. In this context, the present study examined oocyte and cumulus cell quality parameters that have not been investigated before, especially in the ovine species.All chemicals and reagents were purchased from Merck Life Sciences  unless otherwise indicated.2 and maximal humidity before IVM. The rationale behind supplementing the selection and wash media with cAMP modulators before pre-IVM culture was to avoid cAMP levels dropping immediately after COCs’ collection [Ovaries from Manchega or mixed-breed adult ewes of around 6 years old  were collected at a slaughterhouse and transported in saline solution (8.9 g/L NaCl #S9625), supplemented with penicillin (0.1 g/L #P3032) and streptomycin (0.1 g/L #S6501) at 25 °C for approximately 2 h. Immature cumulus-oocyte complexes (COCs) were retrieved from the follicles using a scalpel blade in 2 mL of collection medium (TCM199 medium (#M4530), supplemented with 4 µL/mL of gentamicin (#G1272), 2.38 mg/mL of HEPES (#H4034), 2 µL/mL of heparin , and 4 µL/mL of gentamicin (#G1272)). Immediately, the COCs with clear or moderately granular ooplasm surrounded by at least three layers of packed cumulus cells were selected and homogeneously distributed in two types of selection media: control  and pre-IVM , and 100 µM of forskolin ). Then, the COCs were washed in TCM199 and 4 µL/mL of gentamicin (control) or TCM199, 4 µL/mL of gentamicin, 500 µM of IBMX, and 100 µM of forskolin (pre-IVM). Subsequently, control COCs were subjected to IVM, while pre-IVM COCs were incubated in TCM199, 4 µL/mL of gentamicin, 500 µM of IBMX, and 100 µM of forskolin for 2 h at 38.5 °C, in 5% COllection .n = 100, 5 replicates) by the chemiluminescent immunoassay (CLIA) using the DetectX© High-Sensitivity Direct Cyclic AMP Kit, Acetylated Format . Briefly, after a 2 h incubation with cAMP modulators, some COCs were denuded by pipetting in 500 µL of PBS-PVA and 500 µM of IBMX, and washed to remove debris from the cumulus cells. Denuded oocytes were transferred to a 1.5 mL tube containing 125 µL of Sample Diluent, immediately frozen in liquid nitrogen, and stored at –80 °C until the analysis day. The CLIA assay was performed according to the manufacturers’ acetylated protocol. All reactions were read on a Sinergy HT luminescence plate reader  at 0.1 s reading time per well. The cAMP concentration was calculated by interpolating luminescence units on a standard curve generated from known amounts of cAMP with the MyAssays application.Intra-oocyte cAMP concentration was assessed in pools of 10 oocytes , 10 µg/mL of follicle-stimulating hormone (FSH), and 10% fetal calf serum (#F0804). Then, medium was covered in mineral oil , and COCs were incubated for 24 h at 38.5 °C, 5% COUpon maturation, a total of 371 oocytes (5 replicates) were washed in PBS-PVA (#P4417 and #P8136), denuded from cumulus cells by gentle pipetting, and placed in a glass slide with a 1 µL drop of Slowfade™  and 5 µg/mL of Hoechst 33342 (#14533) under a coverslip. After 10 min at room temperature, chromatin configurations were analyzed by fluorescence microscopy . Oocytes showing a germinal vesicle (GV) chromatin configuration were considered immature, and those showing a metaphase plate and a polar body were categorized as matured metaphase II (MII) oocytes . Early apoptosis was evaluated in 108 mature oocytes (5 replicates) using Annexin V staining , following the manufacturers’ protocol. Denuded oocytes were incubated in Annexin V and 100 µg/mL of propidium iodide  for 20 min at 37 °C on a heated plate in the dark. After washing three times in PBS-PVA, oocytes were mounted on slides in a 1 µL drop of Slowfade™ and 5 µg/mL of Hoechst 33342. Oocyte nuclei were observed with an epifluorescence microscope . Oocyte status was classified into the following categories: viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), and dead  and 10 µM of Cell Tracker Blue  for 30 min at 38.5 °C in the dark and 5% CO2 to detect intracellular reactive oxygen species (ROS) and reduced glutathione (GSH), respectively. The oocytes were then washed thrice in PBS-PVA and placed on slides for evaluation. Fluorescence intensity was observed by epifluorescence microscopy  were incubated in 10 µM of CM-H2 in the dark. The oocytes were then washed thrice under agitation for 5 min and mounted on slides with a 1 µL drop of Slowfade™ and 5 µg/mL of Hoechst 33342. Fluorescent images were recorded on a Nikon A1 confocal microscope . Lasers of 408 and 561 nm were used to excite Hoechst and MitoTracker Red dyes, sequentially. To determine mitochondrial distribution patterns, 102 mature oocytes (5 replicates) were subjected to a double staining with MitoTracker© Red CMXRos , a mitochondrial-specific probe, and Hoechst 33342 to stain the chromosomes. Following IVM, oocytes were incubated for 30 min in PBS-PVA plus 500 nM of MitoTracker© Red CMXRos at 38.5 °C and 5% COMitochondrial distribution patterns were classified as previously reported, with some modifications : (1) finTo identify the distribution pattern of cortical granules, a total of 79 denuded mature oocytes (4 replicates) were incubated in 4% paraformaldehyde (#P6148) for 1 h at room temperature. After washing thrice under a 5 min agitation in PBS-PVA, oocytes were permeabilized in 1% Triton (#T8787) for 1 h at room temperature and stained in 30 µL drops of 100 µg/mL of fluorescein isothiocyanate-conjugated peanut agglutinin  for 30 min at 37 °C on a heated plate and in the dark. After staining, oocytes were washed thrice in PBS-PVA, mounted on slides with 1 µL drops of Slowfade™ and 5 µg/mL of Hoechst 33342, and evaluated using a Nikon A1 confocal microscope . Lasers of 408 and 488 nm were used to excite Hoechst and FITC-PNA dyes, sequentially. Matured (MII) oocytes were classified into two groups according to the observed distributional pattern of the cortical granules, as previously described , with soTerminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to detect DNA fragmentation in mature sheep oocytes (4 replicates). A total of 75 oocytes fixed in 4% paraformaldehyde were permeabilized in 0.5% Triton X-100 for 1 h at room temperature. Then, the In Situ Cell Death Detection kit (#11684795910) was used to detect DNA strand breaks following the manufacturers’ instructions. Briefly, oocytes were placed in 30 µL drops of TUNEL reagent with deoxyuridine 5-trisphosphate (dUTP)-conjugated isothiocyanate fluorescein and incubated for 1 h at 37 °C. The positive control was pre-incubated with DNAse  for 1 h at 37 °C, while the negative control was incubated in the absence of deoxynucleotidyl transferase enzyme. After that, oocytes were washed thrice in PBS-PVA and placed on slides in a 1 µL drop of Slowfade™ with 5 µg/mL of Hoechst 33342. Samples were evaluated by epifluorescence microscopy . Oocytes with DNA damage, e.g., with a fragmented nucleus, were classified as TUNEL-positive, and those without damage as TUNEL-negative .® Kit  in pools of approximately 10 oocytes and following the protocol by Bermejo-Álvarez et al. [−ΔΔCT methods [BMP15), DNA polymerase gamma 2, accessory subunit (POLG2), gap junction alpha-1 protein , growth differentiation factor 9 (GDF9), nuclear respiratory factor 1 (NRF1), and transcription factor A, mitochondrial (TFAM). Quantification was normalized against that of the endogenous control (Peptidylprolyl Isomerase A (PPIA)).The relative quantification of mRNA transcript abundances was measured by quantitative real-time PCR, as previously reported by Sánchez-Ajofrín et al., with minor modifications . The RNAz et al.  , as previously described [2DCFDA  for 30 min at 38.5 °C, followed by 0.5 µM of PI staining. A compensation overlap was performed before each experiment, and 1000 events were acquired per sample. The raw data were analyzed using IDEAS® software (AMNIS), and out-of-focus cells, debris, and cell clumps were excluded from the analysis  were placed in 4-well dishes containing 500 µL of fertilization medium: synthetic oviductal fluid  and oocytes were co-incubated at 38.5 °C in 5% CO2 and maximal humidity. After approximately 18 h post-insemination (hpi), putative zygotes were washed by repeated pipetting and transferred to 25 µL drops (about one embryo per µL) of culture medium ), covered with mineral oil, and cultured until day 8 post-insemination (dpi) at 38.5 °C in a humidified atmosphere and 5% CO2, 5% O2, and 90% N2 in air. Cleavage and blastocyst rates were checked at 48 hpi and 6, 7, and 8 dpi, respectively.After IVM, control and Pre-IMV groups of 40–45 mature oocytes (uid (SOF ), supplep ≤ 0.05 were considered significant, and results are presented as mean ± SEM.Statistical analyses were performed using the IBM SPSS 24.0  software. Data were tested for normal distribution (Kolmogorov–Smirnov and Shapiro–Wilk tests) and homogeneity of variances (Levene test). Data expressed as percentages were transformed into arcsine (or logarithm) for statistical analysis but reported as percentages to make them understandable. Concentration of cAMP, maturation rates, oocyte viability, apoptosis, mortality, oxidative status, mitochondrial distribution, embryo development (5 replicates), and mRNA transcript abundance, DNA fragmentation, cortical granules’ distribution, and cumulus cells’ activity (4 replicates) were analyzed by factorial ANOVA followed by the Bonferroni post hoc test. Differences with probabilities of p < 0.05) intra-oocyte cAMP levels (4.12 ± 0.43 pmol/mL) compared to the control treatment  resulted in higher (p < 0.05) in pre-IVM (2.08% ± 3.51%) oocytes when compared to the control group  in the percentage of live, early apoptotic, and dead oocytes between the control  and pre-IVM . Although not significant (p > 0.05), the intracellular ROS and GSH contents were lower and higher, respectively, in pre-IVM oocytes (ROS = 18.20 ± 16.95 and GSH = 82.23 ± 17.99) compared to the control  were detected for BMP15, GDF9, Cx43, NRF1, POLG2, and TFAM relative abundances between control and pre-IVM oocytes.Cumulus cell-free oocytes were subjected to qPCR analysis to study mRNA transcripts related to meiotic competence and oocyte quality. As shown in p > 0.05) with respect to each other.In most MII oocytes, irrespective of the treatment, the distribution of mitochondria was homogenous granular or heterogeneous clustered  in both experimental groups.The percentage of cortical granule distribution patterns in MII oocytes is presented in To determine the developmental consequences of forskolin and IBMX supplementation, following pre-IVM, COCs were subjected to standard IVM, IVF, and embryo culture to Day 8.p > 0.05) in terms of oocyte maturation and cleavage rates, the percentage of blastocysts from both the initial number of oocytes and cleaved embryos at 48 hpi was significantly increased (p < 0.05) in the pre-IVM group (Although pre-IVM and control oocytes did not show differences (VM group .p > 0.05) to the control treatment.As shown in To the best of our knowledge, this is the first study to examine the effect of cAMP modulators on a series of nuclear and cytoplasmic maturation features of IVM sheep oocytes, including the redistribution of cytoplasmic organelles, DNA fragmentation, transcription of mRNAs, and cumulus cells’ activity. For this, immature sheep oocytes were cultured for 2 h with IBMX, a PDE inhibitor that halts cAMP degradation, and forskolin, a potent adenylate cyclase activator. Following IVM, oocytes were fertilized and cultured in vitro up to the blastocyst stage.Considering that cAMP modulators aim to synchronize nuclear and cytoplasmic maturation events , we focuDuring germinal vesicle breakdown (GVBD), physiological DNA single- and double-strand breaks  form during the displacement and separation of chromosomes. These damaged DNA fractures need to be repaired quickly to ensure the successful completion of maturation and preserve genomic stability in the oocyte . HoweverIn our study, control oocytes reached the MII stage at the end of the culture period, similar to pre-IVM oocytes. These results are in agreement with recent studies in cattle  and horsWith reduced DNA fragmentation, pre-IVM oocytes developed to the blastocyst stage to a greater extent than control oocytes. Previous studies suggested that oocytes may accumulate sufficient factors, such as mRNAs and proteins, to preserve the genome until the zygotic genome is activated ,45. ThesBased on this evidence, the results suggest that the impact of intra-oocyte cAMP modulation on subsequent in vitro embryo development is linked to a decrease in oocyte DNA damage after IVM. This previously unappreciated action of the cAMP modulators forskolin and IBMX may reflect an integral role in ensuring the maternal genomes’ integrity required for the correct initiation of embryonic transcription in vitro. Therefore, ascertaining whether gamete exposure to cAMP modulators before IVM induces specific DNA repair or cell cycle checkpoint genes could help to develop improved methods for assisted reproduction technologies in animal and clinical research. Nevertheless, since post-mortem ovaries were used in the present study, further investigation will be necessary to account for the variability of the oocyte source.


FLT3/FLT3L induction 3 h after TBI, which was enhanced by EI. The FLT3L induction resulted in phosphorylation of FLT3 in CD11c+ dendritic cells, which enhanced protein synthesis, maturation process, and the immunity of dendritic cells, shown by pS6, peIF2A, MHC-II, LAMP1, and CD68 by immunostaining and TNF-α expression by in-situ hybridization. In conclusion, these data indicate that TBI induces a fast maturation and immunity of dendritic cells which is associated with FLT3/FLT3L signaling and which is enhanced by EI prior to TBI.The consequences of systemic inflammation are a significant burden after traumatic brain injury (TBI), with almost all organs affected. This response consists of inflammation and concurrent immunosuppression after injury. One of the main immune regulatory organs, the spleen, is highly interactive with the brain. Along this brain–spleen axis, both nerve fibers as well as brain-derived circulating mediators have been shown to interact directly with splenic immune cells. One of the most significant comorbidities in TBI is acute ethanol intoxication (EI), with almost 40% of patients showing a positive blood alcohol level (BAL) upon injury. EI by itself has been shown to reduce proinflammatory mediators dose-dependently and enhance anti-inflammatory mediators in the spleen. However, how the splenic immune modulatory effect reacts to EI in TBI remains unclear. Therefore, we investigated early splenic immune responses after TBI with and without EI, using gene expression screening of cytokines and chemokines and fluorescence staining of thin spleen sections to investigate cellular mechanisms in immune cells. We found a strong  Traumatic injury to the brain has acute, large-scale systemic consequences  that affThe spleen is one of the most important immune regulatory