TAZD1-TAZD5) via end-capped redistribution of donor moieties by keeping the same π-bridge and central acceptor unit for organic solar cells (OSCs). To analyze the photovoltaic characteristics of these derivatives, DFT estimations were accomplished at B3LYP/6–311 G  functional. Different investigations like frontier molecular orbital (FMO), absorption spectra (UV–Vis), density of states (DOS), binding energy (Eb), open circuit voltage (Voc), and transition density matrix (TDMs) were performed to examine the optical, photophysical and electronic characteristics of afore-mentioned chromophores. A suitable band gap (∆E = 2.723–2.659 eV) with larger bathochromic shift (λmax = 554.218–543.261 nm in acetonitrile) was seen in TAZD1-TAZD5. An effective charge transference from donor to acceptor via spacer was observed by FMO analysis which further supported by DOS and TDM. Further, lower binding energy values also supported the higher exciton dissociation and greater CT in TAZD1-TAZD5. Among all the designed chromophores, TAZD5 exhibited the narrowest Egap (2.659 eV) and maximum red-shifted absorption in solvent as well as gas phase i.e. 554.218 nm and 533.219 nm, respectively which perhaps as a result of the phenothiazine-based donor group (MPT). In a nutshell, all the tailored chromophores can be considered as efficient compounds for promising OSCs with a good Voc response, interestingly, TAZD5 is found to be excellent chromophores as compared to all these designed compounds.Fullerene free organic chromophores are widely utilized to improve the efficacy of photovoltaic materials. Herein, we designed D-π-A-π-D form chromophores ( Among promising and cost-effective substitutes for future sustainable energy are OSCs due to their exceptional advantages. OSCs have been proved as an effective scheme for light manipulation, which is capable of improving the light absorption process2. In this way, larger photocurrent is produced due to the light scattering. The photovoltaic innovation has gained consideration of academic along with industrial communities’ decades-long. The photovoltaic (PV) silicon-based solar cells were known as the foremost and popular energy gadgets owing to their remarkable eco-friendly nature, proficiency and low cost3. Crystalline silicon solar cells have the greatest manufacturing history with over 60 years of progress and their efficiency has increased to above 25% because of the improvements in their architecture5. However, they are fragile and possess non-tunable energy levels due to which the organic-based solar cells have become more popular in recent years. They possess various important characteristics such as; (i) light weight; (ii) low-cost materials; (iii) tunable energy levels; (iv) mechanical flexibility; (v) variety of structural modulations and (vi) compatibility with large manufacturing9. Furthermore, organic–inorganic perovskite photovoltaics have gained impressive power conversion efficiency (PCE) of 22.1% because of remarkable characteristics like intense absorption spectrum, greater charge mobility and long diffusion length of charges12. Another class of OSCs namely dye-sensitized solar cells (DSSC) has also captured significant attention owing to their stability besides tunable visual characteristics e.g. transparency and color15. Components in DSSCs, such as the dye catches special attention on because of light conversion capability to electricity supported by photoexcitation13. The DSSCs that are metal free are of great efficiency and advantageous because of their synthesis, purification and many optical properties by easy chemical modifications16. Although in beginning efforts to commercialize organic photovoltaics (OPVs) were difficult as a result of lower power conversion efficiency (PCE) than the approximated market viability of 15%17. Besides, the photovoltaic domain is developed with other very compelling substances like OSCs based on fullerene derivatives namely PC71BM, ICBA, and PC61BM. Organic solar cells (OSCs) keeping fascinating characteristics like simple processability, light weight, mechanical flexibility, high formulation area and ease made them significant alternative tools21. Due to the exceptional electronic and structural properties of fullerene-based OSCs, they have been widely examined since 198522. Low reorganization energy of excitons24, elevated electron affinity25, and high mobility of electrons27 are some fascinating and distinctive properties of fullerene based OSCs. There also exhibit certain drawbacks in fullerene acceptors (FAs) which include less absorption in visible and near IR regions, poor photochemical and thermal stability28, non-tunable LUMO energies29 and less sunshine assimilation which impelled the researchers to search for some more strongly absorbing analogues30. Therefore, non-fullerene acceptors (NFAs) are utilized in the OSCs in place of FA owing to their flexible nature, higher fabrication area and wide tunability of their energy levels31. The non-fullerene small molecule acceptors (NF-SMAs) are regarded as remarkable constituents for proficient OSCs32. A rapid improvement in PCE is observed (~ 18–19%) in NFAs benefiting from years of research on fullerene-based BHJ materials34. The obvious increase in fill factor (FF) as well as short-circuit current (Jsc) is also seen in NFA-based devices with greater open-circuit voltage (Voc) in comparison to their fullerene counterparts35. Literature is flooded with many examples in which fullerene free donor or acceptors are extensively utilized to improve the efficiencies of photovoltaic materials39. Keeping in view the importance of NF organic systems, herein, we have tried to design benzodithiophene based organic systems for high efficacy photovoltaic devices. For this purpose, we take a synthesized X94FIC40 fullerene free A-π-A-π-A architecture acceptor nature molecule and designed sequence of donor type D-π-A-π-D configured TAZD1-TAZD5 chromophores by structural modification of end capped acceptors with efficient donor moieties. Perhaps, it is first ever systematic comparative study of impact of NF chromophores with five-, six- and seven-membered rings on the electrochemical as well as photophysical characteristics. To check the influence of donor groups on photovoltaic characteristics, DFT method was employed and has anticipated their significance in OSCs.Solar energy has turn into a promising energy source which involve the phenomena of photoelectric effect and overcome the elevating power crisis as the sunlight is non-exhaustible, non-polluting and widely available41 was exploited to understand the photovoltaic response of benzodithiophene based organic (TAZD1-TAZD5). To choose the suitable functional for current investigation, a relative investigation of X94FICλmax outcomes among several TD-DFT functionals and experimental results was performed. For this purpose, the reference chromophore X94FIC was subjected to geometry optimization using four different functionals, including B3LYP42, M0643, MPW1PW9144 and ɷB97XD45 in acetonitrile solvent as range-separated functionals estimates HOMO–LUMO gaps and excited-state energies better49. Then these optimized geometries were applied to execute UV–Vis analysis in acetonitrile solvent and 818.864, 736.686, 676.142, and 495.461 nm values of λmax were obtained at aforesaid functionals, respectively. The λmax values of X94FIC obtained using these functionals were compared to the experimentally determined maximum absorption value of 783 nm39 of X94FIC chromophore. At B3LYP/6-311G  functional, closed harmony was seen with experimental results. Moreover, we also compared the band gap values of X94FIC calculated at aforesaid functional of TD-DFT  with experimental ΔE value (1.41 eV)39 and interesting, good harmony with experimental results was seen at B3LYP, hence, this functional was selected for this study. First of all, structures of designed systems were optimized at B3LYP/6-311G to get true minima geometries in acetonitrile solvent. The absence of any imaginary frequency specified that structures were at true minima potential energy surface. After the successful optimization of geometries, different analyses; FMOs, DOS, UV–Vis, Voc, Eb and TDMs were attained to inspect the optical, electronic and photophysical characteristics of afore-mentioned chromophores at B3LYP/6-311G  level of DFT/TDDFT in acetonitrile solvent. Nevertheless, in order to understand the effect of different media on UV–Vis properties, we performed absorption analysis in gas and acetonitrile at foresaid functional. For the extraction of data from output files, Gauss View 5.0 program50, Avogadro51, Chemcraft52, PyMOlyze 2.053, and Origin54 software were utilized and the data was recorded in the form of graphs and tables.Gaussian 09 package55, A-D-A56 and A-π-A gain significant importance in improving the efficiency of solar cell materials59. Therefore, in current study we formulated a range of donor nature chromophores (TAZD1-TAZD5) with D-π-A-π-D framework from a synthesized system X94FIC (A-π-A-π-A)40 by molecular replacement at the terminals with efficient azacycle donor moieties  keeping the central ‘π-linker’ and ‘A’ same as shown in Fig. TAZD2-TAZD5 are designed by replacing the four member azacycle donor rings unit with three, five and six member ring azacycle donor unit as exhibited in Fig. THC), 5-phenyl-10,11-dihydro-5H-dibenzoazepine (THA), 5-phenyl-5H-dibenzoazepine (TBA), 9,9,10-triphenyl-9,10-dihydroacridine (PTH), 3-methyl-10H-phenothiazine (MPT) and their structures can be seen in Fig. Voc, Eb and TDMs of all the studied compounds. The modifications in derivatives with their respective donor moieties might prove as a significant step towards introducing efficient solar cells.In current era, fullerene free-organic systems (FF-OSs) with some special architectures like D-π-A-π-D, A-π-A-π-Asee Fig. . First oores TAZD-TAZD5 wi60 are investigated via utilizing FMOs63. The band gap of HOMO/LUMO orbitals is closely linked to these parameters64. As HOMO is the electronically filled highest orbital, so it is considered as an electron contributor, whereas, LUMO is considered to be an electron acceptor as it is an empty or unfilled orbital. Molecules having high energy gap (Egap) values are hard, because they resist changes in electronic configurations, resulting in lower reactivity and increased kinetic stability. Conversely, the compounds with low energy gap are attributed as soft molecules owing to their less stability and higher reactivity. These compounds reveal strong intramolecular charge transfer (ICT) possibilities due to their highly polarized nature and are extremely efficient molecules in the production of solar cell materials65. In addition, the HOMO–LUMO band difference is important in calculating a molecule’s total Voc and Eb64. So, FMO analysis is used to compute EHOMO, ELUMO and Egap of TAZD1-TAZD5 and the outcomes are exhibited in Table The optoelectronic properties i.e. charge transfer, electronic features, reactivity, chemical stability and molecular interactionsTAZD1, TAZD2, TAZD3, TAZD4 and TAZD5 as − 5.177, − 5.185, − 5.185, − 5.199 and − 5.104 eV while energies of LUMO are − 2.469, − 2.467, − 2.463, − 2.476 and − 2.445 eV, correspondingly. Egap is used to calculate molecules conductivity and net charge transfer67. The Egap values of designed chromophores (TAZD1-TAZD5) are revealed as 2.708, 2.718, 2.722, 2.723 and 2.659 