Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.The transgenic tobacco ( The main sources of ROS in cells are the plasma membrane NADPH oxidases, or respiratory burst oxidase homologs (Rbohs) [2−, which is then converted into hydrogen peroxide (H2O2). Even though NO and ROS are produced under normal physiological conditions, they are prone to uncontrolled overproduction in stressful situations, resulting in cellular nitro-oxidative damage that can compromise cell survival.Nitric oxide (NO) appears to be a critical signaling and metabolic molecule in a wide range of environmental crises, such as hypoxia. When the ambient oxygen  . They caCucumis sativus [Plant cells have evolved sophisticated defense mechanisms to avoid organelle damage as a result of redundant NO and ROS formation, e.g., excessive electron flow is dissipated by the alternate respiratory pathways, such as AOX, to preserve mitochondrial redox state. AOX maintains energy stability and keeps the flow through the mitochondrial electron transport chain (mETC) by minimizing the generation of mitochondrial NO and ROS ,5. Howev sativus . DespiteArabidopsis and rice [2 sensing [Plants have evolved a plethora of plant-specific transcription factors to precisely control the expression of their genes. The ethylene response factors (ERFs) superfamily is one of the largest families of transcription factors that respond to a variety of biotic and abiotic stressors by binding directly to promoter regions of defense-related genes . Overexpand rice ,9. Sinceand rice . ERF-VIIand rice , while eand rice . ET was  sensing ,13. Stud sensing ,15. In tTobacco leaves, when placed under hypoxia, emitted NO, while NO emission from leaves incubated in air could not be detected by using the method of chemiluminescence, due to technical features of this method . From thTo confirm these results by the second method  and to dS-nitrosylation (R-SNO) was significantly lower in the AOX overexpressors, as compared to the WT and knockdowns  atmosphere. The level of O2− in tobacco leaves was significantly higher in the knockdowns than in other plant lines under normal conditions. The O2− level increased significantly in response to a 6 h hypoxia treatment and was the highest under this treatment in the knockdowns and the lowest in the overexpression plants, with the WT showing an intermediate amount  can also reflect the degree of oxidative damage in tobacco caused by hypoxic condition. AOX overexpressors, particularly B8, displayed less stress damage than other plant lines, according to our findings in RbohB expression was significantly higher in the knockdowns and in the overexpressor B8, as compared to WT  generating ROS, determined by quantitative RT-PCR, revealed the differences between tobacco lines under normoxia and hypoxia. In air, ed to WT B. RbohA essor B7 A. Howeveo the WT C. The tressor B8 A,C. Howent lines B.We monitored the activities of antioxidant enzymes, which play important roles in plants to mitigate oxidative damage. In our study, we investigated the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) . In air,2O2, followed a similar pattern as SOD, with the greatest activity being in the knockdowns under hypoxia  and aminocyclopropane-1-carboxylic acid oxidase (ACO) strongly increased in the WT and AOX overexpression plants to the similar levels, with the knockdowns (particularly RI29) showing slightly lower increases A,B.ERF1 and ERF4 transcript amounts were significantly higher in the overexpression plants and WT, as compared to the knockdowns, showing the lower transcript amount , which perceive low-oxygen signals and play a crucial role in determining survival in low-oxygen environments , also sht amount A,C. All reatment B,D.S-nitrosylation can be linked to an increase in NO generation in this plant line. The presence of AOX can reduce electron transfer across complexes III and IV under normoxia, avoiding electron leakage to nitrite and subsequent NO accumulation in AOX overexpressors; this was directly estimated in planta, using laser scanning fluorescent confocal microscopy and biochemical methods [Nitric oxide (NO) is a crucial signaling molecule in plants since it is quickly produced, stimulates particular activities within cells, and is rapidly scavenged. The AOX knockdown plants have higher levels of leaf NO in normoxia relative to WT tobacco plants. The higher amount of protein  methods .S-nitrosylation of various proteins, including ERF-VIIs, COX, aconitase, and APX, might be connected to flooding signaling and tolerance [S-nitrosylation of important enzymes such ACS and ACO [S-nitrosylation of RbohD reduces its function, limiting the cell death caused by stress-induced oxidative bursts [In contrast to its regular function under normoxia, AOX plays a unique role in hypoxia, where it can increase nitrite-dependent NO production ,18. AOX olerance ,18. Furt and ACO . The S-ne bursts . Given te bursts . Under he bursts .2− , which were also elevated in the knockdowns and reduced in the overexpressors in comparison to WT  in their physiologically active and reduced states through the oxidation of NADH and NADPH . We presPDC1 and ADH1 transcripts increased considerably in all plant lines studied here, with highest level in overexpressors  via 1-aminocyclopropane 1-carboxylic acid (ACC) in the reactions catalyzed by ACS and ACO. Here, the transcript levels of ACS1 and ACO1 were higher in the plants expressing AOX  gene induction, and ERFs participate in Arabidopsis defense downstream of ET signaling [Deprivation of oxygen increases the activity of ACSs, thus increasing ET synthesis, and it activates downstream genes such as and ADH1 . ET accuignaling ,37. ERFsignaling ,39,40. AERF1 and ERF4 positively correlated with the overexpression of AOX under hypoxic stress. Studies show that ERFs’ genes (particularly ERF-VII) are key factors in improving plant tolerance to hypoxia by increasing anaerobic gene expression, ADH, PDC, and ROS metabolizing enzymes [In this study, hypoxia increased the number of ERF transcripts in all plant lines. The examined  enzymes ,41,42. A enzymes , PCD ind2O2 vs. O2−) of the generated ROS signal via modifying the level of Rbohs and antioxidant systems. By damping the electron leak from the ETC and increasing Pgb1, AOX could modulate the intensity of the NO. AOX can induce ET biosynthesis genes and ERFs either directly or indirectly by modulated level of NO and ROS. Produced ET might control the NO production through Pgb1 activation, and induced ERFs can regulate Rbohs and fermentation to keep energy levels stable during anoxia. We propose that AOX regulates the interaction of NO, ROS, and ET in the cell, and these act as signals for the onset of tobacco responses in the absence of oxygen.From the findings presented in this work, a model can be proposed that explains some of the aspects of stress signaling during anoxia . In thisNicotiana tabacum L. cv. Petit Havana SR1) wild type (WT) and transgenic lines with the suppressed levels of AOX protein (RI9 and RI29) and elevated levels of AOX protein (B7 and B8) were used for all experiments. The transgenic lines used in this work have been described previously [Tobacco  through the chemiluminescence detector . The measuring gas was made NO free by a NO scrubber . All gas flows were adjusted by using flow controllers .NO was measured in the gas phase, using the chemiluminescence detection method, as described before . The excw/v) PVP). The homogenates were centrifuged at 15,000× g for 10 min at 4 °C. The supernatant was pretreated for 3 min, at room temperature, with superoxide dismutase  and catalase  to remove ROS. NO was quantified in cleared extracts by spectrophotometrically measuring the conversion of oxyhemoglobin to methemoglobin [NO in the liquid phase was measured by the oxyhemoglobin assay. NO was extracted from frozen leaves of tobacco with 1 mL of cooled buffer , 200 mg of fresh leaf biomass was crushed by using a pestle and mortar in 2 mL of 8 M KOH under chilled conditions, and then it centrifuged for 15 min at 12,000× g at 4 °C. The amount of O2− in the supernatant was then evaluated at 550 nm by reduction of cytochrome c, as described in Ma et al. [2O2 content of leaves was measured as described by Xu et al. [g and 4 °C. Then 0.5 mL of supernatant was added to 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL 1M KI, and the absorbance was read at 390 nm.To measure the amount of superoxide anion  activity was assayed by measuring the inhibition of the photochemical reduction of NBT, following the method of Wu et al. [2O2, using the method of Huang et al. [+ (ε340 = 6.2 mM−1 cm−1) [Fresh leaf tissue (300 mg) was homogenized with 3 mL of ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate, and 2% PVP. The homogenates were then centrifuged for 20 min at 12,000× u et al. . CAT  .2, 0.5 mM TPP, 6 mM pyruvate, 0.67 mM NADH, 1 mM DTT, and 3.5 units mL−1 of yeast ADH . The reaction was initiated by adding protein extracts, and the absorbance was read at 340 nm.Pyruvate decarboxylase  activity was evaluated by measuring NADH oxidation in a coupled reaction with alcohol dehydrogenase (ADH) . The rea+ during ethanol oxidation, using an assay buffer of 0.5 M Tris-HCl, pH 9.0, 0.1 M ethanol, and 2 mM NAD+ [Alcohol dehydrogenase  activity was measured by monitoring at 340 nm the reduction of NAD mM NAD+ .4. The homogenate was then neutralized by using 5 M KOH and then centrifuged at 7000× g for 10 min at 4 °C. The clear supernatant was used for spectrophotometric determination of pyruvate by the enzymatic assay coupled to NADH oxidation, as described in Dinakar et al. [To measure the amount of pyruvate, freshly frozen biomass (100 mg) was gently homogenized in 1 mL 70% HClOr et al. .w/v) SDS, 0.5% (w/v) CHAPS, 1 mM EDTA, and 0.1 mM neocuproine (which inhibits denitrosylation). The homogenate was centrifuged , and protein S-nitrosylation was determined by reducing R-SNO to R-SH in the presence of ascorbate and then measuring free thiol groups, using 5,5′-dithio-bis (2-nitrobenzoic acid) [Leaf biomass (100 mg) was homogenized in 1.8 mL of 50 mM HEPES-KOH buffer (pH 8.0) containing 0.2% (ic acid) .The relative electrolyte leakage rate was measured as described by Guo et al. . The lea® Pro Green Kit . The first strand of cDNA was synthesized from 5 μg total RNA, using the Superscript III reverse transcriptase kit (Invitrogen). Quantitative PCR was carried out with an Applied Biosystems StepOnePlus Real-Time PCR 128 System, using SYBR Green qPCR Master Mixes  and gene-specific primers . Transcripts levels of NtADH1 , PDC1 (pyruvate decarboxylase), NtACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), NtACS1 (1-aminocyclopropane-1-carboxylic acid synthase), NtRbohA,B,D , and NtERF1,3,4,5 (ethylene-responsive factors belong to groups IX and VIII (only ERF3)) were analyzed in this study.Total RNA was extracted from frozen leaf, using the FastRNA primers . Triplicp < 0.05 were discussed. The software package SPSS V. 21.0  was used for statistical analysis. One-way ANOVA with Duncan’s multiple range was used to identify significant differences between different lines of tobacco. The data in the text and on figures are the means of three biological repeats ± SD. The statistically significant differences at The data presented in this study show that AOX expression controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.