organs, involved not only in red blood cell clearance but also in the facilitation of interactions between antigen-presenting cells (APCs) and T and B lymphocytes . Prior iEthanol intoxication (EI) is a frequent comorbidity in brain injury, with almost 40% of patients showing a positive blood alcohol level (BAL) upon admission . NotablyIs acute EI posited to interfere or rather to amplify the TBI-induced systemic immunoregulation, and in particular, is EI modulating TBI-induced immune reactions in the spleen? Given the high prevalence of EI in TBI patients, this question has direct clinical relevance. In this study, we investigated the effect of a single high-dose ethanol exposure prior to experimental TBI in adult mice with a focus on the immediate immunological responses in the spleen.post-hoc analysis of spleen samples obtained from previous studies (ad libitum access to food and water). TBI was performed on wild-type (WT) male mice aged p60–90, in agreement with epidemiological data in human TBI (2), after which the mice were subjected to a closed head weight drop TBI model. Animals were positioned in the weight-drop apparatus, and the TBI was delivered by a weight of 333 g free falling from a height of 2 cm, targeting the parietal bone  had the same treatment and procedures , but without the trauma being administered. Ethanol treatment was performed as previously described  was moved to another tube and precipitated with the same amount of isopropanol. The samples were placed at RT for 10 min and centrifuged for 10 min 12,000×g at 4°C. The isopropanol was removed and 1 ml of 75% ethanol in DEPC-treated dH2O was added and mixed. The samples were centrifuged for 10 min 8,000×g at 4°C, ethanol was removed, and the samples were air dried. The RNA pellet was redissolved in 20 µl RNAse-free dH2O. RNA concentration was determined by NanoDrop. Reverse transcription was performed by adding 5 µl random hexamers  to 0.75μg RNA  and incubated for 10 min at 70°C. The samples were placed on ice and a master mix of 0.5µl reverse transcriptase , 0.5 µl RNase Inhibitor , 2 µl dNTPs , and 12 µl reverse transcriptase buffer . The samples were incubated for 10 min at RT and placed in a water bath for 45 min at 42°C. The samples were incubated for 3 min at 99°C, placed on ice, and frozen until further use.RNA was isolated from the spleen using QIAzol  by disrupting and homogenizing the tissue in 1 ml QIAzol, after which 200 µl of chloroform was added and vortexed for 15 s. The samples were placed at RT for 10 min and centrifuged for 10 min 12,000×Mus musculus) was set. The primer pair with the most optimal Tm, self-complementarity, and self-3′ complementarity was chosen. The chosen primer pairs were double-checked in the primer blast tool, to ensure specificity for the target gene. Primers were ordered from Biomers (Germany) and were validated, by performing a run on test samples together with the corresponding controls (samples without RNA and samples without reverse transcriptase) to verify the Ct value and thereby the selectivity and functioning of the primer before using them for the experiments. The detailed list of the primer sequences for each gene tested is reported in The primers used in the present study were designed using the primer blast tool from NCBI , and sequences were found through the NCBI nucleotide search tool, GenBank. Sequences were copied into the primer blast tool and parameters were set to achieve the most optimal PCR product for the in-house lightcycler (Roche LightCycler 480 II). Briefly, the PCR product size was set from a minimum of 70 to a maximum of 140, the primer melting temperature (Tm) was set at 60°C ± 3°C, the exon junction span was set at no preference, and finally, the correct organism (qPCR was performed on the LightCycler 480 II (Roche) with the Power PCR TB green PCR master mix . Two microliters of sample cDNA was used in a total volume of 10 µl (3 µl primer mix and 5 µl of TB green) in a 96-well plate, all samples were duplicated, and the housekeeping gene GAPDH was used as a control , and 10 μm sections were cut with the cryostat and mounted on glass slides. Slides were stored for 24 h at −80°C and washed in 1× PBS, followed by a 10-min fixation step of the sections in 4% PFA. Target retrieval was performed in sodium citrate buffer pH 8.5, followed by blocking of the sections in blocking buffer  for 2 h at RT. The primary antibodies , with small modifications  equipped with a ×100 oil objective, and a single optical section with 3 × 3 tile scan was made spanning an area covering a splenic follicle and the red pulp. Acquisition parameters were set to avoid hyper- or hyposaturation and kept constant for each experimental set. Images were merged with the BZ-II analyzer software  and analyzed using the ImageJ software. Fluorescence intensity was assessed by manually tracing the CD11c+ cells and measuring the mean gray value. Density was assessed by using the ImageJ plugin cell counter. The analyzed marker was assessed as high or low expressing, by thresholding the signal. Each picture was analyzed by making a ratio between total CD11c+/CD45+ cells and the imaged marker or the ratio between CD11c−/CD45+ cells and the imaged marker.in-situ hybridization images were acquired using an LSM-710  microscope with a ×40 oil objective with optical thickness fitted to the optimum value. A z-stack of 8 images were acquired at 1,024 × 1,024 pixel resolution and 16-bit depth. Acquisition parameters were set to avoid over- and undersaturation and kept the same for each experimental set. TNF-α and beta-2 adrenergic receptor (ADRB2) mRNA density in CD11c+ cells were assessed by using the ImageJ plugin cell counter.Single mRNA λ parameter), because the experiment included multiple dependent variables (cytokines or chemokines) and two independent variables (TBI and ethanol treatment). The post-hoc comparisons were performed with two-way ANOVA with Tukey’s post-hoc correction. All groups were tested for normality using the Shapiro–Wilk test. Correlation matrices of gene expression data were made with the Prism analysis suite (GraphPad Prism version 8), and Pearson r coefficient and p-values for every correlation were assessed. For the histological datasets, two-way ANOVA was performed with Tukey’s post-hoc comparison, since two independent treatments were done . Statistical significance was set at p <0.05 after multiple-comparison correction.Statistical analysis for the gene expression data sets 2 was peSystemic immune functions are quickly modulated by the occurrence of neurological conditions through pathways conceptualized as the “brain–spleen axis” –47. We sAs an entry point, we screened the induction of the mRNA of several cytokines (with the acknowledged limitation that mRNA levels may not directly represent protein levels) in whole-spleen extracts obtained 3 h after TBI (or sham surgery), pretreated (30 min) either with saline or with ethanol (5 g/kg).IL-12, IL-17, IL-23a, and IFN-y); prototypical Th2-B-cell-directed, anti-inflammatory mediators ; and prototypical DC–monocyte mediators  , and the interaction of the two parameters showed no significance in the two-way MANOVA . This could be attributed  to the significantly increased expression of FLT3L upon TBI alone  = 9.771; SS vs. ST; 100 ± 27 vs. 163 ± 44; p = 0.045; FLT3  = 4.266; SS vs. ST; 100 ± 46 vs. 239 ± 119; p = 0.037; CX3CR1  = 6.825; SS vs. ST; 100 ± 16 vs. 134 ± 18; p = 0.01; IL-13 was significantly downregulated  = 7.499; SS vs. ST; 