eV, correspondingly. Highest energy difference between HOMO and LUMO (2.723 eV) is observed in TAZD4 among all the other derivatives which may be due to the 9,9-diphenyl-10-(p-tolyl)-9,10-dihydroacridine (PTH) donor moiety. The Egap value is abridged to 2.722 eV in TAZD3 due to the substitution of PTH with 5-(p-tolyl)-5H-dibenzoazepine (TBA) donor moiety which may be due to the decreased in hindrance in charge transfer of TBA as compared to that of PTH. Furthermore, the replacement of donor of TAZD3 i.e. TBA with 5-(p-tolyl)-10,11-dihydro-5H-dibenzoazepine (THA) in TAZD2 resulted in further reduction of bandgap to 2.718 eV owing to the enhancement in conjugation in the newly introduced donor moiety. TAZD1 is designed via replacing THA with carbazole containing donor moiety i.e. 9-(p-tolyl)-9H-carbazole (THC) in which nitrogen atom of carbazole exhibit the electron donating capability. As a result of this, the energy difference is lessen to 2.708 eV in TAZD1 because of the enhanced push pull mechanism. Moreover, TAZD5 has exhibited minimum energy gap as compared to all of the studied chromophores owing to the use of phenothiazine-based donor moiety such as 3-methyl-10H-phenothiazine (MPT) instead of THC in TAZD1. The extra electron-rich sulphur atom in phenothiazine might give an improved electron-donating capacity compared to donors that just include nitrogen atoms, like carbazole. Overall, the band gap descending order in the studied compounds is; TAZD4 > TAZD3 > TAZD2 > TAZD1 > TAZD5.The above table reveals HOMO energy values for TAZD1-TAZD4 is predominantly located over the center ‘A’ and ‘π-spacer’ parts of the organic systems and minor over some atoms of donor, while in TAZD5 the electron density is dispersed on entire system. For LUMO, the electron density is majorly located over π-bridge and core acceptor in TAZD1-TAZD5. Among all the investigated compounds, TAZD5 is found to be the appropriate candidate for future OSCs with enhanced PV behavior due to less energy band gap and adequate charges transition from terminal donors to center acceptor , transition energy (E), transition type and maximum absorption wavelength (λmax) are shown in Table TAZD1-TAZD5 is displayed in Fig. UV–Vis analysis is significant to investigate the possibility of ICT, kind of configurations of transitions and electronic transitions in a compound. To calculate the absorption spectra of the excited states, the TD-DFT calculations are accomplished in gas and acetonitrile solvent. The observed oscillator strength (TAZD1-TAZD5) exhibit absorption range from 543.361 to 554.218 nm in acetonitrile. In solvent phase, λmax values are found to be more red-shifted contrary to gas phase because of solvent effect. Furthermore, the absorption spectra of studied compounds (TAZD1-TAZD5) are dominated by π-π interactions68. The polar medium results in the stabilization of π-π* state associated with n-π* characteristics by the use of an efficient electronic state69. This indicates that, in the stabilization of first singlet state, hydrogen bonding and dipole interactions are imperative70 and the molecules exhibit red-shifted absorption as a result of enhancement of solvent polarity.In solvent (acetonitrile), all the investigated compounds have revealed maximum absorbance in visible spectrum Fig. . The desmax values are controlled efficiently by end-capped donor moieties which successively drive the red shifted absorption spectra72. The absorption band of TAZD4 is noticed at 543.361 nm having 2.282 eV energy of transition, 1.031 fos by exhibiting 98% molecular orbital contribution from HOMO to LUMO. The computed λmax value is shifted towards bathochromic shift in TAZD3 due to the replacement of PTH donor of TAZD4 by TBA so, TAZD3 has exhibited λmax at 544.101 nm, 2.279 eV transition energy, and 0.964 oscillator strength via showing HOMO → LUMO MO contribution of 98%. Furthermore, the substitution of TBA with THA donor moiety resulted in red-shifted absorption of 544.483 nm in TAZD2 along with lower transition energy (2.277 eV) and 1.006 oscillator strength via same MO contributions. Additionally, TAZD1 absorption spectra further shifted towards bathochromic shift (546.427 nm), owing to the deposition of another donor moiety i.e.,THC in TAZD1 in the replacement of THA in TAZD2. Finally, the substitution of TTC with MPT donor moiety resulted in maximum red shift of 554.218 nm in TAZD5 due to phenothiazine group in MPT by showing 96% HOMO → LUMO and 2% HOMO-2 → LUMO molecular orbital contribution at 1.005 oscillator strength and minimum transition energy (2.237 eV) owing to its lowest band gap. max of all the compounds in acetonitrile solvent is found to be in increasing order as TAZD4 < TAZD3 < TAZD2 < TAZD1 < TAZD5.It is seen that, TAZD5 compound has exhibited maximum absorption wavelength, the lowest transition energy and minimum band gap which implies that, it can be used as an efficient material for photophysical characteristics in non-fullerene OSC materials.In gaseous phase Table , all theVoc) is another significant study that provides insights into the performance of OSCs i.e. their maximum working capability. The total current that can be produced via any optical system can be estimated by Voc55. So, Voc shows direct relation with EHOMO and ELUMO of donor and acceptor molecules, correspondingly. Thus, Voc outcomes of TAZD1-TAZD5 are determined via Eq. , while on HOMO highest density is observed from − 7 to − 12 eV as shown in Fig. TAZD2, TAZD4 and TAZD5 both HOMO and LUMO have comparable charge densities which depict their equal contribution toward FMOs. In TAZD1-TAZD5, the donor contributes 7.7, 5.3, 4.8, 4.6 and 22.0% to HOMO, whereas to LUMO its participation is 5.5, 5.5, 5.2, 5.9 and 4.0%, respectively. In the same fashion, π-spacer participates 72.5, 74.4, 74.8, 74.8 and 61.7% to HOMO, while 35.0, 35.2, 34.2, 35.9 and 32.6% to LUMO in TAZD1-TAZD5, respectively. Likewise, acceptor shows its percentage participation 19.8, 20.3, 20.4, 20.5 and 16.2% to HOMO in TAZD1-TAZD5, respectively, while 59.5, 59.3, 60.6, 58.2 and 63.4% to LUMO, respectively. Overall, the pattern of electronic charge distribution elucidates the delocalization of charges and large amount of charge transfer has taken place in all the modulated chromophores. Interestingly, all the designed derivatives portray almost alike contributions and the electron density is more prominent on the central unit (π-spacer and A units).The maximum density on LUMO is noticed at − 2.5 to 4 eV in all the investigated chromophores  are presented in Fig. TDM is considerably utilized for the evaluation of electronic transitions along with their nature for It has been seen that adequate charge is transmitted out of donor towards acceptor moieties as the electron–hole pair is constituted diagonally on the entire TDM plot and represented by clear red and green spots near the acceptor portion. The electron delocalization is seen in the diagonals of A and π-spacers and very little in the D region. Moreover, the charge coherence and electron–hole pair generation are similarly observed in the off-diagonal portions of TDM heat maps see Fig. .78. In this study, hole-electron analysis is performed at B3LYP/6-311G  to understand the charge transmission in our studied molecules. Figure X94FIC) while, the hole intensity is observed at C36 and C38 of the acceptor region. Furthermore, it is also clear from Fig. TAZD2, TAZD3 and TAZD4 whereas, hole intensity is observed to maximum at sulphur atoms (S15 and S16) of the π-spacer. However, in TAZD5 hole intensity is higher at methyl group of the π-spacer and electron density is intense at nitrogen atoms (N7 and N8) of the acceptor portion. Moreover, electronic cloud is observed to be thick at nitrogen (N7 and N8) and sulphur (S9) atoms of the acceptor in TAZD6 however, hole intensity is maximum at carbon atoms (C12 and C14) of the π-linker. The labeled structures of entitled chromophores without hydrogen atoms are illustrated in Fig. TAZD5, are electron type materials as electronic cloud is observed thick at electronic band in contrast to hole intensity at hole band. In TAZD5, hole intensity is found higher at hole band therefore, it is a hole type material.Hole-electron analysis is popularly accomplished by utilizing the Multiwfn 3.8 software. It is a very useful method for revealing the nature of electron excitations. Moreover, it offers a deep understanding of all different electron transfer propertiesEb)79. The Eq.  to S1 (excited state) during the first electronic transition. EH-L signifies the energy difference among HOMO and LUMO whereas, Eb is the binding energy that is obtained by the difference in band gap between molecular orbitals and first singlet exciton energy. Usually, the lower the value of Eb, greater would be the charge separation and current charge density Jsc which results in higher PCE80. The outcomes for the studied compounds calculated in acetonitrile are formulated in Table Here, Eopt decreases progressively in all the designed compounds and is found as minimum in TAZD5 and the same behavior is observed in the HOMO and LUMO energy gap. The binding energy values in TAZD1-TAZD5 are found to be 0.439, 0.441, 0.443, 0.441 and 0.422 eV, correspondingly. The lowermost Eb value of TAZD5 depicts that it has excessive charges that can be separated into isolated charges. It is noted that TAZD5 exhibits high segregation of charges along with high Jsc which indicates that it is the leading candidate to improve the efficiency of organic photovoltaics. Furthermore, Eb data unveil a good agreement with TDM outcomes.According to our obtained results, the values of TAZD5) and acceptor (PC16BM) polymer and FMO is investigated as shown in Fig. TAZD5 due to its unique properties such as reduced band gap and greater UV–Vis absorption spectra etc. among all fabricated chromophores. According to Fig. TAZD5 donor chromophore and significantly transferred towards the acceptor polymer in LUMO which elucidates the significantly charge transfer from donor towards acceptor.In order to understand the intermolecular charge transfer between donor and acceptor, a complex is developed between a donor molecule ( (X94FIC) fullerene free donor based chromophores (TAZD1-TAZD5) were designed. To comprehend their photophysical properties, the behavior of charge transfer, and structure–activity relationship, various analyses were performed at quantum chemical approach. A reasonable energy gap between LUMO/HOMO (∆E = 2.723–2.659 eV) and significant charge transfer with wider absorption spectra (λmax = 554.218–543.261 nm in acetonitrile) was examined in all non-fullerene donor chromophores. Additionally, the less binding energy outcomes (Eb = 0.422–0.411 eV) in formulated compounds specified higher rate of exciton dissociation that also reinforce the tremendous charge transition out of HOMO towards LUMO as shown by FMOs, DOS and TDMs analyses. Moreover, the Voc values are also determined with regarding to TAZD4 (1.199 V) > TAZD2 (1.185 V) = TAZD3 (1.185 V) > TAZD1(1.177 V) > TAZD5(1.104 V). Consequently, significant photovoltaic materials can be developed by structural tailoring with efficient azacycle donor moieties. Moreover. this study also encourages the experimentalist to synthesize these efficient materials for practical use.In a nutshell, through the molecular engineering with azacycle donor moieties in an organic systemSupplementary Information.