Furthermore, ATP1A4 is essential for male fertility, as it interacts with signaling molecules in both raft and non-raft fractions of the sperm plasma membrane to regulate capacitation-associated signaling, hyperactivation, sperm-oocyte interactions, and activation. Interestingly, ATP1A4 activity and expression increase during capacitation, challenging the widely accepted dogma of sperm translational quiescence. This review discusses the literature on the role of ATP1A4 during capacitation and fertilization events and its prospective use in improving male fertility prediction.An advanced understanding of sperm function is relevant for evidence-based male fertility prediction and addressing male infertility. A standard breeding soundness evaluation (BSE) merely identifies gross abnormalities in bulls, whereas selection based on single nucleotide polymorphisms and genomic estimated breeding values overlooks sub-microscopic differences in sperm. Molecular tools are important for validating genomic selection and advancing knowledge on the regulation of male fertility at an interdisciplinary level. Therefore, research in this field is now focused on developing a combination of in vitro sperm function tests and identifying biomarkers such as sperm proteins with critical roles in fertility. The Na The sustainability of a burgeoning world population demands a concomitant rise in the efficiency of global food production . IncreasAlthough a breeding soundness examination (BSE) can identify bulls that are grossly abnormal, this procedure is inadequate to identify sub-fertile bulls or predict variations in fertility among bulls that are considered fertile. A standard BSE investigates bull fertility based on conventional semen analysis, without considering submicroscopic differences in sperm characteristics affecting fertility . The evaIn recent decades, semen evaluation has shifted towards an objective multi-parametric analysis using advanced techniques and multi-omic studies. This could assist in elucidating the reasons behind compromised semen quality in sub-fertile or infertile males, identifying biomarkers of fertility, and perhaps developing methods for early diagnosis of male subfertility/infertility. It could also be of interest in selecting young fertile bulls for commercial semen production and culling bulls with compromised fertility. Sub-fertile bulls cause considerable economic losses in terms of reduced conception rates, delayed calving-to-conception intervals, increased culling of females, etc. . In seve2+ concentration, and intracellular pH, aiding molecular events such as capacitation, acrosome reaction, and hypermotility   . Ouabainlar [H+] , which wmonensin . Moreovemonensin .2+]i in a limited range is also vital for sperm motility i through a reduced cation clearance, and decreased sperm kinematics in rats in a time-dependent manner [2+]i in bull sperm  Met–[35S] Cys) during capacitation into nascent proteins was clear evidence that sperm are translationally active ,128. RecIn contrast to somatic cells, mature sperm are devoid of cytoplasm , and theMale fertility is regulated by several factors and, therefore, a BSE or conventional semen analysis is inefficient in predicting fertility variations among bulls. It is also noteworthy that a bull’s ejaculate does not represent a uniform, homogeneous sperm population; rather, it consists of subpopulations with different functional characteristics such as motility ,134,135,Male fertility prediction is investigated either as a negative or positive biomarker-based approach. A negative biomarker-based approach is based on identifying proteins or ligands unique to defective spermatozoa and aims to identify and remove infertile and sub-fertile bulls from the breeding herd, whereas the positive biomarker-based approach involves the selection of HF breeding bulls. Proteins such as TMEM95 , PostacrThe prediction of male fertility requires a multifactorial approach, with an assessment of submicroscopic differences in sperm essential to improve our precision. The significance of ATP1A4 in sperm physiology associates well with the current focus on identifying sperm proteins as biomarkers for improved fertility prediction and addressing male infertility. During capacitation, lipid raft aggregation enriches the proteins involved in fertilization events where ATP1A4 interacts with signaling molecules to regulate capacitation-associated events such as sperm motility, tyrosine phosphorylation, and hypermotility, and potentially contributes to the PLC ζ-mediated activation of oocytes. ATP1A4 re-localization in the lipid rafts of the sperm head is essential for interacting raft/non-raft fraction proteins to activate signaling pathways; however, further investigation of the specific mechanisms of action of ATP1A4 in the male gamete physiology leading to fertilization is needed. Moreover, an increase in ATP1A4 protein during capacitation challenges the widely accepted dogma of sperm translational quiescence. The exclusion of translational machinery (ribosomes) with cytoplasm at the end of spermatogenesis demands investigating the underlying mechanisms regulating the translation of this protein during capacitation. An improved understanding of proteins regulating sperm functions at the molecular level may assist in differentiating apparently normal sperm through conventional semen evaluation methods.


Caragana korshinskii Kom., Pediococcus acidilactici and an exogenous fibrolytic enzyme were employed to investigate the fermentation profile, structural carbohydrates degradation, enzymatic saccharification and the dynamics of bacterial community of C. korshinskii silage. After 60 d of ensiling, all additives increased the fermentation quality. The highest lactic and acetic acids and lowest non‐protein nitrogen (NPN) and ammonia nitrogen (NH3‐N) were observed in P. acidilactici and Acremonium cellulase (PA + AC) treated silage. Additionally, all additives significantly increased the ferulic acid content and fibre degradability with the highest values obtained from PA + AC silage. The bacterial community in all silages was dominated by P. acidilactici throughout the entire fermentation process. The bacterial community was also modified by the silage additives exhibiting a relatively simple network of bacterial interaction characterized by a lower bacterial diversity in P. acidilactici (PA) treated silage. The highest 6‐phospho‐beta‐glucosidase abundance was observed in PA‐treated silage at the mid‐later stage of ensiling. PA treatment exhibited lower structural carbohydrates degradation but performed better in lignocellulose conversion during enzymatic saccharification. These results indicated that pretreating C. korshinskii improved its silage quality and potential use as a lignocellulosic feedstock for the production of bio‐product and biofuel.To explore the biofuel production potential of  The present study provides a pretreatment method for preservation and conversion of the lignocellulosic residue of C. korshinskii through sole P. acidilactici treatment or in combination with Acremoniuum cellulase, thereby facilitating the utilization of the C. korshinskii biomass as feedstock for biofuel production. Caragana korshinskii Kom., a forage shrub species for vegetation rehabilitation, is widely planted in the arid and semi‐arid regions of Asia and Europe for prevention and control of desertification as well as ecological restoration. However, to enable fresh regrowth and sustainability of C. korshinskii plantation, old stems are being pruned and cut back frequently which annually generates over 4 million tons of stubbles in China  on 19th June 2020. P. acidilactici and Acremonium cellulase  were supplied as freeze‐dried powders and applied as silage additives. The P. acidilactici bacterial strain was isolated from corn stalk silage and stored in our laboratory. The viable count of P. acidilactici powder was 2.5 × 1011 colony‐forming units (CFU) per gram. Based on the manufacturer's description, Acremonium cellulase activity was more than 1000 U/g, and the patent formula of plant cell wall‐degrading enzymes was composed of β‐Glucanase, α‐Arazyme, α‐Galactosidase, β‐Galactosidase and β‐Xylanase. Before the onset of the experiment, P. acidilactici and Acremonium cellulase were stored at 4°C.Branches (with leaves and pods) of C. korshinskii was taken to a laboratory and chopped into 2 to 3 cm segments using a manual forage chopper . Subsequently, C. korshinskii was mixed into a pile and randomly separated into 84 sub‐samples (every sub‐sample weighted approximately 400 g). Four random fresh sub‐samples were collected and frozen at −20°C pending further analysis. The remaining 80 sub‐samples (4 treatments × 5 time points × 4 replicates) were randomly subject to the following treatments: (1) distilled water (Control); (2) P. acidilactici (PA); (3) Acremonium cellulase (AC); (4) a combination of P. acidilactici and Acremonium cellulase (PA + AC). The application rate of P. acidilactici was 1 × 105 CFU/g fresh weight (FW). The application rate of commercial cellulase was 0.3 g/kg FW as described by Li, Ke, et al.  immediately after measuring the silage pH. The acidized liquid was filtered through a 0.22‐μm filter for the determination of organic acids  according to the method of Zhang et al.  from each silo was squeezed with 180 ml of distilled water in a high‐speed blender, then filtered through four layers of medical gauze. The filtrate was divided into two portions. The first portion was acidized to a pH of around 2.0 by Hg et al. . Anotherf Thomas , and NPNf Thomas .