Cytoplasmic male sterility (CMS) is a very important factor to produce hybrid seeds, and the restoration of fertility involves the expression of many fertility-related genes. Our previous study showed that the expression of CaPIPLC5 was significantly up-regulated in pepper restorer accessions and minimally expressed in sterile accessions, speculating that CaPIPLC5 is related to the restoration of fertility. In this study, we further validated the function of CaPIPLC5 in the restoration of fertility. The results showed that CaPIPLC5 was specifically expressed in the anthers of the restorer accessions with the subcellular localization in the cytoplasm. Furthermore, the expression of CaPIPLC5 was significantly higher in restorer lines and restorer combinations than that in CMS lines and their maintainer lines. Silencing CaPIPLC5 led to the number of pollen decreased, pollen grains wrinkled, and the ratio of pollen germination reduced. In addition, the joint analysis of Yeast One-Hybrid (Y1H) and Dual-Luciferase (dual-LUC) assays suggested that transcription factors such as CaARF5, CabZIP24 and CaMYB-like1, interacted with the promoter regions of CaPIPLC5, which regulated the expression of CaPIPLC5. The present results provide new insights into the study of CaPIPLC5 involved in the restoration of fertility in pepper.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

BACKGROUND: Neurotrophins are essential factors for neural growth and function; they play a crucial role in neurodegenerative diseases where their expression levels are altered. Our previous research has demonstrated changes in synaptic plasticity and neurotrophin expression levels in a pharmacological model of Huntington\'s disease induced by 3-nitropropionic acid (3-NP). In the 3- NP-induced HD model, corticostriatal Long Term Depression (LTD) was impaired, but neurotrophin-3 (NT-3) restored striatal LTD. This study delves into the NT-3-induced signaling pathways involved in modulating and restoring striatal synaptic plasticity in cerebral slices from 3-NPinduced striatal degeneration in mice in vivo.
METHODS: Phospholipase C (PLC), phosphatidylinositol-3-kinase (PI3K), and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways activated by NT-3 were analyzed by means of field electrophysiological recordings in brain slices from control and 3-NP treated in the presence of specific inhibitors of the signaling pathways.
RESULTS: Using specific inhibitors, PLC, PI3K, and MEK/ERK signaling pathways contribute to NT3-mediated plasticity modulation in striatal tissue slices recorded from control animals. However, in the neurodegeneration model induced by 3-NP, the recovery of striatal LTD induced by NT-3 was prevented only by the PLC inhibitor. Moreover, the PLC signaling pathway appeared to trigger downstream activation of the endocannabinoid system, evidenced by AM 251, an inhibitor of the CB1 receptor, also hindered NT-3 plasticity recovery.
CONCLUSIONS: Our finding highlights the specific involvement of the PLC pathway in the neuroprotective effects of NT-3 in mitigating synaptic dysfunction under neurodegenerative conditions.

Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Calcium (Ca2+) is a highly versatile intracellular messenger that regulates several cellular processes. Although it is unclear how a single-second messenger coordinates various effects within a cell, there is growing evidence that spatial patterns of Ca2+ signals play an essential role in determining their specificity. Ca2+ signaling patterns can differ in various cell regions, and Ca2+ signals in the nuclear and cytoplasmic compartments have been observed to occur independently. The initiation and function of Ca2+ signaling within the nucleus are not yet fully understood. Receptor tyrosine kinases (RTKs) induce Ca2+ signaling resulting from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus. This signaling mechanism may be responsible for the effects of specific growth factors on cell proliferation and gene transcription. This review highlights the recent advances in RTK trafficking to the nucleus and explains how these receptors initiate nuclear calcium signaling.

Based on the specific binding of drug molecules to cell membrane receptors, a screening and separation method for active compounds of natural products was established by combining phospholipase C (PLC) sensitized hollow fiber microscreening by a solvent seal with high-performance liquid chromatography technology. In the process, the factors affecting the screening were optimized. Under the optimal screening conditions, we screened honokiol (HK), magnolol (MG), negative control drug carbamazepine, and positive control drug amentoflavone, the repeatability of the method was tested. The PLC activity was determined before and after the screening. Experimental results showed that the sensitization factors of PLC of HK and MG were 61.0 and 48.5, respectively, and amentoflavone was 15.0, carbamazepine could not bind to PLC. Moreover, the molecular docking results were consistent with this measurement, indicating that HK and MG could be combined with PLC, and they were potential interacting components with PLC. This method used organic solvent to seal the PLC greatly ensuring the activity, so this method had the advantage of integrating separation, and purification with screening, it not only exhibited good reproducibility and high sensitivity but was also suitable for screening the active components in natural products by various targets in vitro.

The impact of environmental enrichment (EE) on natural rewards, including social and appetitive rewards, was investigated in male Swiss mice. EE, known for providing animals with various stimuli, was assessed for its effects on conditioned place preference (CPP) associated with ethanol and social stimuli. We previously demonstrated that EE increased the levels of the prosocial neuropeptide oxytocin (OT) in the hypothalamus and enhanced ethanol rewarding effects via an oxytocinergic mechanism. This study also investigated the impact of EE on social dominance and motivation for rewards, measured OT-mediated phospholipase C (PLC) activity in striatal membranes, and assessed OT expression in the hypothalamus. The role of dopamine in motivating rewards was considered, along with the interaction between OT and D1 receptors (DR) in the nucleus accumbens (NAc). Results showed that EE mice exhibited a preference for ethanol reward over social reward, a pattern replicated by the OT analogue Carbetocin. EE mice demonstrated increased social dominance and reduced motivation for appetitive taste stimuli. Higher OT mRNA levels in the hypothalamus were followed by diminished OT receptor (OTR) signaling activity in the striatum of EE mice. Additionally, EE mice displayed elevated D1R expression, which was attenuated by the OTR antagonist (L-368-889). The findings underscore the reinforcing effect of EE on ethanol and social rewards through an oxytocinergic mechanism. Nonetheless, they suggest that mechanisms other than the prosocial effect of EE may contribute to the ethanol pro-rewarding effect of EE and Carbetocin. They also point towards an OT-dopamine interaction potentially underlying some of these effects.

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.