Both subunits participate in steroid-induced modification of BK activity: β1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone’s cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3–1 µM and ≥100 µM for β1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from β1−/− vs. wt mice. Data showed that β1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 μM and ECMAX ≥ 10 μM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in β1−/− mice. Therefore, β1 enables BK activation and MCA dilation by low µM aldosterone.Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory β Iniewed in ). The va  in solution was adjusted to the desired value by adding the appropriate amount of Ca2+ from a 1 mM CaCl2 stock. Nominal free Ca2+ was calculated with MaxChelator Sliders (Stanford University).BK channel currents were recorded from excised I/O patches obtained from individual MCA myocytes. Bath and electrode solutions were identical and consisted of (mM) 130 KCl, 5 EGTA, 1.6 HEDTA, 2.28 MgCl− as main anion (from a stock of 1 M KCl) was used as ground electrode. Patch recording electrodes were pulled as described elsewhere [An agar bridge with Cllsewhere . After tIonic currents at single-channel resolution were recorded under voltage-clamp conditions in gap-free mode using an EPC8 amplifier (HEKA) at 1 kHz. Data were digitized at 5 kHz using a Digidata 1550B A/D converter and pCLAMP 10.6 . As index of channel steady-state activity, we used the product of the number of channels in the I/O patch  and channel open probability (Po). Drug-induced NPo changes were determined by comparing baseline NPo  to the NPo from the same patch/cell exposed to the agent under study  dissolved in bath solution. NPo was automatically determined using Clampfit 10.6/10.7 software (Molecular Devices).Following MCA isolation (see section above), each artery was cut into 1–2 mm long segments. Then, one arterial segment was cannulated at each end in a temperature-controlled perfusion chamber . To remove the endothelial layer of the arterial segment, an air bubble was pushed into the vessel lumen for 90 s prior to vessel cannulation. This method has been shown to adequately remove the endothelial layer based on the differential response of treated arteries to endothelium-dependent vs. endothelium-independent vasodilators ,64.2, 1.2 MgSO4, 0.023 EDTA, 11 glucose, 24 NaHCO3. PSS was maintained at pH 7.4 with a mixture of 21/5/74% O2/CO2/N2 and maintained at 35–37 °C. Arteries were monitored with a charge-coupled camera . The external wall diameter was measured as by us [Chemicals below), and measurement of aldosterone effect was conducted only after the MCA segment reached a maximal, steady-state response to the steroid, upon which washout with PSS was immediately perfused. After washing, maximal contractility was determined with a depolarizing 60 mM KCl solution (mM): 63.7 NaCl, 60 KCl, 1.2 KH2PO4, 1.2 MgSO4, 0.023 EDTA, 11 glucose, 24 NaHCO3, and 1.6 CaCl2 , the chamber housing the cannulated arterial segment was perfused at a rate of 3.75 mL/min with physiologic saline : 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.6 CaClas by us ,66 and oas by us ,68. This.6 CaCl2 . KCl wasAldosterone , calcium chloride , dimethyl sulfoxide , sodium bicarbonate , Phenol Red , and Taurine  were obtained from Sigma-Aldrich. BSA , papain , and soybean trypsin inhibitor  were stored at 4 °C. Collagenase  was stored at −20 °C. Sodium chloride (S271), potassium chloride (P217), potassium phosphate (P285), and magnesium chloride (M33) were purchased from Fisher Scientific . Magnesium sulfate (MX0070-1) was purchased from EMD Millipore Sigma . Ethylenediamine tetra-acetic acid (EDTA)  was purchased from VWR . Clinical blood gas mixture  was purchased from Nexair . A stock solution of aldosterone was first made  in DMSO. Aliquots were made and frozen at −20 °C until needed, and aliquots were only kept up to a year. The DMSO–aldosterone mixture was added to 30 mL of physiologic saline for a final concentration of (µM) 0.03, 0.3, 1, 3, 10, and 100. Aldosterone-containing DMSO was added to physiological saline immediately prior to profusion. The saline mixture was covered with parafilm to prevent any evaporation.p values with significance set at p < 0.05. Validity of the Boltzmann fit results were given by the corresponding reduced chi-square and R-square values; the reduced chi-square statistic was used to represent the goodness-of-fit testing and the R-square results evaluated the scatter of data points around the fitted regression line.Statistical analysis was performed using SPSS v27. When the number of observations in the groups under comparison exceeded 6, and the Gaussian distribution of the data was confirmed by the Kolmogorov–Smirnov test, analysis was performed using an unpaired Student’s t-test. In all other cases, statistical analysis was conducted using the Mann–Whitney nonparametric test. For comparison of multiple experimental groups, the Kruskal–Wallis test with Dunn’s post-test were used. In all cases, testing assumed two-tailed 1 subunits allow μM aldosterone to evoke activation of vascular SM BK channels and endothelium-independent dilation of MCA. These findings constitute a first step towards identification of an aldosterone-recognition site in BK β1-subnits and eventually obtaining novel, more potent compounds that, through BK channel activation, could modify aldosterone-mediated cerebrovascular pathophysiology.Our study documents, for the first time, direct binding of a signaling, circulating steroid molecule  to the subunits that primarily define the phenotype of SM BK channels, and advances the idea that regulatory β


Macrophages have a vital role in phagocytosis and antiviral effect against invading influenza viruses. Previously, we found that methionine enkephalin (MENK) inhibited influenza virus infection by upregulating the “antiviral state” of macrophages. To investigate the immunoregulatory mechanism of action of MENK on macrophages, we employed proteomic analysis to identify differentially expressed proteins (DEPs) between macrophages infected with the influenza-A virus and cells infected with the influenza-A virus after pretreatment with MENK. A total of 215 DEPs were identified: 164 proteins had upregulated expression and 51 proteins had downregulated expression. Proteomics analysis showed that DEPs were highly enriched in “cytokine-cytokine receptor interaction”, “phagosome”, and “complement and coagulation cascades pathway”. Proteomics analysis revealed that MENK could be an immune modulator or prophylactic for the prevention and treatment of influenza. MENK promoted the polarization of M1 macrophages, activated inflammatory responses, and enhanced phagocytosis and killing function by upregulating opsonizing receptors. There are four types of influenza viruses: A, B, C and D. The influenza-A virus is a contagious pathogen responsible for severe respiratory disease in humans, and its prevalence continues to pose a major threat to public health worldwide , 2. VaccA dendritic cell (DC)-targeting vaccine has received global attention recently. It directed hemagglutinin and the chemokine receptor Xcr1 + of the influenza virus to DCs to induce an immune response and confers protection against the influenza virus , 7. MacrMethionine enkephalin (MENK) is an endogenous neuropeptide, composed of Tyr-Gly-Gly-Phe-Met, derived from the prohormones and proenkephalins produced by the adrenal glands, with neuroendocrine regulatory activity, strong immunoregulatory activity and non-cytotoxic , 12. MENOur laboratory data have illustrated that MENK could be working as a non-specific prophylactic to be used for influenza epidemic. In order to further elucidate the molecular mechanism of MENK as immune modulator or prophylactic on macrophages, we employed proteomics analysis and bioinformatics analysis to identify differentially expressed proteins (DEPs) between macrophages infected with the influenza-A virus and cells infected with the influenza-A virus with MENK pretreatment (pre-MENK).The influenza-A virus, A/PR/8/34 (H1N1), was kindly provided by the China Center for Disease Control and Prevention . A murine macrophage cell line (RAW264.7) was purchased from the Cell Resource of Chinese Academy of Sciences .50) of 100 for 1 h and washed with phosphate-buffered saline (PBS). Then, they were cultured in Dulbecco’s modified Eagle’s medium  containing 1% bovine serum albumin (Gibco), penicillin (100 U/mL), streptomycin (100 μg/mL), HEPES , and tosyl phenylalanyl chloromethyl ketone-trypsin  for 72 h. Pre-MENK group cells were treated with MENK  in DMEM containing 10% fetal bovine serum (Gibco) for 48 h. Then, they were washed with PBS, inoculated with H1N1 (100 TCID50) for 1 h, washed with PBS, and allowed to culture for 72 h. Cells were collected 72 h after virus infection and stored at − 80℃.RAW264.7 cells were divided into two groups: influenza A virus-infected