α‐amylase was used during aNDF analysis. Hemicellulose and cellulose contents were calculated in line with Li, Ke, et al.  and ground to pass through a 1‐mm sieve for nutrient analyses according to AOAC . Total n, et al. . Ferulic, et al. .C. korshinskii was performed with a TIANamp Bacteria DNA Kit  according to the manufacturer's protocol. The concentration of extracted DNA samples was determined using a Thermo Fisher NanoDrop instrument . The PCR amplification of the bacterial 16S rRNA gene was carried out by using primers 27F (5′‐GRGTTYGATYMTGGCTCAG‐3′) and 1492R (5′‐RGYTACCTTGTTACGACTT‐3′). The bacterial 16S amplification program was conducted following the description of Zhou et al.  based on cellulose content. A 200 μl of 2% sodium azide solution was added to avoid microbial contamination. Distilled water was supplemented to bring the total volume to 10 ml before the incubation. Another tube was used as a substrate blank with the same dried biomass, the same volume of buffer, antimicrobial agent and distilled water. An enzyme blank was also prepared in another tube with buffer, azide solution, water and enzyme solution. All tubes were incubated in a constant temperature shaker for 72 h at 50°C and 160 rpm. At each 12 h interval, samples were collected and the reaction was terminated at 100°C for 10 min. Glucose and xylose contents were determined by an Agilent high‐performance liquid chromatography 1200  after centrifugation at 10,000g for 10 min and filtered through a 0.22‐μm filter membrane. The cellulose conversion was calculated following the Laboratory Analytical Procedure of the National Renewable Energy Laboratory of Selig et al.  were declared by Tukey's honestly significant difference (HSD) test.All data on fermentation parameters and chemical composition at the end of ensiling (60 d) as well as level 2 KEGG orthologue gene and 6‐phospho‐beta‐glucosidase abundance within the same ensiling time were analysed by one‐way ANOVA . Significant differences among means (Yij = μ + Ei + Tj + (E × T)ij + eij, where Yij = response variable; μ = overall mean; Ei = effect of the ensiling time ; Tj = effect of treatment ; (E × T)ij = effect of interaction between the ensiling time and treatment and eij was the residual error.General linear model (GLM) procedure was used to analyse the bacterial diversity according to 4 × 5 factorial experiment model: Yij = μ + Hi + Tj + (H × T)ij + eij, where Yij = response variable; μ = overall mean; Ei = effect of the hydrolysis time ; Tj = effect of treatment ; (E × T)ij = effect of interaction between the ensiling time and treatment and eij was the residual error. Significant differences among means (p < 0.05) were declared by Tukey's HSD test.The non‐structural carbohydrate content obtained after enzymatic saccharification was also analysed using GLM procedure according to 4 × 6 factorial experiment model: C. korshinskii is shown in Table C. korshinskii exhibited a dry matter (DM) content of 551 ± 7.57 g/kg FW before ensiling, and the pH value was 6.35 ± 0.01. The concentrations of neutral detergent fibre (aNDF), acid detergent fibre (ADF), acid detergent lignin (ADL), hemicellulose and cellulose were quantified before ensiling, which were 335 ± 3.78, 215 ± 3.56, 57.5 ± 1.12, 120 ± 7.34 and 158 ± 2.46 g/kg DM respectively. In addition, ferulic acid concentration was 1091 ± 1.01 mg/kg DM.The chemical composition of fresh C. korshinskii silage are shown in Table p < 0.001). Butyric acid was not detected in any of the treatments. The highest DM content after ensiling was recorded in the PA group, while the DM loss was highest in the Control group. A significant effect (p < 0.001) of additives was also observed on the water‐soluble carbohydrates (WSC) of C. korshinskii silages in the present study. In contrast to the Control, the addition of AC had the highest WSC content while the sole application of P. acidilactici recorded the lowest WSC content. The silages treated with PA and AC had higher crude protein (CP) content compared to the Control silage, but no difference was found between PA + AC and other silages. The lowest contents of non‐protein nitrogen (NPN) and ammonia nitrogen (NH3‐N) were observed from silages inoculated with the combination of P. acidilactici and Acremonium cellulase. The addition of P. acidilactici and commercial cellulase , as well as the ensiling time, had a significant effect on the silage pH . Subsequently, the pH values declined continuously with a significant difference among the treatments until 30 d, and the lowest pH value was observed in PA + AC‐treated silage. The pH values of C. korshinskii silages at 60 d were all below 4.95, and the addition of AC and PA + AC significantly decreased the pH values compared with the Control and PA groups after 60 d of ensiling.The fermentation and chemical characteristics of H Figure . The addC. Korshinskii silage are shown in Figure P. acidilactici on aNDF maintained a plateau from the mid up to the end of the ensiling time, with a lower concentration compared to the Control and AC‐treated silages. The Control silage had the highest ADF content throughout the fermentation period compared to other treatments (p < 0.05). No difference was observed in ADF among PA, AC and PA + AC treatments at 7 d, but a marked difference appeared continuously with the advancing period of ensiling. The PA + AC treatment showed the lowest content of ADF from 14 to 60 d of fermentation. The higher content of ADL was obtained after the addition of P. acidilactici combined with Acremonium cellulase throughout the ensiling process except for the 30 d, and the trend was comparable with the group of PA which declined to the lowest at 30 d. With the advancement in the fermentation period, differences among treatments were obtained in hemicellulose and cellulose contents , while no significant difference appeared among other groups. At 7 and 14 d, the highest concentration of ferulic acid was found in PA‐treated silage, implying that the P. acidilactici strain contributed to the dramatic increase in the concentration of ferulic acid. Meanwhile, the ferulic acid concentration of AC‐treated silage declined at 14 d of ensiling, and there was no difference between the Control and PA + AC‐treated silages. An increasing trend was observed in the AC and PA + AC groups in the mid‐stage of ensiling, and the highest ferulic acid content  was detected when the mixture of P. acidilactici and commercial cellulase was added compared to other silages. The concentration of ferulic acid had been on a plateau during the initial and mid ensiling phases of Control and PA treatments. As the fermentation advances to the end of ensiling, the trends remain similar for the ensiling time at 30 and 60 d except for Control silage where ferulic acid concentration declined. The PA + AC treatment had the highest ferulic acid content  when compared with Control, PA and AC groups after 60 d ensilage.The effects of additives and ensiling time on ferulic acid are presented in Figure C. korshinskii decreased after ensiling. Additives decreased the species richness  compared with Control at the initial‐mid phase of ensiling (3–30 d), whereas PA + AC inoculated silage had the highest richness among the four groups at the end of the fermentation. PA inoculated silage had a lower Shannon value when compared with Control after 3 to 14 d of ensiling, whereas it had a higher Shannon value than the other three groups after 60 d of ensiling. The Shannon value in AC and PA + AC‐treated groups was lower than that of the Control from 3 to 7 d of ensiling, and lower than that of the PA inoculated group after 30 and 60 d of ensiling. In addition, the lowest Shannon value among the four treatments was observed in AC‐treated group after 30 and 60 d of ensiling.As shown in Table Variovorax boronicumulans (5.30%), Methylobacterium goesingense (4.36%), P. acidilactici (2.37%) and others (69.48%) at the species level. Regardless of pretreatment, the relative abundance of P. acidilactici dramatically increased and dominated the bacterial community of all the silages after 3 d. In addition to P. acidilactici, there were undesirable bacteria such as Erwinia tasmaniensis in the Control after 3 d of ensiling. As the ensiling period advanced from 7 to 60 d, P. acidilactici became the predominant species (>97%) in the C. korshinskii silage treated with or without additives. The linear discriminant analysis effect size (LEfSe) analysis was used to explore the differences in bacterial communities of the four C. korshinskii silage groups during the ensiling  pathways were used to observe functional shifts, which were cellular processes, environmental information processing, genetic information processing and metabolism, among the bacterial community of the four treatments Figure . The relC. korshinskii silages are shown in Table P. acidilactici had the highest glucose yield (p < 0.001) when compared with other treatments regardless of the hydrolysis times except at 24 h which had no significant difference with Control (Table p < 0.05) conversion was obtained in PA‐treated silage throughout the incubation period of the enzymatic saccharification, and the lowest cellulose conversion was observed in Acremonium cellulase‐treated silage except that the treatment was not different from the PA + AC group at 36 h  and cellulase as additives before ensiling was considered in the present study. Silages treated with PA were found to have the lowest WSC content compared to Control, AC and PA + AC‐treated silages, whereas the PA + AC group had higher lactic and acetic acid contents than the Control. Previous studies have shown that LAB strains can decline pH value by accelerating WSC transformation into lactic and acetic acids  in C. korshinskii before ensiling was the same as the added inoculant, the differences occurred due to the abundance and function. In the present study, the application of P. acidilactici decreased the relative abundance of Erwinia tasmaniensis and other epiphytic bacteria after 3 d of ensiling, and the P. acidilactici maintained the highest relative abundance throughout the entire fermentation period. This could be attributed to the adaptability as well as the rapid growth and multiplication of P. acidilactici which produces higher lactic acid that swiftly declined the pH to inhibit the growth of spoilage microorganisms throughout the fermentation  degradation during silage fermentation, and the remaining portion of the biomass became harder to be easily hydrolysed during enzymatic saccharification . Additionally, higher 6‐phospho‐beta‐glucosidase abundance was also observed in PA‐treated silage due to the adaptability of P. acidilactici at the initial‐mid stage of ensiling. After enzymatic saccharification, PA‐treated silage had the highest lignocellulose conversion which is evidence of alteration of the lignocellulosic structure. Therefore, the present study provides a pretreatment method for preservation and conversion of the lignocellulosic residue of C. korshinskii through sole P. acidilactici treatment or in combination with Acremoniuum cellulase, thereby facilitating the utilization of the C. korshinskii biomass as feedstock for biofuel production.After 60 d of ensiling The authors declare that they have no competing interests.