100 ± 35 vs. 42 ± 16; p = 0.0005; FLT3L  –51. SurpThese screening data not only point toward a rapid activation of splenic immune cells upon TBI but also reveal a pattern compatible with a selective effect on innate immunity.CCl2, CCL3, CCl4, and CCL12)    and EI , and the interaction of the two parameters showed no significant effect in the two-way MANOVA . However, post-hoc analysis (Tukey corrected) showed that TBI alone significantly increased the expression of CCL3  = 8.910; SS vs. ST; 100 ± 13 vs. 161 ± 42; p = 0.028; CCL1  = 4.156; SS vs. ST; 100 ± 63 vs. 244 ± 116; p = 0.019; CCL22  = 7.483; SS vs. ST; 100 ± 30 vs. 159 ± 47; p = 0.039; CCL3  = 6.093; SS vs. ET, 100 ± 35 vs. 155 ± 54, p = 0.042; CXCL10 expression  = 3.458; SS vs. ET; 100 ± 78 vs. 37 ± 18; p = 0.014; CCL2 in both sham and TBI groups  = 9.679; SS vs. ES, 100 ± 61 vs. 29 ± 25, p = 0.0021; ST vs. ET, 66 ± 37 vs. 17 ± 8, p = 0.014; We sought to confirm and further extend the rapid effect of TBI on splenic immune cells, particularly on DCs. We assessed the expression of a set of chemokines known to be strong modulators of innate immune responses , 53. Thed CCL12) –56, Th c CXCL10) –60, or l CXCL16) –64. We cIL-17, IL-23, IL-19, and IFN-γ (known to constitute a unified pathway)  were significantly correlated in ET but not in ST samples, further underscoring the peculiar immunological milieu determined by the concomitant TBI and EI.As an additional exploration of the complex immunological interactions taking place in the spleen upon TBI with or without concomitant EI, we performed a cross-correlation analysis of the expression levels of the tested cytokines and chemokines (pathway) , 66 in SThese data not only confirm the rapid engagement of the brain–spleen axis upon TBI but also show that EI displays a substantial modulatory effect on innate and adaptive immunity, particularly on APCs.). Phospho-FLT3 immunoreactivity was highly inhomogeneous in the spleen sections, with a comparatively small number of cells highly positive for pFLT3 localized around follicles and a minor number of cells with moderate pFLT3 immunoreactivity scattered in the parenchyma. Initial immunostainings revealed that CD11c density was not altered by TBI or concomitant EI  = 0.9360; p = 0.45; ). Co-immunostaining with CD11c and CD45 revealed that nearly all the pFLT3high cells (upon binning) were CD45+ . However, when investigating the density of pFLT3high cells in CD45+/CD11c− subpopulations (labeling leukocytes other than DC), we found no significant difference among treatment groups  = 0.8446; p = 0.50; ). Although the fluorescence intensity of pFLT3 in CD11c+ DCs was not altered across the four treatment groups  = 2.541; p = 0.06; high/CD11c+ cells was significantly affected by TBI and treatment  = 45.84; p < 0.0001), and post-hoc comparison (Tukey corrected) revealed a significant increase in pFLT3high/CD11c+ cells after TBI alone  = 123.5; p < 0.0001), and a post-hoc comparison (Tukey corrected) showed a significant increase after TBI  = 68.07; p < 0.0001), due to the massive increase of pBTKhigh/CD11c+ cells in the ST compared with the SS group  = 418.3; p < 0.0001). The post-hoc comparison (Tukey corrected) showed a significant increase after TBI  = 26.09; p < 0.0001) due to the substantial elevation of pS6 occurring after TBI  = 8.553; p < 0.0001, post-hoc; SS vs. ST; 48 ± 10 vs. 54 ± 10; p < 0.0001; p = 0.78; high cells with CD11c+ cells reveals a significant difference within treatment groups  = 40.50; p < 0.0001). The post-hoc comparison (Tukey corrected) shows a significant increase after TBI  = 118.0; p < 0.0001). Post-hoc analysis (Tukey corrected) indicated a significant increase after TBI  = 60.83; p < 0.0001) due to a massive increase upon TBI  , 76. MatF = 79.68; p < 0.0001) with a strong increase after TBI  = 41.25; p < 0.0001) due to TBI which resulted in an increase in the amount of CD68high/CD11c+ cells in the spleen  = 214.9; p < 0.0001) due to a significant increase upon TBI  = 43.02; p < 0.0001). The post-hoc comparison within the treatment groups (Tukey corrected) showed a strong significant upregulation after TBI  = 79.5; p < 0.0001), due to a strong increase after TBI  = 18.99; p < 0.0001), due to a significant decrease after TBI  after TBI, splenic DCs undergo a maturation process that involves FLT3/FLT3L signaling, enhanced protein synthesis, increased phagocytotic and lysosome activity as well as upregulated expression of MHC-II, and finally, increased inflammatory properties, shown by TNF-α expression. Most notably, in the case of concomitant high-dose EI, the maturation process is enhanced, with increased expression of FLT3L and larger fractions of CD11c+ cells displaying elevated protein synthesis and signs of immune function activation, however with a simultaneous increased ADRB2 expression. Thus, not only does the TBI set in motion events that influence an important compartment of the systemic immune response, and even though concomitant EI is capable of substantially amplifying these cascades, it also simultaneously shows a rapid autonomic innervation.Maturation of DCs is conceptualized as the phenotypic change from a state characterized by high endocytic capacity, low expression of co-stimulatory molecules and MHC-II, and weak induction of T-cell responses (immature dendritic cells) to a state of downregulated phagocytosis, high expression of MHC-I and MHC-II, and effective stimulation of naive T cells . FurtherWhat drives such an induction of maturation? The maturation process of DCs is set in motion, among others, by PAMPs or DAMPs , 76, i.eWhat are the possible consequences of TBI-induced maturation of splenic DCs on local and systemic immune activation following brain injury? The net effect of the activation of the brain–spleen axis in TBI (either by circulating cytokines or by the dys-autonomia associated with TBI) seems to be detrimental, since immediate splenectomy results in an improved survival, reduced brain, and systemic cytokine response, ameliorated brain edema, and preservation of cognitive abilities in experimental rat TBI , 113. Thin-vitro and in-vivo data about the effects of EI on trauma-associated inflammation suggests an overall immunosuppressive effect of EI, in agreement with the impact of chronic ethanol on DC function. If this extrapolation is sound, then the enhanced maturation of splenic DCs seen in EI/TBI samples may result in a DC phenotype with inflammation-resolution properties. This hypothesis is supported by the observed upregulation of the beta-2 adrenergic receptor in DC. Nevertheless, the combination of increased TNF-α and ADRB2 mRNA (not previously described) may correspond to a peculiar state of activation characterized by high immune-stimulatory properties and, at the same time, quick dampening from autonomic innervation. Thus, the impact of EI-driven expansion of DC maturation may ultimately contribute to the reduced systemic immune reactivity seen in TBI upon ethanol intoxication.What is the contribution of EI-driven enhanced DC maturation upon TBI? EI has been reported to be dose-dependently associated with a reduced inflammatory response  at the injury site –27 in muin vivo or an ex-vivo naive T-cell stimulation assay, which was beyond the technical scope of the present project. Second, the use of CD11c+ marker does not distinguish the subpopulation of myeloid DC or the plasmacytoid DC; we have nevertheless maintained a consistent selection of the region of interest in correspondence of the marginal zone . JZ was supported by the Chinese Scholarship Council (ID No. 202108080183). FR was also supported by the ERANET-NEURON initiative “External Insults to the Nervous System” as part of the MICRONET consortium (funded by BMBF: FKZ 01EW1705A).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.