control (H1N1-C) and pre-treatment with MENK (pre-MENK). H1N1-C group cells were inoculated with H1N1 at a median tissue culture infectious dose  and a 1% protease inhibitor cocktail was added. Cells were lysed by sonication. The remaining debris was removed by centrifugation at 12,000 × For digestion, the protein solution was reduced with dithiothreitol (5 mM) for 30 min at 56 °C and alkylated with iodoacetamide (11 mM) for 15 min at room temperature in the dark. Then, the protein sample was diluted by addition of tetraethylammonium bromide (100 mM) to facilitate a urea concentration < 2 M. Finally, trypsin was added at a mass ratio of trypsin:protein of 1:50 for the first digestion overnight and a mass ratio of trypsin:protein of 1:100 for a second 4 h-digestion.Tryptic peptides were dissolved in solvent A  and loaded directly onto a homemade reversed-phase analytical column . Peptides were separated with a gradient from 4 to 23% solvent B (0.1% formic acid in 90% acetonitrile) over 62 min, 23% to 35% in 20 min, climbing to 80% in 4 min, and then holding at 80% for the final 4 min, all at a constant flow rate of 500 nL/min on an EASY-nLC 1200 UPLC system ./z. The 10 most-abundant precursors were selected for further MS/MS analyses with a dynamic exclusion of 30 s. Higher-energy C-trap dissociation was undertaken at a normalized collision energy of 28%. Fragments were detected in the Orbitrap at a resolution of 15,000. The automatic gain control target was set at 5E4, with an intensity threshold of 2.5E5 and a maximum injection time of 40 ms.Separated peptides were analyzed an a Q Exactive™ HF-X mass spectrometer (Thermo Fisher Scientific) with a nano-electrospray ion source. The electrospray voltage applied was 2.1 kV. The full MS scan resolution was set to 120,000 for a scan range of 400–1500 mwww.maxquant.org/). Tandem mass spectra were searched against the Mus_musculus_10090_SP_20201214. fasta database concatenated with reverse decoy database. Trypsin/P was specified as the cleavage enzyme, with up to two missing cleavages being allowed. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search. The mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification. Acetylation on protein N-terminal, oxidation on Met and deamidation (NQ) were specified as variable modifications. The false discovery rate was adjusted to < 1%. The minimum score for peptides was > 40. We changed the label-free quantitation (LFQ) intensity (I) level of the proteins in different samples through centralization to obtain the relative quantitative value (R) of the protein. The calculation formula is Rij = Iij/Mean(Ij),where i denotes the sample and j denotes the protein. H1N1-C group (Group A) was set as the control sample, and pre-MENK group (Group B) was treated sample. We used three standard biological replicates to test whether the quantitative results were consistent with statistical consistency. Pearson's correlation coefficient (PCC), principal component analysis (PCA) and relative standard deviation (RSD) were used to evaluate repeatability.The resulting MS/MS data were processed using MaxQuant 1.6.15.0 (P < 0.05 were set as the significant thresholds for DEPs. Based on terms set in the Gene Ontology (GO) database (http://geneontology.org/), DEPs were annotated into three categories: biological process, cellular component, and molecular function. Analyses of enrichment of signaling pathways were undertaken using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/).Fold change > 1.50 or < 0.67 and The related protein structures were downloaded from PDB, and MENK 2D structures were downloaded from Pubchem, and it was transformed from SDF formal to MOL2 using OpenBabelGUI. Subsequently, the receptors were dehydration using PyMOL software, with removing the other useless atoms. Next, molecular docking was performed in AutoDock to complete the docking and analysis. Then the docking results were exported in the PDBQT format. PyMOL software was used to calculate the length of hydrogen bonds and exported the ligand and the overall pictures.In total, 462,118 spectrograms were obtained by MS. The number of available effective spectrograms was 203,221, and 44.0% of them were utilized. A total of 41,119 peptides were identified by spectrogram analysis, among which 39,679 were specific peptide segments. A total of 4,147 proteins were identified, of which 3,290 were quantifiable  and complement and coagulation cascades (mmu04610), both of these pathways indicated that complement receptors and Fc receptors played a crucial role in receptor-mediated endocytosis. In Fc receptor-mediated endocytosis, downstream STX 13, STX7, and Rab7 were involved, while αvβ5 and αvβ3 were involved in complement-mediated endocytosis , “mildly downregulated” , “mildly upregulated” , and “severely upregulated”  Fig. A.Fig. 8HDEPs with severely upregulated expression were enriched for the “phagosome, complement and coagulation cascades”, “viral protein interaction with cytokine and cytokine receptor”, “cytokine–cytokine receptor interaction”, and “chemokine signaling pathway” Fig. B, which According to confidence score > 0.7 (high confidence), the differential protein interactions were extracted from 215 differential proteins. Visualization of the differential protein interaction network was performed using the R package "networkD3" tool. In order to clearly demonstrate the protein–protein interaction, we screened proteins with the closest interaction relationship and mapped the protein interaction network. In Fig. Based on the data of PPI, and combined with KEGG pathway(mmu04061 and 04145), we evaluated the interaction between MENK and 8 small-molecule core proteins. CCL3, CCL4, TNFR2, CR3, FcγR, Rab7, STX13 and STX7 proteins crystal structures were downloaded from the PDB database, improved by PyMOL, and molecular docking of the target proteins to the related ingredients was performed in AutoDock. Affifinity was the score used by the software to calculate the binding ability. It is generally believed that affinity < -7 kcal/mol indicated stronger binding activity, -7 < affinity ≤ -4 kcal/mol indicated moderate binding activity, affinit > -4 kcal/mol indicated week binding activity . After cMacrophages play a leading part in pathogen recognition, and protect host defense through inflammation initiation and clearance of pathogens in the airways. Macrophages can be subdivided into classically activated (M1) and alternatively activated (M2) phenotypes according to their functional characteristics , 20. In Orientia tsutsugamushi infection [Consistent with those findings, we revealed significant enrichment of leukocyte chemotaxis, as well as the migration and activation of cells involved in the immune response, whereas the small cytokine (integrin/chemokine)/IL-8-like domain was enriched markedly according to clustering analysis of the protein domain. Furthermore, several processes related to the cytokine response, such as “viral protein interaction with cytokine and cytokine receptor (mmu04061)” and “chemokine signaling pathway” (mmu04062), were found to be enriched. MENK in prophylactic administration upregulated expression of the cytokines CCL3, CCL3L1, CCL4, CCL4L1, CCL4L2, and tumor necrosis factor receptor type (TNFR)2. CCL3, CCL3L1, CCL4, and CCL4L1 are family members related to chemokines . CCL3 ennfection . PPI shoCytokines secreted by M1 macrophages have a regulatory role in expression of the receptor FcγRs. In general, proinflammatory cytokines increase the level of activation of FcγRs . FurtherKEGG enrichment pathway mmu04145) showed that FcγR, CR3, Rab7, STX13 and STX7 related to the processing of endosomes and phagosome formation was upregulated, and there were upstream and downstream relation among these molecules displaying by the PPI. “Phagosome formation” refers to an internalization process in which larger particles  are engulfed and processed within a membrane-bound vesicle . Vesicle45 showedOur proteomics-analysis data on macrophages demonstrate that prophylactic administration of MENK may play an important part in upregulating antiviral state of macrophage before influenza viruses infection. In addition to our previous study reporting that MENK can promote macrophage polarization and increase cytokine secretion, our proteomic results suggest that macrophage receptor-mediated endocytosis and phagosome formation may be regulated by pre-MENK administration. Besides, MENK promotes the adaptive immune response indirectly by improving the antigen presentation of macrophages. Therefore, we speculate that MENK acts on innate immune cells with a non-specific, antigen-eliminating function. Prophylactic MENK should improve the antiviral status of macrophages in advance, which is conducive to the clearance process of all strains of influenza viruses. Hence, MENK could be used as an immune modulator or non-specific prophylactic for the prevention and treatment of influenza.