We focused on the recent findings with regard to the expression pattern and levels in regulating neocortical formation in mammals and its interactions with other known signaling pathways, including Slit–Robo signaling and Shh signaling. Finally, we review the functions of Notch signaling pathway in different species as well as other developmental process, mainly somitogenesis, to discuss how modifications to the Notch signaling pathway can drive the evolution of the neocortex.Expansion of the neocortex is thought to pave the way toward acquisition of higher cognitive functions in mammals. The highly conserved Notch signaling pathway plays a crucial role in this process by regulating the size of the cortical progenitor pool, in part by controlling the balance between self-renewal and differentiation. In this review, we introduce the components of Notch signaling pathway as well as the different mode of molecular mechanisms, including  Drosophila melanogaster  can replace OSM-11 during the development of C. elegans, suggesting the presence of another mechanism that activates Notch signaling by using non-canonical ligands with the DOS domain, such as Dlk1/2, in invertebrates and vertebrates  genes in drosophila, her1 and hey1 in zebrafish, and Hes1 and Hes5 genes in mice  protein family such as  in mice . The clucapacity . Moreovell cycle  and the er cells .2+-binding sites observed in the X-ray structure (trans-activation), the interaction between Notch ligands and receptors within the same cell can inhibit Notch signaling (cis-inhibition)  . Althougch other . del Álahibition . Becausehibition , some Nohibition . Solublehibition . This phormation ; howeverormation . Beyond hibition ; Nandagoin vitro . They foucidated . Thus, tucidated . Howeverucidated .At the early beginning of embryo development, the telencephalon originates from the most anterior part of the neural tube arising from a single layer of epithelial cells. On the basis of the anatomical position and composition of cell types, the telencephalon can be categorized into dorsal and ventral compartments. The neocortex, which is believed to be responsible for higher cognitive functions, is a major part of the dorsal telencephalon. The neocortex is formed by a six-layer laminated structure composed of glutamatergic excitatory neurons. Here, we focus on the involvement of the Notch signaling pathway in the formation of the laminated structure.Hes1/5 double-knockout mice and control mice at the NEC stage E8.5, whereas the number of RGs decreased due to prematuration at later stages (E9.5–10.5) when NECs begin to transform to RGs , RGs, and intermediate progenitor cells (IPCs). NECs are the earliest type of neural progenitor cells that are highly polarized in a pseudostratified pattern . Becausem to RGs . These sNeurogenesis from RGs to neurons can occur in a direct or an indirect manner. Direct neurogenesis is one RG divides to generate an RG and a neuron in the ventricular zone (VZ), and indirect neurogenesis is one RG may generate two RGs or two other types of progenitor cells, such as IPCs. Subsequently, IPCs symmetrically divide to generate two neurons. Indirect neurogenesis is beneficial for the increase in the final neuron pool and is more common in the mammalian neocortex compared with direct neurogenesis, which is the predominant neurogenesis manner in the developing cortex of other vertebrates, such as birds and reptiles .Mib1 gene can impair Notch signaling. Conventional Mib1 knockout mice exhibited deficient Notch signaling that led to prematuration at E9.0–E9.5, resulting in the death of embryos before E12.5 , a RING-type E3 ubiquitin ligase, promotes the endocytosis of canonical Notch ligands, knocking out the re E12.5 . Furtherat E13.5 , resultixpressed ; this fixpressed , the evoIn gyrencephalic species, such as ferret and primates, a large population of proliferative cells can be noted in the basal region of the VZ. They are a subtype of RGs, called basal RGs (bRGs). These bRGs, unlike IPCs, have radial fibers but lose the apical attachment to the ventricular surface, unlike their apical cohorts, apical RGs (aRGs). bRGs can undergo self-renewal to expand the progenitor pool in the SVZ region. In the developing primate neocortex, the majority of bRGs are positioned in the outer SVZ (OSVZ), which is separated from the inner SVZ (ISVZ) by an inner fiber layer. During neocortical expansion, the thickness of the OSVZ gradually increases with the expansion of the VZ . Except Hes1 can maintain the pool of neural progenitor cells. Concurrently, the expression of Notch ligand Dll1 and the proneural gene Ngn2 were fluctuated in a manner which is coordinated but opposite to the oscillated expression pattern of Hes1. The fine balance of the oscillating gene expression pattern is orchestrated by several elaborate transcriptional regulatory mechanisms. The oscillating pattern of Hes1 expression can be regulated through a negative feedback loop. After the activation of Hes1 by the Notch ligand–receptor interaction, Hes1 protein cis-represses its own transcription by directly targeting its promoter. Another key is the short half-life of Hes1 mRNA and Hes1 protein. The half-life of Hes1 mRNA and Hes1 protein is as short as 20 min. As both Hes1 mRNA and Hes1 protein are degraded soon after their production, the Hes1 promoter can be released from autoinhibition. Also Hes protein represses proneural genes such as Mash1 , proneural genes (Mash1 and Ngn2), and cell cycle regulators (cyclin D1 and cyclin E1)  technique. Another study showed that activation of Notch signaling maintains the neural progenitor cell pool by overexpressing the NICD fragment  . This reyclin D1 . Furtherain size  similar ll1 gene . Notablyogenesis . Howeverfragment . Thus, wRobo1/2 knockout mouse, neural progenitor cells in the VZ underwent a premature asymmetric division and increased the generation of IPCs, thus reducing the brain size. This deficiency was found to be mediated by Robo-mediated transcriptional activation of the Notch effector Hes1, which suggested the interplay between Robo and Notch signaling is crucial to regulate neurogenesis precisely  are responsible for regulating axon guidance which contributes to cortical circuits . Moreoverecisely .CNS evolution across species has been investigated for decades; however, it still remains largely unclear. The differential regulation of direct and indirect neurogenesis in different species is one of the most prominent hypotheses. Recently, a study examined the switch between Dll1–Notch and Robo–Slit signaling in corticogenesis to determine the predominant mode of indirect or direct neurogenesis along with its effects on the neuron number, brain size, and neural circuit complexity across amniotes. To elucidate the involvement of Notch–Dll1 and Robo–Slit signaling, the expression level in the neural progenitors of each representative species among diverse amniotes  was analyzed. Data indicated a high Robo expression level and a low Dll1 expression level in brain structures including the dorsal telencephalon of snake, the medial dorsal telencephalon of chick, and the olfactory bulb, hippocampus, and spinal cord of mammals, but a high Dll1 expression level and a low Robo expression level in the advanced brain structures including the lateral dorsal telencephalon of chick and the neocortex of mammals . In brieShh gene. Initially, hedgehog gene was identified from Drosophila melanogaster. Mutations in hedgehog gene lead to abnormal segmental patterning and polarity in flies , which is an autosomal dominantly inherited disorder. Patients with HPE have intellectual disability, microcephaly, and epilepsy (Ptch), which inhibits Smoothened (Smo) in the absence of Shh (Sonic hedgehog (Shh) is a secreted protein encoded by in flies . Shh sigin flies . In the in flies  and chicin flies . Shh is in flies . Mutatioepilepsy . Shh proe of Shh . When Sme of Shh .Hes1-overexpressing mice could be rescued from their defects by crossing them with transgenic mice expressing constitutively active Smo, an effector of Shh signaling. This result suggested that dysfunction in Notch signaling can be complemented by promoting Shh signaling  and the downstream target hairy/E(spl) genes (her1 and her7) with an oscillatory expression pattern. Both DeltaD and DeltaC are essential for somitogenesis, and DeltaC, as one of the oscillators, is critical for proper somite segmentation , gecko (Poekilocerus pictus), turtle (Pelodiscus sinensis), and chick . Compared with the neural progenitors of other species, neural progenitors in geckos required a longer period to differentiate, twice as those required by mice and chicks. Although cortical progenitors stay in the progenitor stage for a longer period in geckos than in mice, the number of mitotic cells was lower in geckos, suggesting that the size of the neural progenitor pool may not be associated with the duration in the progenitor stage. Furthermore, they used a CBF1-driven reporter to monitor Notch activity in neural progenitors. Neural progenitors of geckos exhibited higher Notch activity than those of other species. Notably, the distribution of neural progenitors with active Notch signaling differed among species: mosaic in mice, turtles, and chicks but homogenous in geckos (Nomura found that neural progenitors in the pallium of geckos required a long time to differentiate into neurons compared with other species of amniotes . They apn geckos . Two posn geckos . Second,n geckos . In eithNOTCH2 partial duplicated paralog, NOTCH2NL gene, was found to be expressed in both human aRG and bRGs and to improve the expansion of cortical progenitors by activating NOTCH signaling through interrupting the cis-inhibition of DLL1 (NOTCH2NL in embryonic mice or human organoids prolonged the self-renewal stage and delayed neuronal differentiation, resulting in clonal expansion in neural progenitors (NOTCH2NL knockout accelerated neuronal differentiation and reduced neurogenesis (NOTCH2NL genes in humans induced microcephaly and megacephaly, respectively, suggesting the crucial role of NOTCH2NL in human neocortical development (Notch signaling is essential for self-renewal in RGs to maintain the progenitor cell pool during cortical development. Expansion of the neural progenitor pool and a prolonged neural progenitor self-renewal period are believed to be critical events in cerebral cortex evolution . Recentl of DLL1 . Overexpgenitors . By contogenesis . Investiogenesis . In addielopment . Hence, Notch signaling is highly conserved among species and regulates a wide range of developmental processes. It had been demonstrated that the activity of the canonical Notch signaling pathway determines the size of the neural progenitor pool and the initiation of neural differentiation during the telencephalon development in amniotes . HoweverF-SN and P-SH collected the relevant research for the review and wrote the manuscript. Both authors contributed to the article and approved the submitted version.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.