This product has been prepared from silveroxide, H2quin, and 1,3,5-triaza-7-phosphaadamantane(PTA), and fully characterized by standard methods including single-crystalX-ray diffraction. Compound 1 has revealed distinctivebioactive features, namely (i) a remarkable antiviral activity againstherpes simplex virus type 1 (HSV-1) and adenovirus 36 (Ad-36), (ii)a significant antibacterial activity against clinically importantbacteria , and (iii) a selective cytotoxicity againstHeLa (human cervix carcinoma) cell line. The present work widens agrowing family of bioactive coordination polymers with potent antiviral,antibacterial, and antiproliferative activity.Coordination polymershave emerged as a new class of potent biologicallyactive agents due to a variety of important characteristics such asthe presence of bioactive metal centers and linkers, low toxicity,stability, tailorable structures, and bioavailability. The researchon intermediate metabolites has also been explored with implicationstoward the development of selective anticancer, antimicrobial, andantiviral therapeutic strategies. In particular, quinolinic acid (H A new silver(I) coordination polymer driven by aminophosphineand quinolinate ligands was self-assembled and fully characterizedand revealed a potent antiviral, antibacterial, and antiproliferativeactivity. Even now, we aredefenseless facing the COVID-19 outbreak due to limited antiviraltreatment options.3 Therefore, the identification and development ofnew antiviral chemicals and treatment approaches is a subject of paramountimportance for modern society.Infectious diseasesand particularly those of a viral genesis areresponsible for igniting most of the global pandemics. Influenza,smallpox, rabies, and Ebola viruses have been harvesting a death tollfor centuries and contributed to the deaths of millions of peoplearound the globe.8 and especially coordination polymers(CPs)12 built from biocidal metal nodesand/or bioactive organic ligands represent a promising route for developingnew metal-based antiviral materials and metallodrugs. Because of somestructural and physicochemical fractures, CPs can exhibit an interestingtherapeutic outcome with a potential synergic effect of its variouscomponents. An assembly of biologically active metal ions and biorelevantorganic pillars (metabolic intermediates) into metal–organicnetwork provides an appealing way for designing new bioactive materials.12Coordination compounds2quin, pyridine-2,3-dicarboxylicacid), which is the main downstream metabolite in the kynurenine pathwaysin mammals and in aspartate pathways in bacteria and plants.13 An appropriate level of this bioligand in cerebrospinalfluids is crucial for correct functioning of the nervous system andpreventing the development of multiple sclerosis Huntington’sand Alzheimer’s diseases. On the other hand, perturbationsin kynurenine pathways involving a production of H2quinand alterations of its concentration are observed in specific neoplasticand viral diseases.21A particularly interesting example of such a biorelevant pillarconcerns quinolinic acid  pose obstacles on the development of new silver(I)coordination compounds and their medicinal applications.26In the inflammatory changed cellular microenvironment, quinolinicacid can potentially exert a poisonous effect on the abnormal cancercells and viruses initiating their programed death. This propertyof H29 In particular, water-soluble cagelike aminophosphineligands such as PTA  and itsvarious derivatives constitute an appealing class of linkers for designingbioactive coordination polymers.32 Notable examples ofsuch silver(I)-based compounds with potent and diverse bioactivitywere reported in our previous studies.40 Within this context and following our major goal in exploring thechemistry and biofunctional applications of silver–PTA coordinationpolymers, herein we report the self-assembly synthesis, characterization,spectroscopic and structural features, and antiviral, antimicrobial,and antiproliferative properties of a new silver(I) coordination polymerderived from PTA and quinolinic acid, [Ag(Hquin)(μ-PTA)]n·H2O (1).In contrast to discrete metal complexes, some silver(I)-basedcoordinationpolymers represent improved stability and release properties, thusalso gaining an impetus for applications in anticancer and antimicrobialtreatment.n·H2O (1) wasself-assembled by using a mixed-ligand synthetic approach (2quin), and 1,3,5-triaza-7-phosphaadamantane(PTA) in water/methanol medium at ∼25 °C. The productwas isolated in a microcrystalline form and characterized by standardmethods, including the single-crystal and powder X-ray diffractionanalyses. The latter shows a good match between the experimental andsimulated patterns, thus confirming a phase purity of the obtainedproduct .A new aqua-soluble silver(I)CP [Ag(Hquin)(μ-PTA)]approach 1, namely1 indicates the characteristicνas(COO) and νs(COO) bands of carboxylategroups in the 1700–1365 cm–1 region . The Δν value of 229  is in good agreement with the data observedfor unidentate carboxylate ligands.41 Thereare also typical ν(C–X)  bands at 1400–500cm–1 associated with the coordinated PTA moieties.The 1H NMR spectrum of 1 in D2Oexhibits a set of signals expected for the aromatic protons of quinolinicacid along with the methylene protons of PTA ligands . The 31P{1H}NMR spectrum reveals a broad singlet at δ – 75.4 thatis consistent with the presence of the coordinated PTA in solution.The IR spectrum of 1 is air and light stable in the solid state.It is also stable in the D2O solution for at least severaldays. The solution stability of 1  was monitored over time intervals of 0, 1, and 7 daysby 31P{1H} and 1H NMR spectroscopy,indicating no appreciable spectral changes under these conditions. However, 1 is less stable in DMSO-d6 andslowly decomposes, as attested by an evolving PTA=O peak inthe 31P{1H}NMR spectrum after 1 day . The observed molar conductivitiesof 1 measured in H2O and DMSO solutions are14.35 and 2.07 mS m2 mol–1, respectively,and thereby indicate its electrolyte nature.Compound Figures S8 and S9)reveled the presence in solution of two characteristic cationic species,namely [Ag(PTA)2]+ and [Ag2(PTA)2(Hquin)]+. Both fragments exhibit high intensityand expected isotopic distribution. On the basis of these observations,we can conclude that the 1D polymeric chain structure of 1 is maintained in solid state, while in solution the compound dissociatesinto stable lower mass fragments. This is also in agreement with thedata observed for other reported [Ag(PTA)(carboxylate)]n coordination polymers.38The ESI-MS data(1 comprisesa silver(I) center, a μ-PTA linker, a terminal Hquin– ligand, and a crystallization water molecule  Å that is equal to the b unit cellparameter 6]Cl3 was selected as a positive control on account of being themost promising compound in the therapy of adenoviral infections.4 However, under the influence of [Co(NH3)6]Cl3, the virus level decreases only by 3log (99.9% reduction). Although quinolinic acid is a neurotoxin becauseof its highly virucidal properties, it seems appropriate to considerthe use of H2quin to treat viral infections.Both compounds ectively 1, indica2quin