Background: Myocardial infarction is one of the leading causes of mortality worldwide; hence, there is an urgent need to discover novel cardioprotective strategies. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway, has been previously reported to have cardioprotective effects. However, the mechanisms by which KYNA may be protective are still unclear. The current study addressed this issue by investigating KYNA’s cardioprotective effect in the context of myocardial ischemia/reperfusion. Methods: H9C2 cells and rats were exposed to hypoxia/reoxygenation or myocardial infarction, respectively, in the presence or absence of KYNA. In vitro, cell death was quantified using flow cytometry analysis of propidium iodide staining. In vivo, TTC-Evans Blue staining was performed to evaluate infarct size. Mitochondrial respiratory chain complex activities were measured using spectrophotometry. Protein expression was evaluated by Western blot, and mRNA levels by RT-qPCR. Results: KYNA treatment significantly reduced H9C2-relative cell death as well as infarct size. KYNA did not exhibit any effect on the mitochondrial respiratory chain complex activity. SOD2 mRNA levels were increased by KYNA. A decrease in p62 protein levels together with a trend of increase in PARK2 may mark a stimulation of mitophagy. Additionally, ERK1/2, Akt, and FOXO3α phosphorylation levels were significantly reduced after the KYNA treatment. Altogether, KYNA significantly reduced myocardial ischemia/reperfusion injuries in both in vitro and in vivo models. Conclusion: Here we show that KYNA-mediated cardioprotection was associated with enhanced mitophagy and antioxidant defense. A deeper understanding of KYNA’s cardioprotective mechanisms is necessary to identify promising novel therapeutic targets and their translation into the clinical arena. Myocardial infarction (MI) remains one of the leading causes of morbidity and mortality in developed countries . MI is aThe kynurenine pathway (KP) is the major route of tryptophan degradation ,6. ApproRecently, alteration of KP has also been implicated in the pathophysiology of cardiovascular diseases (CVD) [es (CVD) ,16. Sevees (CVD) . Kynurenes (CVD) . In serues (CVD) . Anotheres (CVD) . Moreovees (CVD) ,21,22. Ies (CVD) . KYNA is a key metabolite of the kynurenine-tryptophan pathway with pleiotropic effects . KYNA hap < 0.001). Relative cell death was significantly reduced in the KYNA-treated groups compared to the H/R group  (Ψm) (46.8 ± 7%) compared to the control . The KYNA treatment was able to significantly increase the number of cells with a conserved ΔΨm   A. H/R lectively) B.p = 0.023), whereas the AAR%LV was similar in the two groups . To ensure consistent mitochondrial content between groups, we assessed the CS activity in the same hearts used for the complex activity studies. The CS specific activity did not differ among the sham, MI, and MI + KYNA groups. Next, we evaluated the specific activities of complexes I, II, III, and IV, normalized to the CS activity. Despite no significant differences among sham, MI, and MI + KYNA groups in each complex’s specific activity, the equilibrium between respiratory complexes (complex ratio)  and significantly increased for complex ratio III/I in the MI and MI + KYNA groups compared to sham group . The AMPKα’s, a metabolic sensor regulating PGC1α’s activity, phosphorylation levels were not significantly different among the groups. PGC1α protein expression, a transcription factor mediating mitochondrial biogenesis and oxidative phosphorylation, was not different among the sham, MI, and MI + KYNA groups (As shown in A groups C.p = 0.044), whereas the KYNA treatment significantly reduced FOXO3α phosphorylation levels  (p < 0.001). The KYNA treatment was associated with a significant decrease in ERK1/2 phosphorylation , as well as a significant decrease in Akt phosphorylation compared to the MI group  . After 15 min of reperfusion, FOXO3α phosphorylation levels were significantly increased in the MI group compared to the sham group  A. FOXO3α= 0.047) B.p = 0.004; 17.21 ± 1.39 in MI vs. 26.08 ± 1.24 in sham for catalase, p < 0.001) whereas SOD2 and SOD3 mRNA remained unchanged. The KYNA treatment induced a significant increase in SOD2 mRNA expression  and an increase, though not significant, in SOD3 mRNA levels  (p < 0.001). Catalase protein levels were also significantly higher in the MI (0.42 ± 0.03) vs. the sham group . Finally, the KYNA treatment exhibited a trend towards increasing catalase protein levels compared to the MI group  (KYNA has been reported to exert reactive oxygen species (ROS) scavenger properties. Thus, we evaluated mRNA as well as protein expression of SOD1, SOD2, SOD3, and catalase. The SOD1 and catalase mRNA levels were significantly decreased following MI  A. The SO= 0.055) B.p = 0.004), decreasing even further following KYNA treatment . In addition, PARK2 protein levels were significantly increased in the MI group compared to the sham group . The KYNA treatment tended to increase PARK2 protein levels compared to the MI   .Myocardial infarction is a major cause of death and disability in the world . ReperfuThe role of the kynurenine pathway, the major route of tryptophan degradation, in the heart is not clear. The first metabolite generated is kynurenine, which in turn can be metabolized into KYNA. Previous studies have demonstrated the neuroprotective abilities of KYNA. In a neonatal rat cerebral ischemia/reperfusion model (left carotid artery ligation), KYNA was administrated at 300 mg/kg 2 h following cerebral hypoxic-ischemia. This induced a reduction in brain lesions . In anotIschemia/reperfusion injuries are associated with a dysfunction in oxidative phosphorylation . Indeed,FOXO3α is a transcription factor known for its role in promoting the transcription of genes implicated in mitophagy and antioxidant defense ,46. FOXO−/− mice, cardioprotection by KYNA administered either 2 or 24 h prior to injury in vivo or 10 or 30 min prior to ischemia/reperfusion in ex vivo hearts was completely abrogated [i/0 mediated responses, confirmed ERK1/2 phosphorylation stimulation by GPR35 [KYNA has been identified as a ligand of the recently de-orphanized G protein-coupled receptor 35 (GPR35) . The emebrogated . Moreovebrogated . Similarbrogated ,59. Indeby GPR35 . TherefoTaken together, our present study further indicates KYNA as a potential novel and promising cardioprotective therapeutic agent to reduce myocardial ischemia/reperfusion injuries. We improved our understanding of underlying KYNA-mediated cardioprotection mechanisms by exploring several possible pathways. Future directions would be to translate this novel cardioprotective therapy that targets the kynurenine pathway into a clinical setting, where there is a lack of effective cardioprotective strategies and unmet needs of acute MI patients. The role of KYNA should be more comprehensively investigated to promote its clinical applications.Our in vitro work demonstrated the cytoprotective effects of KYNA in H9C2 cells, a widely employed immortalized cardiomyoblast cell line. These cells present a phenotype differing from that of adult cardiomyocytes. We confirmed KYNA-induced cardioprotection in an in vivo rat myocardial ischemia/reperfusion model and proposed underlying pathways. In order to explore them, we chose to work with a pretreatment model using high KYNA dosing. GPR35 is a well-documented receptor for KYNA. Its implication in the studied signaling pathways remains unclear. A pharmacological antagonism or genetic inhibition of GPR35 could confirm its implication in the activation of potential downstream effectors such as ERK1/2 and Akt. Thus, further studies are needed to further translate our findings into the clinical arena.An experimental study was conducted to explore KYNA-induced cardioprotective pathways using both in vitro hypoxia/reoxygenation and in vivo ischemia/reperfusion models. In vitro, H9C2 cells were subjected to hypoxia/reoxygenation and cell viability as well as the mitochondrial membrane potential were evaluated. In vivo, rats were subjected to a preclinical myocardial ischemia/reperfusion. Left ventricular tissue samples collected at the end of the reperfusion period were used to assess infarct size, mitochondrial respiratory chain complex activity, mRNA expression and protein levels.2 1, CaCl2 1.8, pH 7.4). The cells were placed into a hypoxia chamber flushed with a stream of pure nitrogen for 4 h and 50 min at 37 °C. The oxygen rate was kept at 0.5%. Reperfusion was mimicked by replacing Tyrode’s solution with the complete medium in a standard incubator under normoxic conditions for 2 h. The groups were defined as follows (–Control group: Cells did not undergo any intervention and were kept in normoxic conditions and a culture medium for 7 h.–Hypoxia/reoxygenation (H/R) group: Cells underwent H/R with the DMSO (vehicle) treatment 10 min before hypoxia and throughout the procedure.–H/R + KYNA group: Cells underwent H/R with the KYNA treatment (1 µM) either 10 min before hypoxia (pre), pre + during hypoxia (pre + per), or pre + per + during reoxygenation (pre + per + post).H9C2-SV40 cells, an immortalized rat cardiomyoblast cell line, were employed for in vitro hypoxia/reoxygenation  in order follows A:–ControAt the end of reoxygenation , the cells were detached from the plates using Accutase . Cell death was quantified by flow cytometry  using 1 µg/mL propidium iodide (PI)  . Cell death in the H/R group was considered equal to one, and other data were expressed as relative mortality compared to H/R. ΔΨm was appreciated using 20 nM DilC1   , a cationic dye that accumulates in potentiated mitochondria. Results were expressed as the percentage of cells that conserved mitochondria with high ΔΨm. Triplicate samples were prepared for each condition, and a total of 10,000 events were acquired by fluorescence-activated cell sorting (FACS) for each sample .Male adult Wistar rats, aged 8 to 10 weeks and weighing 250 to 300 g, were used in this study. They were kept in a temperature-controlled room (22 ± 2 °C) with an adequate 12-h light/12 h dark cycle. Food and water were available ad libitum. All experiments were conducted in agreement with the guidelines from EU Directive 2010/63/EU, French Decree no. 2013-118 of the European Parliament on the protection of animals used for scientific purposes. The protocol was approved by the Ethics Committee in Animal Experimentation of Pays de la Loire and by the French Ministry of Higher Education and Research (APAFIS#8668-20 170 12417473589 v3).®, Axience, France). Afterwards, in one set of experiments, hearts were excised for infarct size assessment and, in another set of experiments, freeze-clamped and stored at −80 °C for mRNA expression analysis, protein analysis, and assessment of the mitochondrial respiratory chain complex activity.Hearts were excised under deep anesthesia , orotracheally intubated, and mechanically ventilated with room air by means of a small animal ventilator , as previously described [–Sham group: animals undergoing all the surgical procedure except ligature of the coronary artery.