Scaphoideus titanus and Halyomorpha halys, in natural conditions. Biotremology techniques could be profitably used to monitor not only the presence of target pest species but also the biodiversity associated with vibrational signaling insects. In particular, the method implemented in this study could be used as a tool to compare the quality of cultivated areas under different management systems.Environmental conditions are crucial factors that influence communication systems and affect animal behavior. Research in the field of biotremology has improved our understanding of insect behavior, ecology, and evolution. However, the interactions between vibrational signaling and environmental factors are less studied, mainly because of technical issues faced in field trials. We therefore developed and tested an approach to investigate the effect of abiotic factors on insect vibrational signaling and explored its implementation as a monitoring tool for insect vibrational signals, using a vineyard as an agroecosystem model. Our results showed a significant decrease in insect signaling activity during unsuitable conditions of high temperature and wind velocity. We determined for the first time, the daily signaling pattern of the two insect pests,  Recording sites were chosen randomly in the vineyard, the distance between neighboring sites ranged between 6–18 m (see −1), air pressure (hPa), relative humidity (%) and air temperature at 2m height (°C)) were obtained from the automated weather station website of Fondazione Edmund Mach in San Michele all’Adige , which automatically stored data every 10 s and averaged measurements over an hour.Trials were conducted from the beginning of July to the end of August 2019, in an organic vineyard of 0.28 ha on the campus of Fondazione Edmund Mach . The vineyard was limited by a wooded area  and semi-field (known population density). We excluded females from the experimental cages, and studied only the signaling behavior of males, because females emit signals only when elicited to respond by male calling songs and the presence of active females can trigger males rivalry behaviors (Vitis vinifera L. cv. Pinot noir grafted on Kobber 5BB) and recorded only once. Five minutes before initiating recordings, the 6 males were collected from the rearing cages and placed on a grapevine plant inside a mesh cage that was positioned outdoors. Similar to the vineyard experiment, trials were performed in two recording sessions (S1: from 7:00 to 14:00 and S2: 14:00 to 21:00). Each session was replicated 6 times , resulting in a total duration of 84 h of recording. For uniform environmental conditions, sessions were divided into periods of the day in both experiments: S1 was called “morning” while S2 was divided in “afternoon” (from 14:00 to 18:00) and “evening” (from 18:00 to 21:00).This experiment served as a control for the daily vibrational activity of ehaviors . The latHalyomorpha halys , were identified in our recordings by comparisons with our signal library , as well as those resulted from abiotic and anthropogenic sources and from air-borne sounds  were excluded from the analysis.Vibrational signals were recorded using a portable laser doppler vibrometer , which measures vibrational velocities. The laser was plugged into a rechargeable battery  through a charger . The laser beam was focused onto a reflective sticker, attached to a green stem of the grapevine plant (diameter ca. one cm) in both the vineyard and the semi-field experiments, at approximately 20 cm from the plant stem. Signals were acquired using the software BK Connect  at a sampling rate of 44.1 kHz and a resolution of 16 bit, through a multichannel calibrated data acquisition device , and stored on a portable computer hard drive. To accommodate the processing of signals to manageable proportions for a more convenient analysis, recordings were automatically saved as 10-minute wav files. When an insect vibrational signal overlapped two recordings it was considered to be one vibrational signal , and the duration of the signal was calculated as the sum of the two pieces. Spectrogram analysis was performed with the software Raven Pro 1.5  using Fast Fourier Transform, with a 512 samples Hann window and 75% overlap. Each recording was screened, by visual and aural inspection, to characterize the vibrational signal types (VST) which were defined as different types of vibrational signals, classified according to distinct temporal and spectral characteristics , focusin library , whereasStatistical analyses were performed with R version 4.0.2  run in tij is the jth recording in Sitei (i = 1 to 26), and Sitei is the random intercept, which is assumed to be normally distributed with mean 0 and variance σ2.where SAH. halys, S. titanus and other unidentified insects was compared between periods of the day  using a Wilcoxon rank sum test.The SA of S. titanus in the vineyard and in semi-field conditions, the relationship between the hour average SA recorded in the vineyard (field) and in semi-field conditions, in the different recording periods, was assessed with Pearson correlation coefficients.To compare the daily SA of The morning (07:00–14:00) and evening (18:00–21:00) of the recording period were characterized by low temperature and low wind velocity, with the occurrence of many VSTs (range 0–13) . While tR2 = 0.42) and together with the random factor  had a high explanatory power in the used model. Most signaling occurred in the morning and in the evening, with a significant decrease in the afternoon  and the peak of SA started from 17:00 while it started from 18:00 in field conditions. The correlation between the average signaling activity per hour recorded in the vineyard and semi-field .The daily pattern of  evening . Howeverield see  was signThe present study investigated the daily vibrational signaling of insects in field conditions in a vineyard and provided novel information of the effect of environmental conditions on their signaling activity. Although the total measured signaling activity resulted from vibrational signals of several different insect species, a common pattern of insect activity during a summer day in a vineyard was obtained. As expected, the signaling activity increased with the number of VSTs, which however are not necessarily associable to different species but in some cases could be part of the signal repertoire of the same species .Nilaparvata lugens (Hemiptera: Delphacidae) in controlled conditions (Enchenopa binotata (Hemiptera: Membraciade), where the daily vibrational signaling activity was associated with wind velocity , when temperatures were below 20 °C. Cold conditions slow down the insect biological processes and for this reason many species can start calling only when a certain temperature is reached . On the nditions . A similvelocity . Our stuvelocity . Often, ty e.g.,  to meet ty e.g., . An incrty e.g., . For insty e.g., . The perty e.g., , which wty e.g., . Althougty e.g., . Resultsty e.g., . Even atty e.g., . As a rety e.g., . High teScaphoideus titanus, in particular, concentrates the flight activity early in the morning and at dusk (−1) and the temperature is between approximately 18 and 30 °C. In semi-field conditions, sporadic signals of S. titanus were recorded also in the morning and one hour earlier in the evening in contrast to field conditions. Beside the fact that in semi-field conditions a higher number of individuals were consistently present on the same plant, it must not be excluded that in a cage the presence of several individuals in proximity may have triggered their interaction through accidental body contact or the detection of incidental signals derived by body movements  of other individuals. However, such differences are negligible when considering the general signaling pattern and peak of activity, indicating that vibroscape monitoring could reliably monitor the actual presence of S. titanus  in a vineyard. at dusk , althoug at dusk . We founE. vitis were not detected in our recordings. The signals could have been significantly attenuated before reaching the plant stem because of heterogeneity of the plant parts , while in the case of S. titanus (a bigger species: 5 mm vs 3 mm) the active space can include different leaves of the same plant and even leaves not in physical contact but sufficiently close (up to 7 mm) to the one where the insect is calling from   and this information would be important not only to fill an important biological knowledge gap, but also to better setup a strategy of pest management based on the use of vibrational traps. Such traps are currently the object of study and field testing   whose sivelocity , that is titanus . In the the area , the sig testing  and we ce.g., sticky traps, sweeping nets) and not highly specific . In the absence of an updated reference library of vibrational signals, we cannot yet make precise assumptions about species composition, thus we cannot provide reliable ecological information in terms of biodiversity. Nevertheless, it would be possible in perspective to make comparisons between similar environments. In the case of vineyards or other orchards, beside the presence of key species, it seems to be feasible to quantify the occurrence of a complex of vibrational signals which could be used as a proxy of environmental quality. For example, as an instrument to measure the impact of pest management practices both on an insect community normally present in a certain site and on specific target species. In this regard, it becomes crucial to setup protocols of recording that aim at optimizing the signal recording efforts. For instance, enlarging the matrix of sampling points at plant and vineyard level, to detect insects that are characterized by a very narrow active space such as E. vitis. This would also increase the system sensitivity and fill the gap observed between field and semi-field tests. However, because moving from a plant to another during the recording sessions was challenging in our study, the use of light equipment such as accelerometers, would facilitate recording in multiple spots during shorter periods. A better option would be using fixed vibrational sensors deployed in a regular grid to record vibrational signals simultaneously and continuously, which could also enable evaluating the seasonal signaling variation and vibroscape composition, once a large library of vibrational signals is developed. A matrix of sensors may also point out the transition of insects through the agroecosystem such as the case of H. halys in the vineyard. Since vibrational signals are species and sex specific  and corresponding parameters included in the model and the The field measurements of signaling duration of the identified insects used for The data of Scaphoideus titanus signaling duration in field and semi-field measurements that were used for Click here for additional data file.