in 1 results in even superioractivity against HSV. Because 1 can inactivate the envelopedherpes virus, it may also be effective against other enveloped viruses,such as orthomyxoviridae , coronaviridae(including SARS-CoV), flaviviridae, and poxviridae as well as blood-borneviruses including HBV, HCV, and HIV. Moreover, the virucidal efficacyof 1 against the nonenveloped adenovirus, which is moreresistant to disinfection than the enveloped viruses, makes this compoundpromising as a potential drug or disinfectant.It is also interestingto note that a combination of silver ionswith H2quin derivatives is completelyinnovative. So far, the role of quinolinatephoshoribosyltransferase (QPRT), an enzyme that catalyzes theformation of nicotinate mononucleotide from 5-phosphoribosyl 1-pyrophosphate(PRPP) and quinolinic acid, has been considered.42 Wang et al. described QPRT as an antiviral host factoragainst virus infection in the course of hepatitis C.43 H2quin along with 3-HK (3-hydroxyanthranilicacid), 3-HAA (3-hydroxyanthranilic acid), or KYNA (kynurenic acid)are intermediate products of the kynurenine pathway. Interestingly,the concentration of H2quin increases with sustained activationof the immune system. However, no clear explanation for this correlationhas been found so far.44 It is undeniablethat higher levels of H2quin appear as a result of a viralinfection, and at the same time, we have proven in vitro that H2quin is virucidal. This may indicate that an increasedconcentration of H2quin is a form of defense against thepathogen.Hence, an approachto treat viral infections with H1 were evaluated by using strains of three species representingclinically important bacteria, namely Staphylococcusaureus, Escherichia coli, and Pseudomonas aeruginosa. Allthe mentioned species were reported as expressing increasing resistanceto clinically established antimicrobial therapies,46 though the tested strains were not characterizedas resistant or multiresistant. The normalized MIC values obtainedfor compound 1  revealed a higher susceptibility to 1 if compared to a Gram-negative one (S. aureus). However, even in the latter case, the activity of 1 is almost 2-fold superior vs silver(I) nitrate. None of the ligands showed antimicrobial activity at a maximumconcentration tested. This is also in agreement with prior data forPTA (MIC > 600 μg mL–1)37 and H2quin (MIC > 1024 μg mL–1)47 established for the same species ofbacteria. The MIC values shown by 1 are comparable tothose reported for other Ag-based CPs.40 On the basis of the aforementioned findings, we can hypothesizethat the antimicrobial properties of 1 are mainly associatedwith the action of Ag+ ions, which might be strongly influencedby the type of coordination environment present and binding affinityof O-, N-, and P-donor ligands. However, further studies are requiredto shed light on the actual mechanism of antimicrobial action of compound 1.Antibacterial properties of mpound 1 2 suggest1 against NHDF , A549 (humanlung carcinoma), and HeLa (human cervix carcinoma) cell lines wasinvestigated by using MTT assay along with cisplatin and AgNO3 as positive controls. After incubation period (72 h), a half-maximalinhibitory concentration (IC50) was determined (1 againstHeLa cells (IC50 = 68.6 ± 7 μM) and its inactivityagainst human lung carcinoma (A549). The NHDF, used as a normal bodytissue model, was significantly resistant when treated with 1. A lower cytotoxicity of 1 (93.8 ± 10.5μM) in comparison with cisplatin (16.7 ± 2 μM) fornormal body cell line shows that 1 might be a promisingcompound in anticancer treatment because its cytotoxicity againstHeLa is higher than in normal body cell culture and is still lowerthan the same parameter of cisplatin  but alsounaffected body tissues. Moreover, Walczak44 suggested that an increase of the quinolinic acid concentrationin tissues can be the result of the immune system activation and responseagainst pathogen. Hence, it can be hypothesized that a specific coordinationof quinolinate ligand by silver ion plays an important role in thecytotoxicity of 1.The lack of blood–brain barrier crossing togetherwith thelocal administration of 1 was investigated inthe peroxidation of LinMe (methyl linoleate)in the micellar system; plots of oxygen uptake are shown in 1, PTA, and H2quin . The kinetic plots aresubstantially the same, which indicates that the tested compoundsare kinetically neutral during the peroxidation of micelles initiatedwith ABAP, thus not exerting antioxidant or prooxidant activity.The antioxidant behavior of 1 and HSA, mode of action,and structural changes, we used circular dichroism (CD) and fluorescencespectroscopy techniques.Human serum albumin (HSA) is oneof the most important blood componentsthat plays a main role in the binding and transport of numerous exo-and endogenous ligands such as fatty acids, hormones, toxic metabolites,metals, and drugs. Analysis of the protein interactions with drugsis very important from the pharmacological point of view. To determinethe binding parameters between 1 system are shown in 1 causes a decline of fluorescencesignal intensity without a significant shift of the emission peak.This indicates that the interaction between HSA and 1 occurs, and there is no alteration of the microenvironment aroundthe HAS’s chromophore.The fluorescence emission spectra ofthe HSA–1 system.F0 and F are therelative fluorescence intensities of protein inthe absence and presence of a quencher, respectively, KSV is the Stern–Volmer quenching constant, Kq is the quenching rate constant, [Q] is thequencher concentration, and τ0 is the average lifetimeof a biomolecule without quencher . The corresponding quenching constants KSV for the interaction between HSA and 1 are 5.15 × 103 and 5.42 × 103 M–1 at 310 and 298 K, respectively. It is clearthat the KSV values decrease when temperatureincreases, and therefore we can conclude a static quenching fluorescenceof albumin by 1  decrease with an increase in temperaturewhen the protein interacts with 1.All calculated binding parameters are collected in 1 forms an unstablecomplex with the protein, which also undergoes a partial decompositionat higher temperatures. The N value approximatesto 1, indicating that only one binding site in protein is accessibleto compound 1. Under physiological conditions the bindingconstant of the system is 1.73 × 103 M–1. It points a rather weak affinity of 1 to HSA, whichis comparable with the Ka value of theHSA–cisplatin system (Ka = 8.52× 102 M–1) and is lower in comparisonto other metal complexes.55This relationshipindicates that 1 system:H°, ΔS°, and ΔG° are the enthalpy, entropy, and free energy, respectively, Ka is the binding constant at the correspondingtemperature T [K], and R is thegas constant.The Ross–Subramanian theory and H° and ΔS°. The hydrogen bonds and van der Waals interactions may decreasethem, whereas the electrostatic forces usually make H° ≈ 0 and S° > 0. In this context,both negative values of ΔH° and ΔS° indicate that intermolecular interactions play animportant role in the binding process of 1 with HSA.Moreover, the negative values of ΔG° showthat spontaneous processes occur in this system.Usually four types of the binding forces betweensmall moleculesand biological macromolecules can be present in the interaction. Theseinclude electrostatic forces, hydrophobic and ionic interactions,and van der Waals forces as well as hydrogen bonds. Nevertheless,the contribution of the metal ion in the interaction cannot be