–MI group: animals undergoing myocardial ischemia/reperfusion and injected 10 min before ischemia with NaOH 1 M (vehicle).–MI + KYNA group: animals undergoing myocardial ischemia/reperfusion and injected 10 min before ischemia with 300 mg/kg KYNA . The dose was chosen based on previously published data .The rats were anesthetized by means of an intraperitoneal injection of 60 mg/kg of sodium pentobarbital  was outlined after a retrograde perfusion with Evans blue (1%). From the apex to the base, the heart was cut into five equal slices, then incubated with a 1% solution of 2.3.5-triphenyltetrazolium chloride (TTC)  in phosphate buffer at pH 7.4 and 37 °C. TTC staining enabled the distinction between infarcted myocardium in white and viable myocardium colored brick red. The slices were photographed. Infarct size was quantified using planimetry with Image J software . The area of necrosis (AN) was expressed as a percentage of the AAR (AN%AAR), and AAR as a percentage of total left ventricular (LV) area (AAR%LV) .We quantified the expression of genes encoding catalase, and superoxide dismutase 1, 2, 3  following 120 min of reperfusion . Genes e® 480 II Thermocycler . The thermal cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The results of the target genes’ mRNA were normalized on the mean Ct value of the reference genes mRNA and expressed as 2ΔCt, where ΔCt is defined as the difference between the Ct of reference genes and Ct of target genes.Total RNA was extracted using the RNeasy MiniKit  according to the manufacturer’s instructions from approximately 30 µg of frozen LV tissue samples from the ischemic zone of the MI and MI + KYNA animals or non-ischemic zone in the sham animals. The cDNA was synthesized using the Quantitect Reverse Transcription Kit  following the manufacturer’s instructions. In a total volume of 20 μL reaction system (10 ng of cDNA), SYBR™ Select Master Mix  was applied to perform qPCR using a LightcyclerThe freeze-clamped ischemic  and non-ischemic (sham) LV rat hearts were employed following 15 min or 120 min of reperfusion for WB analysis as previously described . Brieflyg) while discarding the pellet.The activities of lactate dehydrogenase (LDH), citrate synthase (CS), and the electron transport chain complexes (complexes I–IV) were spectrophotometrically measured at 37 °C with a UV spectrophotometer  in the sham, MI, and MI + KYNA LV muscle homogenates following 2 h of reperfusion ,66. HomoThe CS activity was measured in a reaction medium consisting of 100 mM Tris·HCl pH 8.1, 150 µM 5.5′-dithio-bis (2-nitrobenzoic acid) (DTNB), 50 µM oxaloacetate, 30 µM acetyl-CoA, and 0,1% Triton X-100. After 2 min of incubation, the reaction was initiated by adding 10 µL/mL homogenate, with the change in optical density at 412 nm recorded over 1 min.2PO4 buffer pH 7.5, 3.75 mg/mL bovine serum albumin (BSA), 100 µM decylubiquinone, and 10 µL/mL homogenate, with  or without 10 µM rotenone. After 2 min of incubation at 37 °C, the reaction was initiated by adding 0.1 mM NADH. The activity was measured at 340 nm by monitoring NADH oxidation over 2 min.NADH ubiquinone reductase (complex I) activity was assayed in KH2PO4, 2.5 mg/mL BSA, 6.5 µM rotenone, 5 µmg/mL antimycin, 25 mM succinate, 1 mM KCN, and 100 μM DCPIP, pH 7.5. After 2 min of incubation at 37 °C with 15 µL of homogenate, the reaction was initiated by adding 100 μM decylubiquinone, with the optical density recorded for 2 min.The succinate dehydrogenase (complex II) activity was measured after the reduction of 2,6-dichlorophenolindophenol (DCPIP) at 600 nm in a buffer containing 50 mM KH2PO4 pH 7.5, 250 µM ethylenediaminetetraacetic acid (EDTA), 1 mg/mL BSA, 1 mM KCN, and 100 μM oxidized cytochrome c, with or without 5 µg/mL antimycin (non-enzymatic reduction of cytochrome c). The reaction was initiated by adding 100 μM decylubiquinol, and the optical density was measured over 40 s. The specific complex III activity was calculated by subtracting the activity of the non-enzymatic reaction from that of the total activity.The ubiquinone-cytochrome c reductase (complex III) activity was determined by monitoring the reduction of cytochrome c at 550 nm. Overall, 10 µL/mL homogenate was incubated for 60 s in a reaction medium consisting of 100 mM KH2PO4, pH 7.0 was pre-incubated over 2 min. The reaction was initiated by adding 10 µL/mL of homogenate, with the change in optical density measured over 40 s.The cytochrome-c oxidase (complex IV) activity was measured by monitoring the oxidation of reduced cytochrome c at 550 nm. An 80 μM solution of reduced cytochrome c (92–97% reduced using dithionite) in 55 mM KHA control with beef heart mitochondria was executed in parallel with each set of samples to ensure proper running of the experiments. The cellular protein content was determined using the BCA protein assay kit  with BSA as standard. Specific complex activities were expressed as a ratio of and normalized to CS activity.t-test. For multiple group comparison, either a one-way ANOVA followed by an LSD post hoc test was performed or a Kruskal–Wallis test followed by a Pairwise Comparison’s post hoc test. Data are expressed as mean ± standard error of the mean (SEM); a p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS Statistics v.17.0 software . After verifying data’s distribution’s normality using Shapiro–Wilk test, the appropriate statistical test was performed. Differences between the two groups (for AAR%LV and AN%AAR) were evaluated using Student’s KYNA reduced ischemia/reperfusion injuries in both in vitro and in vivo models. KYNA-mediated cardioprotection was associated with decreased ERK1/2 and Akt phosphorylation levels, which may have contributed to a decrease in FOXO3α phosphorylation, possibly linked with increased mitophagy and antioxidant defense. Additional studies are needed to establish the exact link between these different downstream effectors. For instance, inhibition of underlying signaling pathways would validate their implication in the conferred cardioprotection. Moreover, GPR35′s implication would require confirmation by testing KYNA’s cardioprotective effects in GPR35 knockout mouse model or using antagonists of GPR35. Further in-depth investigations of KYNA’s cardioprotective mechanisms using other rodents and mammalian models are required. Thus, supplementation of KYNA might be helpful to improve acute myocardial infarction management.


Here, CeO2 NPs at concentrations lower than 50 mg L−1 have been applied to regulate the transformation of plasmid-borne ARGs to competent Escherichia coli (E. coli) cells. Three types of exposure systems were established to optimize the regulation efficiency. Pre-incubation of competent E. coli cells with CeO2 NPs at 0.5 mg L−1 inhibited the transformation (35.4%) by reducing the ROS content (0.9-fold) and cell membrane permeability (0.9-fold), thereby down-regulating the expression of genes related to DNA uptake and processing . Importantly, CeO2 NPs exhibited an excellent binding capacity with the plasmids, decreasing the amounts of plasmids available for cellular uptake and down-regulating the gene expression of DNA uptake . Altogether, pre-exposure of plasmids with CeO2 NPs (10 and 25 mg L−1) suppressed the transformation with an efficiency of 44.5–51.6%. This study provides a nano-strategy for controlling the transformation of ARGs, improving our understanding on the mechanisms of nanomaterial-mediated ARGs propagation.The direct uptake of extracellular DNA (eDNA) via transformation facilitates the dissemination of antibiotic resistance genes (ARGs) in the environment. CeO Consi−7–10−9) ,12. On t2 NPs are known as effective ROS scavengers and can remove excess ROS (by 52%) in Arabidopsis thaliana without nano-toxicity [2 adsorbent exhibited a high adsorption capacity for the removal of ARGs (eDNA), with a maximum adsorption efficiency up to 83.4% [2 NPs, we hypothesize that CeO2 NPs may inhibit transformation through eDNA binding, eliminating ROS, and maintaining cell membrane permeability.Transformation of eDNA includes the development of cell competence, eDNA binding to cell membrane, uptake, and processing . The comtoxicity ,26. A Ceto 83.4% . Conside2 NPs on plasmid binding and on membrane permeability, three transformation systems were established to identify the optimal stage and concentration of CeO2 NPs for controlling the transformation of plasmid-borne ARGs. Escherichia coli (E. coli) was used as a recipient strain, considering its frequent occurrence in manure and biosolids; it is also known to be persistently (≤231 days) competent in soils [9 copies mL−1, equivalent to the concentration reported in sludge, livestock manure, and household wastewater [2 NPs, ROS content, cell membrane permeability, and expression of genes involved in the transformation process were measured and analyzed to reveal the CeO2 NPs-mediated mechanisms. Our findings could advance the understanding of CeO2 NPs-regulated transformation of ARGs, providing an alternative approach for the management of antibiotic resistance in the environment.To investigate the roles of CeOin soils . pAC plastewater ,29, weremCherry fluorescent protein carrying a chloramphenicol resistance gene (RCm), were purchased from GenScript Biotech Corporation . E. coli cells harboring the pAC plasmids were incubated in Luria-Bertani (LB) medium  containing 10 μg mL−1 chloramphenicol at 37 ℃ overnight on a shaker (220 rpm). The plasmids were extracted with SanPrep Column Plasmid Mini-Preps Kit , and the concentration and quality (A260/A280 ratio > 1.8) of plasmids were determined using a Nanodrop spectrophotometer .The pAC plasmids (3184 bp), marked with E. coli JM109 strain, a non-resistant bacterium, was selected as the recipient strain to assess the efficiency and frequency of plasmid transformations. For the transformation of plasmid-borne ARGs into E. coli cells, an Ultra-Competent Cell Preps Kit  was used to ensure E. coli JM109 was competent for transformation.2 NPs  were purchased from Sigma-Aldrich . CeO2 NPs suspensions (1 mg mL−1) were prepared by dispersing 0.5 g of CeO2 NPs in 50 mL sterile Milli-Q water  via ultrasonication (80 W) for 1 h in a water bath at ambient temperature, followed by dilution to working concentrations in a range of 0–50 mg L−1 for subsequent experiments. The morphology of CeO2 NPs was observed by transmission electron microscopy  were prepared and placed for 2 h followed by filtration using Amicon Ultra 3 kDa MWCO tubes (2348× g for 15 min). The Ce3+ content in supernatants was analyzed using inductively coupled plasma mass spectrometry .CeOE. coli cells as recipients. To investigate the roles of CeO2 NPs on the process of transformation, CeO2 NPs were incubated with plasmids or E. coli alone or in combination.Three transformation systems were set up by using pAC plasmids as extracellular plasmid-borne ARGs and competent E. coli cells with CeO2 NPs. Briefly, 25 ng free plasmids and competent E. coli cells (107 cfu mL−1) were exposed to CeO2 NPs at concentrations of 0, 0.5, 1, 5, 