UA, a natural pentacyclic triterpenoid and RA, a phenolic ester are major bioactive constituents of Rosmarinus officinalis, which is a medicinal herb belonging to family Lamiaceae and exhibiting significant biological properties including neuroprotection. Donepezil, a second generation cholinesterase inhibitor approved for the treatment of mild, moderate and severe Alzheimer’s disease (AD) is used as control. Out of eight groups of male BALB/c mice, stereotaxic surgery was performed on four groups (n = 6 each) to introduce Aβ1–42 in the hippocampus followed by treatment with vehicle ), donepezil, UA or RA. The other four groups were given vehicle, donepezil, UA and RA only. Behavior analysis for social interaction was performed which constitutes the social affiliation and the social novelty preference test. Presence of Aβ plaques and expression of neurogenesis markers i.e., doublecortin (DCX) and Ki-67 were also assessed. Results revealed the neuroprotective effect of UA and RA observed through substantial reduction in Aβ plaques as compared to the Aβ1-42- and donepezil-treated groups. The neuronal density was also restored as evident via DCX and Ki-67 immunoreactivity in Aβ1–42 + RA and Aβ1–42+UA-treated groups in comparison to Aβ1–42-treated and Aβ1–42+donepezil-treated groups. The social affiliation was reestablished in the Aβ1–42 administered groups treated with UA and RA. Molecular docking studies further validated the comparable binding of UA and RA with Ki-67 and DCX to that of donepezil. Our findings suggest that UA and RA are potential neuroprotective compounds that reverses the histological hallmarks of AD and ameliorate impaired social memory and hippocampal neurogenesis.Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder characterized by substantial neuronal damage which manifests in the form of deficits in memory and cognition. In spite of the debilitating nature of Alzheimer’s disease (AD), a dearth of treatment strategies calls for the need to develop therapeutic agents that stimulate neurogenesis and alleviate the associated cognitive deficits. The present study investigates the therapeutic potential of two major phytochemicals Alzheimer’s disease (AD) is the most common form of dementia accounting for more than 80% of the cases diagnosed. With a prevalence that continues to grow as the world population ages, it has emerged as a leading health problem. The debilitating disorder affects nearly 50 million people worldwide . It is cAdult hippocampal neurogenesis, is a unique phenomenon hosted by the hippocampus, which confers significant levels of plasticity to the hippocampal circuitry improving pattern separation and spatial memory . AD leadAmong the various markers of neurogenesis, Doublecortin (DCX) is a brain-specific protein associated with the microtubules which regulates neuronal migration through the polymerization and stabilization of microtubules in migrating neuroblasts . DCX is N-methyl d-aspartate (NMDA) antagonists function mainly by treating the symptoms of AD and do not possess preventive or curative effects .Male BALB/c mice were maintained in the animal facility of Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST). Mice were raised under 12 h natural light-dark cycles and had free access to food and water 1–42  into the hippocampus (CA1 region) from bregma: anteroposterior, 2.3 mm; mediolateral, 1.8 mm; dorsoventral, 2.0 mm, using a stereotaxic apparatus using a Hamilton syringe. Similarly, the control mice were administered phosphate-buffered saline (PBS). The mice were kept in individual cages until further use.BALB/c mice  were used for experimentation. A previously described method was used to establish the animal model . The micn = 6 each). The groups 1–4 were pre-treated with Aβ1–42. The groups 2, 3 & 4 received donepezil (15 mg/kg) (1–42 administration. Group 5 received vehicle (PBS) while groups 6, 7, 8 received the similar dose of donepezil, RA and UA, respectively for similar duration. All the groups were orally administered with normal feed and water. The purity of UA and RA was >99% and >95% respectively while donepezil was taken as a positive control. After the end of treatment period the mice were subjected to behavioral tests and subsequently brain tissue harvesting.The male BALB/c mice were segregated into eight groups (5 mg/kg) , RA (16 5 mg/kg) , or UA (5 mg/kg) , respectThe procedure was performed as reported previously . The appAfter habituating the test subjects (5 min) inside the box, they were introduced back into the box with a cage having mouse A on one side and an empty cage on the other side. The test subject was then left in the box for 10 min and allowed to move in both the cages. The discrimination index (DI) or the interaction time or with the empty cage and mouse A was evaluated and the difference between the time spent interacting with the mouse A or empty cage and the total time was scored.The mouse B was released in the empty cage while the mouse A remained unaltered. The test subject was again allowed an exploration time of 10 min. DI or interaction time was measured as the difference between the time spent interacting with mouse A or mouse B and the total time spent with mouse B.2O). Congo red stain (1%) was applied to the tissue for 25 min after which it was thoroughly rinsed with ddH2O, followed by counter Haematoxylin and eosin staining. The sections were rinsed again with ddH2O, dried and incubated for 20–25 min before clearing in xylene solution.Histology was performed according to the protocol used by 2O2 (1%) was applied to quench the peroxidase activity and blocked with BSA (5%) in PBS for 1 h. After an overnight incubation with primary antibodies at 4°C the sections were incubated for 1 h with a secondary antibody followed by washing thrice with TBST and visualised with 0.025% 3, 3′ diaminobenzidine . Ki-67  and DCX  diluted in the block solution 1:200 and 1:100 respectively, were used as primary antibodies while anti-rabbit IgG-HRP conjugated  diluted in block solution 1:100 was used as secondary antibody. The images were visualized using B-150, OPTIKA microscope  at 4× and 10× resolution. The images were captured using Optika Vision Lite 2.1 image analysis software. Quantitative analysis was performed for cell count in an area of 10,000 μm2 from three randomly selected areas and the average values were calculated and plotted.Immunostaining was performed in accordance with the protocol previously described by https://www.rcsb.org/). The PDB IDs of Ki-67 and DCX were 5J28 and 2BQQ, respectively. The 3D structures of RA, UA and donepezil were taken from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The automated docking models generated were further assessed using FireDock  with Bonferroni’s multiple comparisons as post hoc test using GraphPad Prism 5.0.Data are presented as mean ± SEM and statistical significance was set at 95% confidence level and a 1–42 treated group  exhibited a significant reduction in interaction with mouse A, a conspecific placed in one of the cages, in comparison to the control  and other experimental groups exhibiting a significant decrease in social affiliation. The Aβ1–42 + RA  and Aβ1–42+ UA-treated groups  performed significantly better than the diseased group and comparable to the Aβ1–42+donepezil-treated group . Aβ1–42+ UA-treated group exhibited improved social affiliation in comparison to all of the other groups  as well as empty cage . Conversely, the control and experimental groups interacted more with the mouse A in comparison to the empty cage  exhibited significantly low DI in comparison to the control  and the treated groups. A significant improvement in DI was observed in Aβ1–42 + RA  and Aβ1–42+ UA-treated  groups depicting restoration of social affiliation  of interaction with an unfamiliar mouse B in comparison to the control group (21.38 ± 4.261). The treated groups did not show preference with any mouse cage.In the session II, the mice were assessed for novelty preference and social memory. The mice treated with Aβ1–42 -treated group  relative to the control  and the other experimental groups, thus indicating alterations in social memory. A significant improvement in DI was evident in Aβ1–42 + RA (0.7300 ± 0.06258 p = .0007) and Aβ1–42+ UA-treated (0.6867 ± 0.03783 p < 0.0001) groups in comparison to the control. The DI in RA- and UA-treated groups was comparable to that of the standard drug donepezil  demonstrating their protective effects against social memory deficit in AD  and UA  post Aβ1–42 administration as compared to the mice treated Aβ1–42 only (19.50 ± 0.6455). It was found that RA and UA have greater improvement activity than donepezil , evident through the restoration of the Aβ1–42 deteriorated neurons. No obvious neuronal loss was encountered by the control mice (27.65 ± 1.525), donepezil, RA and UA in Aβ1–42 untreated groups  relative to control (29.33 ± 1.856) indicated the intensity of neurotoxic effects of Aβ. RA  and UA  treatment post Aβ1–42 administration showed improved immunoreactivity with DCX with increase DCX positive cells relative to control and donepezil-treated group   .1–42 was substantially decreased with the treatment of RA and UA indicating a reversal of the plaque formation. RA and UA treatment significantly rescued the cellular density and morphology in Aβ1–42 -treated groups and comparatively have greater neuronal restoration than donepezil. The control littermates showed a normal neuronal pattern  is depictive of higher binding affinity, therefore, the ligand molecules that had the lowest ACE were considered better ligands to Ki-67 and DCX. The docking scores of the complexes are shown in in vivo results  which was comparable to that of donepezil (−293.37). Interactions with DCX also demonstrated a comparable binding energy of UA (−154.2) and donepezil (−157.92). These binding energy values suggest an interaction of UA with Ki-67 and DCX validating the present  results .Global energy depicts the binding energy of the complex while the ACE is based on its contribution to global energy. Low values of ACE correspond to more stable complexes. UA exhibited an ACE of −10.96 in its interaction with Ki-67 which is comparable to that of donepezil (−10.92). UA (−10.06) also showed a comparable ACE to that of donepezil (−11.84) in its interaction with DCX. Attractive and repulsive vdw  are a quantification of the input of van der waal’s forces to the global binding energy. The values are tabulated in R. officinalis, RA and UA on Aβ1–42 -induced neurotoxicity in comparison to donepezil. The data revealed the restoration capability of RA and UA of altered social memory in AD mouse models. Social engagement with the surrounding environment is associated with an improved angiogenesis, synaptogenesis, and neurogenesis which are crucial in delaying the progression of AD -induced neurotoxicity through the formation of an original ternary association between Aβ and Cu (II) (Caenorhabditis elegans transgenic model (The reduction in the plaque formation by RA and UA post Aβ Cu (II) . Also, t Cu (II) . Interes Cu (II) . It also Cu (II) . Consistic model . These r1–42 that represents pathological hallmarks of AD. Our findings revealed that UA and RA can rescue the AD like alterations characterized by accumulated amyloid plaques, impaired social memory and neurogenesis induced by Aβ, thereby reiterating their potential as promising therapeutic agents against AD.This study suggests the pro-neurogenic potential and neuroprotective effects of UA and RA on neurotoxicity induced by Aβ