excludedin the case of coordination compounds. Moreover, the hydrophobic forcesmay increase Δ1 on the protein secondary structure. In the intrinsicregion (180–250 nm), the CD spectrum of the protein is characterizedby two negative bands . A moreintense band at 209 nm and a broad and weak band around 221 nm canbe assigned to α-helical structure and π → π*/n→ π* transitions for the peptide bond, respectively.56 Under the experimental conditions, 1 does not generate the CD signal, but as evident from Figure S14, the presence of the silver compoundhas an effect on the secondary structure of the albumin, causing somechanges in the intensity of the protein negative band at 209 nm. Theα-helical content of free HSA at 209 nm is equal to 67.23%.A decrease in α-helicity to ∼65.30% was observed afteraddition of 1 in the molar ratio of [HSA]/[Ag] = 1:10or 1:20. This indicates that the binding of the ligand with the proteindestroys a hydrogen binding network of HSA.We used thecircular dichroism technique to determine the effectof 1 and HSA. The band intensities ofHSA at 209 and 221 nm decreased with the negative Cotton effect throughthe binding of 1 without causing significant shifts ofthe peaks, thus clearly indicating that the compound induced a slightdecrease in the α-helical structure content of the protein.Reagents concentrations were properly low, and any concentration effectcan be excluded. Moreover, no changes in the negative bands at 209and 221 nm on increasing the concentration of 1 wereobserved. From these results, it is evident that the effect of 1 on HSA caused secondary structural changes to the proteinwith a slight loss of the helical stability.The changes in theprotein secondary structure result from theinteraction between n·H2O (1). By combination of biocidal metal nodesand water-soluble aminophosphine linker with quinolinate(1−)ligand as an important metabolic intermediate, the present compoundextends the family of bioactive silver(I) coordination polymers.In summary, we described the self-assembly synthesis as well asstructural and biological features of a new silver(I)-based coordinationpolymer [Ag(Hquin)(μ-PTA)]1 was investigated in detail, demonstrating its highantimicrobial and antiproliferative potential. In particular, antiviraltests revealed an unprecedented ability of 1 to killherpes simplex virus type 1 (HSV-1) and adenovirus 36 (Ad-36) witha low toxicity to normal cells. Taking into account the antioxidantmeasurements, we can conclude that compound 1 is kineticallyneutral, while its cytotoxic activity is not a result of its prooxidantproperties.A multifaceted antiviral, antibacterial, and cytotoxicity behaviorof 1 can inactivate envelopedherpesvirus, its efficacy against other enveloped viruses can be envisaged,including such families as orthomyxoviridae , coronaviridae, flaviviridae, poxviridae, and blood-borneviruses . Moreover, the virucidal efficacyof the CP 1 against the nonenveloped adenovirus, whichis more resistant to disinfection than the enveloped viruses, makesthis compound promising for consideration as a potential drug or disinfectant.In further research, it would be particularly interesting to investigatea mechanism that would explain the action of 1 as a virucidalagent.Because compound 1 with human serum albuminwasalso investigated by using fluorescence spectroscopy and CD methods.The results prove that the compound interacts with human serum albuminwith moderate affinity and forms an unstable complex with the protein.Furthermore, the CD analysis demonstrates that 1 exertssome effect on the protein secondary structure and reduces the α-helixcontent.The interaction of 32 the present work also provides an important contributionto betterunderstanding of different bioactivity features of such compounds,also revealing their significant potential toward development of novelantiviral and antimicrobial agents. These research lines deserve furtherdevelopment and extension to various combinations of silver centerswith other types of carboxylic acid metabolites.Apart from widening the family of PTA-driven coordinationpolymers,1 (833 μM) wasprepared by dissolving the compound in demineralized water. Proteinconcentration was calculated by using εHSA = 42864M–1 cm–1 at 280 nm.57 PTA was synthesized according to a published method.59All standard chemicals and solvents wereobtained from commercial suppliers. Human serum albumin was acquiredfrom Sigma-Aldrich . The protein stocksolution with a concentration of 2 μM was prepared in PBS buffer(pH = 7.40). A stock solution of –1) were used to measure the IR spectra . SolutionNMR spectra were recorded in D2O and DMSO-d6 by using a Bruker 500/600 AMX spectrometer  at ambient temperature . 1H chemical shifts(δ) are expressed in ppm relative to Si(Me)4, whileδ(31P) shifts are relative to an external H3PO4 (85% aqueous solution). ESI-MS(±) spectra wererun on a Bruker MicroTOF-Q mass spectrometer with an ESI source byusing ∼10–3 M solutions of 1 in H2O/MeOH. The elemental analyzer Vario ELCube  was used for the determination ofC, H, and N contents .Bruker IFS 1113v (Germany) or BIO-RADFTS3000MX  instruments , and PTA  were combined in a CH3CN/H2O (5 mL/5 mL) solution and stirred in air for 1 hto produce a white suspension. This was dissolved by a dropwise additionof an aqueous 1 M solution of NH3 . The obtained solution was filtered off, and the filtrate wasleft in a vial to slowly evaporate in air at room temperature. Colorlesscrystals  were formed in 1 week.These were then collected and washed with water and methanol and driedin air to give 1 in 40% yield, based on Ag2O. Compound 1 is soluble in H2O (S25° ≈ 0.5 mg mL–1) and DMSO (S25° ≈ 0.6 mgmL–1). Elemental analysis: Calculated for C13H18AgN4O5P (MW 449.15):C, 34.76; N, 12.47; H, 4.04. Found: C, 34.96; N, 12.73; H, 3.43; mp160 °C (dec). Molar conductivity : Λ 14.35 and 2.07 mS m2 mol–1, respectively. IR : 3469 (s br) ν(H2O + OH), 3071 (w), 2961 (wm)νas(CH), 2923 (m), 1700 (m), 1594 (m) and 1558 (m)νas(COO), 1473 (m), 1454 (m), 1421 (m), 1365 (m)νs(COO), 1301 (m), 1293 (m), 1241 (s), 1163 (m),1109 (s), 1001 (w), 1038 (w), 1013 (vs), 988 (w), 974 (vs), 958 (m),948 (s), 898 (w), 853 (w), 807 (s), 795 (s), 751 (s), 735 (w), 699(w), 643 (m), 605 (vs), 581 (m), 568 (m) 506 (w), 470 (w), 457 (w),449 (w), 400 (w). 1H NMR : δ8.53 , 8.23 , 7.60 , 4.66 and 4.79, 4.35 . 31P{1H} NMR : −75.3. ESI-MS(±) (H2O/MeOH), selected fragmentswith relative abundance >10%, MS(+), m/z: 421 (100%) [Ag(PTA)2]+, 696 (65%)[Ag2(PTA)2(Hquin)]+; MS(−) m/z: 166 (80%) [Hquin]−, 439 (85%) [Ag(Hquin)2]−, 976 (20%)[Ag3(PTA)(quin)(Hquin)2]−.Silver(I) oxide, quinolinic acid .In this procedure, compound 60 The structure was solved by direct methods withSHELXT-2014/5 and refined with full-matrix least-squares techniqueson F2 with SHELXL-2018/3.62 Direct solution in the correct space group P21/n was unsuccessful. The structure was solvedin the space group Pn, and then the symmetry was transformed to P21/n by using the ADDSYM feature of the PLATONprogram.64 The H3 atom on the COOH functionality wasplaced on O3 atom on the basis of the C–O carboxylate distances.The O3–H3 distance was restrained to 0.97 Å. The O–Hbond distances in the water molecule O1W were restrained to 0.85 Åand H1···H2 separation to 1.38 Å. All other hydrogenatoms were placed at calculated positions and refined by using themodel with Uiso = 1.2Ueq.Single crystaldata collectionwas performed on a Xcalibur diffractometer (Oxford Diffraction) witha Sapphire2 CCD detector, equipped