10, 25, and 50 mg L−1 (100 μL transformation system), respectively. The mixture was placed in an ice-water bath and incubated for 2.5 h.System 1 was established by mixing plasmids and E. coli with CeO2 NPs followed by an addition of plasmids for transformation. Specifically, 100 μL competent E. coli cells (107 cfu mL−1) were exposed to CeO2 NPs at concentrations of 0, 0.5, 1, 5, 10, 25, and 50 mg L−1, respectively. After 2 h exposure in an ice-water bath, 25 ng plasmids were added into the mixture, followed by 30 min of incubation in an ice-water bath.System 2 was conducted by pre-incubation of 2 NPs followed by an addition of E. coli cells. Briefly, 25 ng pAC plasmids were exposed to CeO2 NPs at concentrations of 0, 0.5, 1, 5, 10, 25, and 50 mg L−1, respectively. After incubation on ice for 2 h, the mixture was transferred into 100 μL suspensions of competent E. coli (107 cfu mL−1) and incubated in an ice-water bath for 30 min.System 3 was carried out by pre-incubation of plasmids with CeO2 NPs, each mixture was heat-shocked at 42 °C for 90 s. After resting in the ice-water bath for 3 min, the bacteria suspensions were incubated in 900 μL LB medium at 37 °C, 220 rpm for 1 h. The amount of total culturable cells was calculated by plating samples on LB agar plates containing no antibiotics. Transformants were selected by dispersing another 100 μL of bacterial suspensions on LB agar plates containing 10 μg mL−1 chloramphenicol. The transformation efficiency and transformation frequency were calculated with the following formulas [In all systems, after exposure to CeORCm and mCherry (2651 bp) was detected with long amplicon PCR  . The target gene containing both –3000 bp .2 NPs, 25 ng pAC plasmids were exposed to CeO2 NPs , followed by incubation on ice for 2 h. The mixture was dropwise loaded on the crystal surface of attenuated total reflectance (ATR) attachment for FTIR analysis . CeO2 NPs-bound plasmids were separated from free plasmids by centrifugation at 9391× g and 25 °C for 10 min. The loss rate of plasmids after 2 h exposure was determined using a NanoDrop spectrometer and calculated with the following formula:To characterize the binding of plasmids with CeOE. coli cells were incubated with 10 μM of 2′,7′-dichlorofluorescein diacetate  for 30 min at 37 °C in darkness, with gentle shaking every 5 min. The mixture was washed three times with PBS to remove extra DCFDA. Afterwards, the bacteria cells were exposed to CeO2 NPs as described in System 1–3. The fluorescence intensity of each sample was measured using a microplate reader  with an excitation wavelength of 488 nm and an emission wavelength of 525 nm [The competent E. coli cells sampled from System 1–3 were incubated with 0.5 μM propidium iodide  dye for 15 min at 37 °C in darkness. The fluorescence intensity was measured by a microplate reader with an excitation wavelength of 535 nm and an emission wavelength of 615 nm [For the measurement of cell membrane permeability, the competent f 615 nm .E. coli cells exposed to CeO2 NPs or CeO2 NPs combined with plasmids were washed three times with PBS and centrifuged at 2348× g, 25 °C for 20 min. The collected bacteria were transferred into a 5 mL centrifuge tube for enzymatic digestion with 3 mg L−1 pancreatin mixed with 3 g L−1 lipase in PBS. After incubation for 12 h at 35 °C, the supernatant was obtained by centrifugation  [The competent  15 min) . The sup−ΔΔCT method [The total RNA of bacteria was extracted using RNApure Bacteria Kit  according to the manufacturer’s instructions and quantified using a Nanodrop spectrometer. Reverse transcription of extracted RNA was performed using the EasyQuick RT MasterMix  following the manufacturer’s protocol. Primers used are listed in T method .p < 0.05 was considered statistically significant.All experiments were performed with three biological replicates, and each biological replicate involved three technical replicates. Data are presented as mean ± standard deviation, and the significant differences among treatments were tested by one-way analysis of variance (ANOVA) tests followed by a least significant difference (LSD) test where a E. coli cells with CeO2 NPs  the transformation, while 1 mg L−1 of CeO2 NPs distinctively promoted (by 55.6%) the transformation  and the smallest particle size were observed in System 3 (average size 35.6–37.2 nm), while the maximum amount of CeO2 particles was detected in System 2  may promote the transformation process, while CeO2 NPs at 50 mg L−1 can inhibit the transformation regardless of the presence of plasmids. Pre-incubation of plasmids with CeO2 NPs at 10 or 25 mg L−1 may reduce the bioavailability of plasmids; this will be discussed in the following section. High concentrations of CeO2 NPs might attach to the surface of E. coli cell after the incubation, and a small part of CeO2 NPs entered the bacterial cell via endocytosis. Particles with a positive charge will bind to the negatively charged cell surface and 20–50 nm size particles are taken up more rapidly than smaller or larger particles [Considering the various effects of CeOe-ICP-MS . In genearticles .2 NPs at low concentrations (0.5–5 mg L−1) remained relatively low and was similar to that of control group  elevated the loss rate to 30.7–44.3%  , the hydrodynamic diameter reached 1386.3 ± 82.9 nm, 1437.2 ± 146.5 nm, and 2046.4 ± 173.9 nm for CeO2 NPs exposure at 10, 25, and 50 mg L−1, respectively . Thus, it seems that the self-aggregation of CeO2 NPs (50 mg L−1) may reduce the reactive sites of NPs for plasmid binding. Moreover, the increased membrane permeability resulted from 50 mg L−1 CeO2 NPs exposure  d. Howevenchanged d. The loexposure a; this cfter 2 h , indicat0 mg L−1 b,c. Aggrectively . The sigexposure  may elev2 NPs-plasmids aggregation as almost no dissolution (<0.1%) was observed for CeO2 NPs suspensions . CeO2 could act as a Lewis acid and bind with Lewis bases, such as phosphate backbone or electron-rich aromatic rings [2 NPs-exposed plasmids (P-O and C-O) at 1042.8 cm−1 shifted slightly to 1042.3 cm−1 and became weaker; the band at 1085.2 cm−1 shifted to 1081.8 cm−1  can reduce the availability of plasmids for transformation.It is known that DNA molecules can condense into compact structures when the valency of counter-ions ≥ three . Howeveric rings . Nanomatic rings ,40. Thus1.8 cm−1 e. The sholecules . The zet2 NPs at low concentrations exhibited an efficient ROS scavenging capacity [2 NPs at 10–50 mg L−1 increased the ROS level by 1.1-fold,  remained unchanged in the presence of 0.5 and 10 mg L−1 of CeO2 NPs, while 50 mg L−1 CeO2 NPs induced the expression of umuc by 3.2-fold (−1 CeO2 NPs (ompA and ompC) decreased by 0.6–0.7 fold after exposure to 0.5 mg L−1 CeO2 NPs  was observed in the presence of 50 mg L−1 CeO2 NPs  was consistently detected  could reduce the membrane permeability, inhibiting the transformation of free plasmids  in the presence of 10 mg L−1 CeO2 NPs (System 1 and 2)  or did not affect (System 1) the expression of bhsA (−1 CeO2 NPs (System 3) significantly inhibited the expression of bhsA by 0.7–0.8 folds (ybaV encoding protein ComEA had been significantly down-regulated (by 0.8–0.9 folds) after exposure to 0.5 mg L−1 CeO2 NPs  (2 NPs at 10 or 50 mg L−1 down- or up-regulated the ybaV expression by 0.7- or 1.2-fold (bhsA or ybaV was unaffected (despite 50 mg L−1 for System 1), while inhibition or stimulation of transformation occurred only if both genes were simultaneously down- or up-regulated (2 NPs at relative low concentrations (0.5 or 10 mg L−1) down-regulated the gene expression for DNA binding to the membrane surface, thus inhibiting the transformation  c,d. Howe of bhsA c,d. Pre-.8 folds e. ComGC ctively) c,d. In S1.2-fold e. Proteiegulated a,b. Altoormation a; convernfsB), which is essential for the formation of duplex circular plasmid and homologous recombination of incoming ssDNA [nfsB was up-regulated in the presence of 10 mg L−1 CeO2 NPs for all three systems  and System 3  ; additionally, Deinococcus radiodurans (D. radiodurans) exhibited a 21-fold reduction of plasmid transformation frequency in the absence of the DprA protein [ybaV remained unchanged in System 2 (50 mg L−1 CeO2 NPs), the down-regulation of nfsB and bhsA may contribute to the decreased transformation (recJ in E. coli strains [recF and recJ were up-regulated in E. coli cells exposed to 10 mg L−1 CeO2 NPs (System 1 and 2) (−1 CeO2 NPs in System 1 (2 NPs (0.5 or 50 mg L−1) in both System 1 and 2 inhibited the DNA uptake and integration  might have contributed to the significant reduction in transformation. CeO2 NPs (particularly 10 mg L−1) may inhibit the transformation of eARGs by suppressing the replication of DNA. However, for System 1 and 2, a high concentration of CeO2 NPs (50 mg L−1) was required to generate same inhibiting effect on DNA replication, thereby reducing the transformation frequency.The retraction of pseudopilus facilitates the contact of double-stranded DNA (dsDNA) with protein ComEA, after which one strand of DNA is degraded by EndA and the remaining single-stranded DNA (ssDNA) is translocated into cytoplasm through the ComEC transmembrane channel ,54. The ng ssDNA ,56. The  systems c–e, whilctively) d,e. Muta protein ,58. Thusormation d. DNA re strains . The exp1 and 2) c,d, wherSystem 1 c. Thus, egration a,b, withtrations e. Howevelication . In addi2 NPs for controlling the transformation process. Particularly, pre-incubation of plasmids with CeO2 NPs at 10 or 25 mg L−1 could effectively inhibit the transformation by reducing plasmid availability, inhibiting DNA replication, and down-regulating the gene expression of DNA uptake. Incubation of competent cells with CeO2 NPs at a low concentration (0.5 mg L−1) can inhibit the transformation via decreasing the ROS content and cell membrane permeability, down-regulating the expression of genes related to DNA uptake and processing; CeO2 NPs at relatively high concentrations (10 and 25 mg L−1) led to an opposite effect. Still, incubation of CeO2 NPs with plasmids and competent cells in complex environmental conditions, such as soil and aquatic environments, is needed to make this nano-strategy more practical. Our findings provide insights on the application of nanomaterials for regulating transformation of extracellular ARGs.ARGs are persistent in natural aquatic and soil environments. Transformation enables the evolution of competent bacteria into resistant species. Nanomaterials exhibit unique bioactivities due to their nanoscale size, high specific surface area, and precise nanostructure, significantly influencing the interactions between materials and biological systems to regulate cellular behaviors and functions . Here, w


Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins , hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors . Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI.Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry.Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels.Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms. Perinatal brain injury (PBI) is a leading cause of long-term morbidity and neurodevelopmental impairment in children . In recein utero with inflammation/infection (chorioamnionitis/CHORIO) and/or hypoxia-ischemia. CHORIO is the most common abnormality found in placentae from very preterm infants  injury begins  infants . The pla infants . CHORIO  infants . In pret infants . PBI lea infants  and last infants .Inflammation throughout the placental-fetal-brain axis and neural-immune aberrations after CHORIO are prominent factors in the pathophysiology of PBI. The associated signal transduction and specific molecules have been identified as therapeutic targets . For exaEPO is an important molecule for neurorepair and immunomodulation as it has anti-apoptotic, antioxidant, and anti-inflammatory properties , alongsiIn the present study, we evaluated EPO and MLT signaling throughout the placental-fetal-brain axis and modulation by CHORIO. We also evaluated SIRT1, HIF1α and HIF2α as upstream signaling partners and molecular convergence points between EPO and MLT pathways. . We aimead libitum. Animals were subject to a 12-h dark/light cycle, with lights on at 0800 h. All experiments were performed in strict accordance with protocols approved by the institutional Animal Care and Use Committee (ACUC) at the Johns Hopkins University School of Medicine. Protocols were developed and performed consistent with National Research Council and ARRIVE guidelines (Sprague Dawley rat dams and litters were maintained in a temperature- and humidity-controlled facility with food and water available idelines .Pregnant Sprague Dawley rats underwent laparotomy on embryonic day 18 (E18) as previously reported . Under iBlood was collected on postnatal day (P) 2, P7, and P21 as a terminal procedure and centrifuged at 6,000 g for 15 min at 4°C. Serum was then removed and stored at −80°C for later analysis. Care was taken to avoid freeze-thaw cycles .Placentae and forebrain were harvested at E19 (24 h following induction of CHORIO). Forebrain was also collected at P2, P7, and P21. Gene-of-interest primers and cDNA synthesized from 0.9 μg RNA were added to PowerUp™ SYBR™ Green Master Mix , and run in triplicate on a Quant Studio™ 3 Real-Time PCR System . To standardize transcripts from samples between experiments, cycle thresholds from samples of interest were compared to cycle threshold values from pooled E19 naïve samples, with gene-of-interest transcription normalized to ribosomal 18s endogenous control as previously published . Primer Polr2a), and one negative (E. coli DapB) control probe, and probes against the mRNA of interest. In Situ hybridization (ISH) was performed according to manufacturer’s specifications and the RNAscope Multiplex Fluorescent Reagent Kit V2 protocol . Signal was detected with Opal 570. Confocal z stacks were acquired by blinded observers in the somatosensory cortex at the level of the dorsal hippocampus using a Nikon ECLIPSE Ti2 microscope  with a C2 system . Gain parameters, zoom, pinhole size, step size, scan speed and resolution were uniform across scans.To localize single mRNA molecules of EPO, HIF1α, and HIF2α, RNAscope was performed on 12 μm paraformaldehyde-fixed frozen brain slices at the time points listed above. For each experiment, we used one positive . In accordance with the manufacturer’s specifications and as previously described , serum wProteins from serum were precipitated by the addition of 200 μL of LCMS grade acetonitrile to 50 μL of serum followed by vigorous vortexing for 30 s and centrifugation at 13,000 x g for 15 min at 4°C. The supernatant was pipetted off and dried with vacuum centrifugation (SpeedVac). The dried extract was resuspended in 20 μL of LCMS grade methanol followed by water bath sonication for 5 min at room temperature. The solution was again centrifuged at 13,000 x g for 15 min at 4°C and the supernatant was transferred to tapered autosampler vials for LCMS analysis.6 charges or a maximum injection time of 50 milliseconds at a resolution of 17,500 at m/z 200. For the PRM 2 × 105 charges were acquired or a maximum of 50 milliseconds attempting to reach that charge number. All collected ions were fragmented at 20 eV.Absolute quantification of melatonin was performed using a Dionex U3000 UHPLC system coupled to a Q Exactive “Classic” Mass Spectrometer using a 7-min gradient of 0.1% formic acid in LCMS grade water (Buffer A) and 0.1% formic acid in LCMS grade acetonitrile (Buffer B). All buffers were purchased from Fisher Scientific. A Poroshell 120 EC-18 2.1 × 50 mm column with 1.9 µm particles (Agilent) was used to separation with a consistent flowrate of 400 μL/min across the column maintained at 40°C. The gradient ramped from 5% B to 45% B in 3 min with a rapid ramp to 98% B in 0.5 min where it remained for 0.5 min before returning to baseline conditions for the remainder of the gradient. Eluting molecules were directly ionized into the Q Exactive at 3,500 V with a capillary temperature of 262°C and an accessory probe temperature of 425°C. Sheath gas and auxiliary gas pressures were set at 50 and 12.5 arbitrary units, respectively. A two-step method utilizing one full scan and one parallel reaction monitoring (PRM) scan were performed with the latter centered on the mass 233.1 with an isolation window of 2 Th. For the MS1 scan, all ions from 150 to 500 m/z were collected with a target of 3 × 10The Thermo.RAW files were processed in the freely available Xcalibur QuanBrowser software (Thermo Fisher) with the area under the curve extracted for the PRM fragment ion of 233.1, 174.0915 using a 10 part per million (PPM) mass extraction window and a 15 s extraction window around the target elution time of 2.15 min. For concentration calculation, the area under the curve was compared to a serial dilution of melatonin obtained from the Johns Hopkins Hospital pharmacy diluted in methanol to an approximate concentration range of 1 picogram–60 picogram on column. A linear regression was automatically performed by the program and peaks were manually adjusted only in the case of obvious failures of the program to correctly integrate peak baselines.t-test, and non-parametric differences between two groups were established with a Mann-Whitney U-test. GraphPad Prism 9.3.1 software was used for statistical analysis.Data are represented as mean ± standard error of the mean (SEM). Data were tested for normality with the Shapiro-Wilk Test. Parametric statistical differences between two groups were established with a n = 11, CHORIO: 0.10 ± 0.05, n = 10, p < 0.05, Mann-Whitney U-test)  or EPOR mRNA expression . Acute reductions in these key signaling molecules is commensurate with profound inflammation from CHORIO  . Interespression . Like EPm CHORIO .n = 6, CHORIO: 3.73 ± 0.46, n = 10, p < 0.001, Mann-Whitney U-test, n = 6, CHORIO: 3.38 ± 0.44, n = 10, p < 0.001, Mann-Whitney U-test; n = 9, CHORIO: 0.41 ± 0.19 pg/mL, n = 7, n.s., t-test). However, cerebral MLTR1 levels were also significantly increased in CHORIO brains compared to sham controls . Unlike the increases in EPO, EPOR and MLTR1 mRNA, no significant change in SIRT1, HIF1α, HIF2α mRNA were observed in CHORIO brains compared to sham  with increased HIF1α  and HIF2α mRNA  in CHORIO brains compared to sham.After assessing acute changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α in the fetal brain, we expanded our time course to the postnatal period from P2-P21 to assess cerebral injury-induced mRNA expression dynamics. At P2, a preterm equivalent time point approximately 5 days after CHORIO, EPO, EPOR, and MLTR1 mRNA expression were all stable and unchanged between CHORIO and sham groups. SIRT1 mRNA expression in the brain, however, was significantly decreased . This occurred without changes in EPOR, MLTR1, SIRT1, and HIF1α mRNA expression . To add rigor, localize regional differences, and to examine dynamic cell-specific expression of the mRNAs, we performed RNAscope. Assessment of individual mRNAs revealed widespread increases in EPO with CHORIO, in the presence of decreased HIF2α throughout the somatosensory cortex  and persistently reduced HIF2α mRNA expression . Thus, over the developmental time course from the third trimester through infancy and toddler stages of development, brain mRNA expression increases and then declines in the setting of CHORIO, emphasizing the complex pattern of changes in the injured, developing brain.Notably at P21, a toddler equivalent time point, the pattern shifted considerably. Compared to sham, EPO mRNA , despite no observed significant difference between sham and CHORIO  from HIF1α to HIF2α . SIRT1 dThe data presented herein emphasizes the importance of EPO ligand-receptor mismatch and the increased demand for MLT following CHORIO. In addition to the changes in EPO, EPOR, SIRT1, HIF1α, and HIF2α observed, we also found that CHORIO induced placental reductions in EPO and MLTR1 mRNA expression. This was distinct from the time-dependent changes in cerebral MLTR1 mRNA expression. Maternal MLT plays a key role in fetal growth and in the early stages of neurodevelopment . TypicalThis study is not without its limitations. Melatonin levels are difficult to measure, and LCMS -based evaluation of serum melatonin was used here. Future studies should include expanded time courses and direct brain melatonin measurements. MLT signaling also occurs independent of membrane bound MLTR1, and likely involves nuclear receptors. Since both EPO and MLT signaling occur in the placenta, immune cells, and brain, further studies are needed to elucidate the perinatal immune dynamics of EPO, MLT, SIRT1, and HIF2α signaling and to exploit the potential of endogenous therapeutic agents. The present study was conducted in a genetically homogeneous rodent background after a single stimulus and highlights the need to tailor diagnostic and therapeutic approaches to human infants on an individual basis with attention to initiating insults. In addition, since mRNA may not directly reflect actual protein expression or activity, degradation, phosphorylation, acetylation or other post-translational modification in the context of brain and placental injury, more detailed interpretation of homeostasis after CHORIO would be possible with additional studies of protein dynamics and function.In conclusion, EPO and MLT drive signaling pathways that enhance sustained neuroimmunomodulation and promote microenvironmental homeostasis in the developing brain. CHORIO induces lasting effects on EPO, MLTR1, SIRT1, and HIF2α mRNA and protein expression suggestive of differential effects on EPO and MLT neurorepair capacity dependent on injury and developmental stage. Maintenance of MLT, SIRT1, and HIF2α homeostasis could modulate endogenous EPO levels and may drive signal transduction that maximizes neurorepair compared to EPO dosing in restricted, acute, and isolated regimens. Given the ligand-receptor mismatch found in preterm infants, and the supplementation of endogenous MLT signaling, the combination of EPO and MLT could be a reasonable and indispensable therapeutic strategy for infants with brain injury, as could therapies that support HIF2α.