JCI, Cai, Zhao and colleagues demonstrate that mitochondrial function in macrophages governed the resolution of inflammation and tissue repair by modulating the phagocytic removal of apoptotic cells  as well as myofibroblast activation. These findings provide important mechanistic insights into the potential relevance of metabolic modulation of macrophage functions following MI, which might lead to alternative therapeutic strategies for MI.Cardiac healing following acute myocardial infarction (MI) involves the mobilization and activation of immune cells, including macrophages. In the early phase after MI, macrophages adopt a proinflammatory phenotype, while polarizing toward a reparative one in the late stage. Although metabolic reprogramming has been observed during this transition, the mechanistic links to macrophage differentiation are still poorly understood. In this issue of the  Cardiac repair following myocardial infarction (MI) is the result of a finely orchestrated series of events, which includes an initial inflammatory phase, characterized by intense sterile inflammation and subsequent mobilization of innate and adaptive immune cells, in particular neutrophils, monocytes, and macrophages, to the infarcted area . This reDuring the resolution phase, macrophages transition from the proinflammatory to a reparative phenotype , promotiEmerging evidence suggests that metabolic remodeling is central to macrophage polarization and its effector functions . In proi+ redox state, in turn driving macrophage reparative polarization. These findings are indicative of a role for efferocytosis-derived metabolic signaling in inflammation resolution , but they are also involved in the initiation of different signaling pathways, including those for cell growth, proliferation, and differentiation . Further1F0 ATP synthase) , complex II (or succinate dehydrogenase), complex III (or cytochrome bc1 complex), and complex IV (or cytochrome c oxidase), as well as the electron carriers ubiquinone and cytochrome c. Together, they couple electron flux along the ETC to translocate protons across the inner mitochondrial membrane. This electrochemical gradient drives ATP synthesis at complex V (Fynthase) . Complexynthase) , 15.In fact, excessive mROS activate the NF-κB signaling pathway, promoting proinflammatory macrophage polarization , 16. FurJCI, Cai, Zhao, and colleagues (Ndufs4 (mKO) had reduced mitochondrial function and an increased glycolytic rate, recapitulating the metabolic profile of proinflammatory macrophages  the gene deletion technique used in the work of Cai, Zhao, and colleagues affects both neutrophils and monocytes/macrophages; (b) the data showed an increased amount of neutrophils in the early post-MI phase in mKO mice; and (c) CXCL1/2 levels were increased in the infarcted area of mKO hearts (which can mobilize inflammatory monocytes), it is possible that mitochondrial dysfunction in other immune cells  also affected the functions of those cells and contributed to the poorer post-MI outcomes observed in the mKO mice. Therefore, similar studies focused on the role of mitochondrial dysfunction in other immune cells, such as neutrophils, will expand our understanding of the mechanisms described in the current work .Taken together, the findings of Cai, Zhao, and co-authors  illustra


Remarkably, the finely engineered NiSx catalyst synthesized via thermal‐evolution for 24 h (NiSx‐24 h) exhibits an exceptionally low cell voltage of 1.59 V  for neutral water splitting, which represents the lowest value ever reported. The enhanced performance of NiSx‐24 h is a multi‐synergized consequence of the simultaneous enrichment of oxygen and hydrogen evolution reaction catalyzing species, accompanied by an optimum electrocatalytic surface area and intrinsic high conductivity. Overall, this innovative work opens a route to engineering the active material's electronic structure/morphology, demonstrating novel Ni3+/pSn2−‐enriched NiSx catalysts which surpass state‐of‐the‐art materials for neutral water splitting.Efficient neutral water splitting may represent in future a sustainable solution to unconstrained energy requirements, but yet necessitates the development of innovative avenues for achieving the currently unmet required performances. Herein, a novel paradigm based on the combination of electronic structure engineering and surface morphology tuning of earth‐abundant 3D‐hierarchical binder‐free electrocatalysts is demonstrated, via a scalable single‐step thermal transformation of nickel substrates under sulfur environment. A temporal‐evolution of the resulting 3D‐nanostructured substrates is performed for the intentional enhancement of non‐abundant highly‐catalytic Ni x bi‐functional electrocatalyst exhibiting the best ever reported performance for neutral water splitting applications is presented. This remarkably enhanced performance is a multi‐synergized consequence of the method's capability to finely engineer the active material's surface electronic structure and morphology, thus surpassing the reported performance of state‐of‐the‐art materials for neutral water splitting.Here, the simple and highly scalable preparation of earth‐abundant NiS Even so, water electrolysis still suffers from high overpotentials due to the sluggish oxygen evolution (OER) and hydrogen evolution reactions (HER), thus demands the development of highly active water oxidation/reduction electrocatalysts. Pt and IrO2/RuO2‐based noble‐metal electrocatalysts are benchmarked for HER and OER, respectively, nevertheless, their enormously high cost hinders their industrial scalability and widespread applicability. Various transition metal‐based earth‐abundant electrocatalysts were recently reported in the literature with superior OER and HER performance separately, under alkaline and acidic conditions, respectively. Regardless, bifunctional electrocatalysis for overall water splitting still suffers due to the tradeoff of better OER and moderate HER performance in alkaline media, and better HER and only moderate OER performance under acidic conditions. Only recently, major advancement in the development of transition metal‐based bifunctional electrocatalysts for overall water splitting under alkaline conditions was demonstrated in several reports, with cell voltages of ˂1.5 V at 10 mA cm−2. However, water splitting technologies still suffer due to the extreme pH conditions of the highly acidic or alkaline electrolytes required for their optimum electrochemical performance. These corrosive conditions are not environmental friendly, and limit the usage of the various catalytic electrodes and cell components in the electrolyzer due to limited stability issues, thus increasing the final device and process costs.The ever increasing energy demand around the globe requires suitable green energy production alternatives for sustainable development. The cleanest form of energy; hydrogen, is considered a well‐known alternative to the conventional nonrenewable sources, with a high gravimetric energy density.1 Elec Furthermore, various biological electrolysis systems  can be integrated under the same umbrella, as they require neutral media working conditions for their operation. However, in spite of the tremendous research efforts on the application of transition metal‐based earth‐abundant electrocatalysts, only a scarce amount of reports on water splitting under neutral conditions can be found in the literature. Cobalt‐ and nickel‐based catalytic systems gained major interest in the pioneering research for neutral water‐based electrolytic systems. Xie et al. reported the electrocatalytic oxidation of water in neutral medium using Co2P and cobalt borate with an overpotential of 320 and 290 mV, respectively. Zhang et al. and Wang et al. explored CoO‐modified CoSe and Co4N as bifunctional electrocatalysts for overall water splitting in neutral water, with a cell voltage of 2.18 and 1.79 V at 10 mA cm−2, respectively. Chen et al. explored Ni3N@Ni‐Bi as a bifunctional water splitting electrode with a cell voltage of 1.95 V at 10 mA cm−2. Recently, Cho et al. explored the application of high‐entropy CuCoNiFeMn alloys as electrocatalysts under neutral conditions, exhibiting an overpotential of 320 and 680 mV for HER and OER, respectively. In an interesting recent work, Sun et al. applied activated LDH with multi‐vacancies by memory effect for efficient water splitting under neutral conditions. It can be observed that the highly‐scalable cost‐effective neutral water splitting process using earth‐abundant catalysts is still an immense mostly unmet challenge, as the current state‐of‐the‐art catalytic materials still suffer from overpotentials considerably higher than the theoretical cell voltage of 1.23 V. Furthermore, the synthetic procedures applied in literature reports for creating highly catalytic materials are far from economically and scalability‐wise worthy. Hence, there is an urgent current need for the development of earth‐abundant electrocatalytic electrode materials for the efficient hydrogen production under neutral conditions, that can further be economically scaled up for mass production applications.To overcome these handicapping limitations, water splitting under neutral conditions is a plausible technological solution, as it can be performed without the requirement of special membranes, expensive catalytic electrodes, and complex cell setups.14 Fur In a recent report, Zeng et al. investigated the enhanced OER and HER performance of NiS2(1−x)Se2x in comparison to NiS2. For this purpose, water splitting under neutral conditions was performed using NiS2(1−x)Se2x as a bifunctional electrocatalyst. The NiS2(1−x)Se2x electrolyzer exhibited a cell voltage of 1.87 V at 10 mA cm−2, as presented in our literature comparison of cell voltage for overall water splitting using various electrocatalysts in neutral medium. It is well known that the morphology and chemical states of catalytic centers at the surface of the catalytic substrates can dramatically affect their electrocatalytic performance. In this work, for the first time, the combination electronic structure engineering and surface morphology tuning of highly catalytic earth‐abundant 3D substrates, by a single‐step temporal thermal‐evolution process, is demonstrated for the optimum and exceptional water splitting performance in neutral medium. Novel 3D hierarchical nickel sulfides‐based nanostructured electrodes, with a controlled varying ratio of NiS and NiS2 species are synthesized directly from nickel foil substrates at different transformation times in a single‐step thermal process. A systematic temporal thermal‐evolution of the 3D nanostructured substrate is demonstrated, with a temporal modulation of non‐abundant Ni3+ and pSn2− species at the electrode surface. Conventionally, nickel‐based electrocatalysts are majorly present in the form of Ni2+‐species, that oxidizes to Ni3+‐species in a peroxidation step during water electrolysis, with Ni3+‐species being the catalytically active sites for the electrochemical reaction. Hence, in spite of starting with the Ni2+ as the active species, the electrocatalytic surface engineering for enhancing the Ni3+ species concentration at the surface can be applied as a key route for the water splitting enhanced electrochemical performance. This can eliminate the pre‐oxidation overpotential, and thus decrease the cell voltage for water oxidation. However, the abundance of Ni3+ species is scarce, and is only purely present in some organometallic compounds which possess high catalytic activity. Furthermore, the polysulfide (pSn2−) species are known to exhibit an optimum free energy for hydrogen adsorption leading to efficient hydrogen production at the cathode. Herein, a systematic electronic structure engineering for multi‐synergized high surface area Ni3+/pSn2−‐enriched 3D conducting electrodes is performed, which demonstrates the best reported performance as a bifunctional electrocatalyst for overall water splitting in neutral medium. Furthermore, the ease of the production process of these electrocatalytically active 3D binder‐free electrodes makes this synthetic approach economically highly scalable to industrial applications.In this context, transition metal‐based sulfides are known in the literature for their efficient catalytic properties. Specifically, nickel sulfides have gained special interest in the field of electrocatalysis due to their high intrinsic electrical conductivity, low cost, and environmental benignity.24 In 2x) is performed for engineering the enhanced intrinsic catalytic activity and electrocatalytically active surface area toward overall water splitting under neutral medium . It was observed that the different transformation time has a major impact on the electronic environment of the