with an Oxford Cryosystems open-flownitrogen cryostat, by using ω-scan and a graphite-monochromatedMo Kα (λ = 0.71073 Å) radiation. Cell refinement,data reduction, analysis, and absorption correction were performedwith CrysAlis PRO (Rigaku Oxford Diffraction) software.13H18AgN4O5P, M = 449.15, a = 13.5460(8) Å, b = 6.8252(3) Å, c = 17.1564(8) Å,β = 92.148(5)°, V = 1585.07(14) Å3, T =85(2) K, monoclinic, space group P21/n, Z = 4, Mo Kα, 10636 reflectionsmeasured, 3720 independent reflections (Rint = 0.0778), R1 = 0.0766 (I > 2σ(I)), wR(F2) = 0.1408, GoF(F2) = 1.114.CCDC 2079371 (1).C1.NHDF and A549 cell lines were cultured in Dulbecco’s ModifiedEagle Medium , while HeLa cells were culturedin Minimum Essential Medium . The supplementationof 10% fetal bovine serum , 4 mM l-glutamine , 100 U/mL of penicillin, and 100 μg/mLof streptomycin  was routinely used in all media.The cell cultures of normal human dermalfibroblasts , human lung carcinoma, and human cervix carcinoma  were used to evaluate a cytotoxicity of 1 was dissolved in culturemedium and diluted to reach the concentration of 100, 50, 20, 10,5, 2, and 1 μg/mL and then added to cell cultures and incubatedin standard conditions (37 °C with a constant flow of 5% CO2) for 72 h. After a proper incubation period, the cytotoxicityof 1 was evaluated by using MTT assay .In the MTT procedure, 20 μL of 3--2,5-diphenyltetrazoliumbromide (1 mg/mL) was added to each well and subsequently incubatedfor 4 h at 37 °C. The lysis buffer (80 μL) provoked a destructionof cell membranes, which was monitored by spectrophotometric analysismeasuring the optical density (OD) of solution. The following formulawas used to estimate the viability of investigated cell cultures:cell viability (%) = (average OD for test group/average OD for controlgroup) × 100. The untreated cells were used as a negative controland doxorubicin (a cytostatic routinely used in therapy) was appliedas a positive control.65The scheme of cytotoxicity assayconsisted in enzymatic reduction of soluble tetrazolium salt in metabolicallyactive cells into insoluble purple formazan (MTT test). The concentrationof tetrazolium dye was measured colorimetrically by using a spectrophotometricmicroplate reader . The cell cultureswere inserted into 96-well plates  in a concentrationof 105 cells per well. Compound 1, H2quin,acyclovir, and [Co(NH3)6]Cl3 wereconducted by using human adenovirus 36 (Ad-36virus-ATCC VR-1610) and herpes simplex virus type 1 (HSV-1-ATCC VR-1493).The above compounds were tested by using EN 14476 . This standard describes a quantitative suspensiontest for the evaluation of virucidal activity in the medical area(phase 2/step 1), mixing 1 part by volume of test virus suspension(0.1 mL of 1 × 108 TCID50 Ad-36 virus orHSV), 1 part by volume of interfering substance (0.1 mL of PBS), and8 parts by volume of disinfectant (investigated compounds in a concentrationof 10 mg/mL). At specified contact times (60 min), aliquots were taken,and serial dilutions up to 10–8 of each mixturewere prepared. In eight repeats, 50 μL of each dilution wasadded to the microtiter plate containing a monolayer of confluentA549 or HeLa cells. The plates were observed daily for up to 4 daysfor the development of viral cytopathic effect by using an invertedmicroscope . Additionally, according to the same procedure, acyclovir (againstHSV) or [Co(NH3)6]Cl3 (against Ad36)was tested as a control. Then, residual infectivity was determined.According to PN-EN 14476, a disinfectant is considered as having virucidaleffectiveness if within the recommended exposure time the titer isreduced by ≥4 log 10 steps (inactivation ≥99.99%).Antiviral activity tests of 1 and ligands (PTA and H2quin) was evaluated byusing the method of serial dilutions according to Grove and Randall.68 Two reference strains, Staphylococcus aureus PCM 2054 (= ATCC 25923) and Escherichia coli PCM 2057 (= ATCC 25922), were obtained from the Polish Collectionof Microorganisms of the Institute of Immunology and ExperimentalTherapy in Wroclaw. A clinical strain of Pseudomonasaeruginosa of veterinary origin, identified as previouslydescribed, was also used.37 Microbial inoculawere prepared by using an overnight culture of each strain. Thesewere diluted (1:1000) by using an antibiotic broth (AB). Working dilutionsof the tested substances in AB were prepared in 48-well plates toachieve the final concentrations of the substances  after combining 0.9 mL of the working dilution and 0.1 mL ofmicrobial inoculum. Broth sterility and growth controls were performed.The inoculated 48-well plates were incubated at 37 °C for 24h. The minimum inhibitory concentration  was defined as the lowest concentration of the compound that fullyinhibited the growth of bacteria. For comparison, the MIC values werenormalized for the molar content of silver and are represented ina nmol mL–1 scale. PTA and H2quin showno antimicrobial activity at the maximum concentration tested.The antibacterial activity of 69 Toa glass test tube, 10 μL of methyl linoleate (LinMe) and 5.5mL of 16 mM Triton X-100 were added and shaken on Vortex for 1 min.Next, 5.5 mL of buffer (pH 7.0) was introduced, and the mixture wasshaken again for 1 min. The final concentration of lipid and surfactantin the micellar system was 2.74 mM LinMe and 8 mM Triton X-100. Buffercomposition: pH 7.0 phosphate .Micelles were prepared by usinga previously described procedure.1 and H2quin was measured by monitoringthe rateof peroxidation in a heterogeneous model system (micelles). The uptakeof dissolved oxygen in micelles during peroxidation was performedat 37 °C by using a Biological Oxygen Monitor equipped with aClark-type oxygen electrode. To a glass chamber containing a magneticstirrer, 5 mL of a micellar solution (pH 7.0) was added and then saturatedwith oxygen. Next, the electrodes were placed inside the chambers,and a peroxidation was initiated by injecting the ABAP solution . After 10% of oxygen consumption, 10 μLof the solution of studied compounds was added . The influence of tested compounds on the peroxidationwas determined graphically. Because of the neutrality of studied compounds(no changes in plots after addition of compounds) to peroxidationof micelles, kinetic parameters were not determined.The antioxidantbehavior of 1. The samples were measuredafter 5 min incubation at 298 and 310 K. The final concentrationsof the protein and 1 during titration experiment werecalculated by using 0 and V0 arethe initial concentration and volume of the protein solution, respectively,the [lig]0 is a concentration of stock solution of 1, and ∑VL is total volumeof the solution of 1 added during the titration experiment.Fluorescence measurementswere performed by using a Jasco 8200 spectrofluorometer and a 1.0cm quartz cell. The widths of excitation and emission slits were setat 5.0 nm. The excitation wavelength was set at 280 nm, and the emissionwas recorded in the 300–500 nm range. Phosphate-buffered saline was used as a blank. The fluorescence studies wereperformed by manually titrating 2 mL of HSA solution with 10 μL of 1] variedfrom 1:0 to 1:10 and 1:20.CD measurements wereperformed by using a Jasco J-1500 CD spectrometer in a 0.98 mm quartzcuvette. Spectra were recorded after 24 h incubation at 310 K in the200–250 nm range. A bandwidth of 1 nm, a response time of 2s, and a scanning speed of 50 nm/min were fixed to obtain final spectraas an average of three scans. The final concentration of the proteinwas 4 μM, and the molar ratio [HSA]:[