Ni and S atoms along with a significant change in the resulting surface morphology. Since, both the electronic structure and the morphology of the material determine the overall performance of an electrocatalyst, we have successfully optimized the material with synergized contributions of both electronic surface states and morphology.Overall water splitting urges for an efficient electrocatalyst with high intrinsic electrocatalytic activity and sufficient catalytic surface area for enhanced overall performance. In this work, the temporal thermal‐evolution of nickel sulfides‐based substrates NiSx is per2 (01‐078‐4702) with an evident enhancement in the concentration of NiS2 phase upon increase in the thermal‐evolution time. NiSx‐2 h exhibits 64% elemental composition of NiS2 which increases to 91% and 95% for NiSx‐24 h and NiSx‐40 h, respectively  to NiSx‐12 h (≈126 S cm−1), a systematic increase in the conductivity is observed with value of ≈223 and ≈298 S cm−1 for NiSx‐24 h and NiSx‐40 h, respectively. The trend in conductivity values is a mutual effect of change in morphology and phase change from NiS to NiS2. The initial decrease in the conductivity could be related to the introduction of holes in the NiSx‐12 h morphology and the systematic increase in conductivity observed henceforth can be due to increase in the concentration of intrinsically high conductivity NiS2 from NiSx‐12 h to NiSx‐40h. These 3D temporally evolved hierarchical nanostructured substrates of intrinsically high‐conducting nickel sulfides can be explored as an electrocatalytically high surface area platform for the electrochemical oxidation and reduction of water under neutral conditions.The measured X‐ray diffraction (XRD) data Figure  shows thy Figure . Furthery Figure . Notablyy Figure . This cax substrates was tested as a catalyst for OER under neutral conditions (0.5 m PBS). The voltage polarization curves in the OER region (1.2–2.1 V vs reference hydrogen electrode [RHE]) show that all the nickel sulfide electrocatalysts  exhibit enhanced behavior toward oxidation of water in comparison to the Ni‐foil  can be seen in Figure ). As expected, a systematic decrease in the RCT upon increasing transformation time of the nickel sulfides‐based substrates was observed, with NiSx‐24 h and NiSx‐40 h exhibiting the lowest RCT values of 3.49 and 3.29 Ω, respectively  in the non‐Faradic region  of the different substrates. It can be observed that the TOF values increase with the increasing transformation time for the electrode preparation till 24 h (sample: NISx‐24 h), and then slightly decrease for NiSx‐40 h, corresponding well with the ECSA‐normalized water oxidation behavior. Hence, the NiSx‐24 h shows the best OER performance because of the mutual contribution of enhanced intrinsic catalytic properties and suitable electrocatalytic surface area. However, in NiSx‐40 h the detrimental effect of the considerable decrease in surface 3D nanostructuring and reduced ECSA decreases the overall OER performance in comparison to NiSx‐24 h. The chronoamperometric and cyclic voltammetry stability measurements for NiSx‐24 h were performed in the OER region. NiSx‐24 h exhibits an insignificant change in its performance after 1000 CV cycles, and 12 h of chronoamperometric measurements for OER  show an enhanced performance of all the NiSx substrates in comparison to the Ni‐foil  state located ≈1 eV above the Fermi level . The high resolution Ni2p XPS spectra for all the sulfides‐based samples exhibit the characteristic Ni 2p3/2 and Ni 2p1/2 peaks along with their corresponding satellite bands  oxidation state of Ni2+. In spite, a twofold increase in the relative concentration of Ni3+ in NiSx‐24 h is observed in comparison to NiSx‐2 h which is noteworthy. This measured increase in the Ni3+:Ni2+ ratio is in correlation with the TOFOER values, which show a similar trend of highest TOF value for NISx‐24 h, with a slight decrease at NISx‐40 h. Figure 3+:Ni2+ ratio following the same trend, thus, sufficiently evidencing the reason for enhancement in the intrinsic OER catalytic activity due to the enriched Ni3+ species concentration at the catalyst surface. Furthermore, it is well established that the pSn2−are the highest catalytically active sites for HER, because the H* adsorption energy for the pSn2− is closer to zero, thus facilitating easy adsorption and desorption of H* during HER  and bridged (pSn2−) polysulfide species and lower (≈162.4 and ≈163.6 eV) and higher binding (≈163.1 and ≈164 eV). Additionally, a doublet for the electrocatalytically dormant sulfate (SO42−) species is observed at high binding energy. The deconvoluted area under the peak and the exact ratios are tabulated in Table pSn2−/SO42−) was observed upon temporal evolution of NiSx. Interestingly, an enhancement in the HER‐active pSn2− species surface concentration in comparison to the HER‐dormant SO42− is observed with the highest pSn2−/SO42− ratio of 0.65 for NISx‐24 h. Additionally, NiSx‐24 h exhibits the highest S/Ni ratio of 2.9 indicating the sulfur rich catalyst surface due to relative enhancement in pSn2− species. A clear correlation between the pSn2−/SO42− ratio and the TOFHER can be observed in Figure x‐24 h due to enriched concentration of polysulfide species at the surface. It is to be noticed that the ratios of Ni3+/Ni2+ for sample NiSx‐12 h and NiSx‐40 h are nearly same  of NiSx‐40 h (≈298 S cm−1) in comparison to NiSx‐12 h (≈126 S cm−1)  sulfur and nickel mapping of NiSx‐2 h shows a non‐uniform distribution of nickel and sulfur, with domains of NiS and NiS2  , corroborating well with the XRD analysis  Figure . Additiox samples for OER and HER in a three‐electrode setup, a full electrochemical cell for overall water splitting under neutral conditions was assembled. The polarization curves for all the nickel sulfide samples show a steep increase in the current density upon increasing the cell voltage  and more than twofold lower than that of NiSx‐2 h (RCT = 25.4 Ω) substrates  species at the catalyst surface  species at the surface induces a change in the morphology of the post‐OER electrode, and an insignificant change upon HER treatment is observed , HER active sites (pSn2−), ECSA, and conductivity as displayed in Figure Inspired by the exceptional performance of our NiSh Figure . NiSx‐s Figure . Noteworh Figure . The cyc3+) and hydrogen evolution species (pSn2−), independently. 4) The optimal electrocatalyst exhibits the lowest cell voltage (1.59 V) as a bifunctional electrocatalyst for overall water splitting in neutral medium ever reported. The measured overpotentials are even lower than the electrochemical cell assembled using the benchmarking precious catalysts IrO2 and Pt‐C. 5) The synthesis process is simple, highly economic, and industrially scalable. It is indeed remarkable to observe a finely engineered NiSx multi‐synergized bifunctional electrocatalyst for exceptional overall water splitting in neutral medium.Hence, the rational engineering of surface electronic structure and morphology is shown here to deeply affect the catalytic performance of the resulting electrocatalysts. In this work, nickel sulfides with finely engineered electrocatalytically active surface moieties are successfully synthesized, which possess the following advantages over conventional reported nickel sulfides, or any other earth‐abundant electrocatalysts, in general. 1) The methodology developed gives a fine control over the morphology and the electronic structure of the metal center and the anion species by a simple temporal modulation of the process. 2) This is the first report on nickel sulfide as an electrocatalyst where the surface active sites for both OER and HER are observed and analyzed in depth, apart from the general physical and electrochemical characterization usually observed in the literature. 3) The systematic evolution of a highly‐conducting electrocatalytic material is observed, with an enhancement in the surface concentration of electrocatalytic oxygen evolution species  for oxygen and hydrogen evolution, respectively. The two‐electrode bifunctional assembly of the NiSx‐24h||NiSx‐24 h electrolyzer exhibits a cell voltage of 1.59 and 1.94 V at 10 and 50 mA cm−2, respectively, the lowest reported cell voltage for any earth‐abundant catalytic material applied as a bifunctional electrocatalyst for overall water splitting under neutral conditions. Remarkably, the reported cell voltage is even lower than benchmarked Pt/C‐IrO2‐based electrolyzer under neutral conditions. The enhanced performance of the electrocatalyst developed in this work is a synergized contribution of electrocatalytic activity (dominated by the surface electronic states) and the active sites (dominated by the surface morphology). The NiSx‐24h||NiSx‐24 h cell shows insignificant performance changes after 12 h of continuous chronoamperometric tests, and after thousand CV cycles. Hence, a methodology for engineering the intrinsic catalytic properties and morphology demonstrated in this study, with the newly developed catalyst sample NiSx‐24 h exhibiting exceptional electrocatalytic performance, can further be explored in a plethora of other electrochemical applications.In conclusion, the electronic structure engineered 3D hierarchical nanostructures of highly conducting NiS4m) and further cleaned with isopropyl alcohol and acetone several times before use. Nickel foil was then heated in an elemental sulfur environment, in a pre‐evacuated tubular furnace, at a fixed temperature of 450 °C. The desired temperature was ramped at 10 °C min−1 till a temperature of 450 °C was reached, and maintained for different time durations of 2, 12, 24, and 40 h, respectively. Upon completion of the thermal process the furnace was cooled naturally. The sulfur source used in this work was molecular sulfur powder (99.99% Sigma), and the nickel‐to‐sulfur weight ratio was optimized to 2:1. All the reagents used in this work were of analytical grade, and used as purchased without any further purification.Nickel foil (99.99%) was sonicated in dilute hydrochloric acid . The HAADAF elemental mapping and transmission electron microscopy (Fei Themis Z G3) were performed by cutting a lamella sample from the surface of the material using a Thermo Fisher Helios 5 UC focused ion beam (FIB) system, further applied for the analysis of the crystallographic structure and lattice spacing. The surface chemical composition was analyzed using the XPS system, and measurements conducted on an ESCALAB QXi X‐ray Photoelectron Spectrometer Microprobe. The XPS data was fitted using the Fityk software.m) as electrolyte. All the potentials were converted to the RHE, and were presented without performing any iR corrections. A working area of 0.5 cm2 was fixed for the working electrode. The cyclic voltammetry measurements were conducted at a scan rate of 5 mV s−1 in the OER and HER regions, and the reverse scan was considered for calculating the overpotential values. The ECSA was calculated using the formula, ECSA = CdlCs cm2ESCA, where, Cs = 40 µF cm−2 (specific capacitance of flat surface). The TOF was calculated using the formula TOF = 12nF where, F: Faraday constant, and n: number of active sites; n = Q/2F, calculated by performing CV in the non‐Faradic region. Nyquist analysis was performed in the frequency range 0.1–106 Hz at the overpotential in the OER and HER region and the plots were fitted using the EIS spectrum analyzer. The activity of the NiSx electrocatalysts was compared with the commercial IrO2  for OER, Pt‐C  for HER and IrO2||Pt‐C for full cell. For electrode preparation, 10 mL dispersion of active material was prepared via sonication in a water‐ethanol (4:1) mixture with 50 µL Nafion (of 5%) in ethanol. The electrodes were prepared by drop‐casting the dispersion of active material on Ni foam with a mass loading of ≈1 mg cm−2.All the electrochemical measurements were conducted by a Metrohm Autolab potentiostat. The three‐electrode measurements were conducted using a platinum mesh as counter electrode and Ag/AgCl as reference electrode, in pH 7 potassium phosphate buffer (0.5 The authors declare no conflict of interest.Supporting InformationClick here for additional data file.