Endogenous Activity Research Articles

Atrial natriuretic peptide (ANP) modulates stress-induced autophagy in endothelial cells

Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.

Methionine oxidation of actin cytoskeleton attenuates traumatic memory retention via reactivating dendritic spine morphogenesis

Post-traumatic stress disorder (PTSD) is characterized by hypermnesia of the trauma and a persistent fear response. The molecular mechanisms underlying the retention of traumatic memories remain largely unknown, which hinders the development of more effective treatments. Utilizing auditory fear conditioning, we demonstrate that a redox-dependent dynamic pathway for dendritic spine morphogenesis in the basolateral amygdala (BLA) is crucial for traumatic memory retention. Exposure to a fear-induced event markedly increased the reduction of oxidized filamentous actin (F-actin) and decreased the expression of the molecule interacting with CasL 1 (MICAL1), a methionine-oxidizing enzyme that directly oxidizes and depolymerizes F-actin, leading to cytoskeletal dynamic abnormalities in the BLA, which impairs dendritic spine morphogenesis and contributes to the persistence of fearful memories. Following fear conditioning, overexpression of MICAL1 in the BLA inhibited freezing behavior during fear memory retrieval via reactivating cytokinesis, whereas overexpression of methionine sulfoxide reductase B 1, a key enzyme that reduces oxidized F-actin monomer, increased freezing behavior during retrieval. Notably, intra-BLA injection of semaphorin 3A, an endogenous activator of MICAL1, rapidly disrupted fear memory within a short time window after conditioning. Collectively, our results indicate that redox modulation of actin cytoskeleton in the BLA is functionally linked to fear memory retention and PTSD-like memory.

Rational Molecular Design of a Fluorescent Probe for Selectively Sensing Human Cytochrome P450 2D6.

Cytochrome P450 2D6 (CYP2D6) is a key enzyme that mediates the metabolism of various drugs and endogenous substances in humans. However, its biological role in drug-drug interactions especially mechanism-based inactivation (MBI), and various diseases remains poorly understood, owing to the lack of molecular tools suitable for selectively monitoring CYP2D6 in complex biological systems. Herein, using a tailored molecular strategy, we developed a fluorescent probe BDPM for CYP2D6. BDPM exhibits excellent specificity and imaging capability for CYP2D6, making it suitable for the real-time monitoring of endogenous CYP2D6 activity in living bio-samples. Therefore, our tailored strategy proved useful for constructing the highly selective and enzyme-activated fluorescent probes. BDPM as a molecular tool to explore the critical roles of CYP2D6 in the pathogenesis of diseases, high-throughput screening of inhibitors and intensive investigation of CYP2D6-induced MBI in natural systems.

7657 Single Cell RNAseq And Omics Revealed Novel Role Of Liver Group 2 Innate Lymphocytes In Regulation Of Hepatic Gluconeogenesis

Abstract Disclosure: M. Fujimoto: None. T. Tanaka: None. Background: The liver is an important organ that maintains homeostasis of glucose metabolism in the body through gluconeogenesis. It is considered that gluconeogenesis in hepatocytes is particularly active around the portal vein. However, since excessive gluconeogenesis aggravates the pathogenesis of diabetes, it is necessary to elucidate the regulatory mechanism of gluconeogenesis. Although several studies revealed that group 2 innate lymphocytes (ILC2) contribute to blood glucose maintenance in adipose and pancreatic tissues, the contribution of liver ILC2s to glucose metabolism is unknown. This study aims to elucidate the contribution of liver ILC2s to glucose metabolism and its molecular mechanism. Methods: We administered IL-33, an endogenous ILC2 activator, to BALB/c and NSG mice, and measured fasting blood glucose levels and blood glucose elevation by gluconeogenic substrate pyruvate, and blood glucose levels in liver ILC2s transplanted NSG mice. Molecular mechanisms were analyzed by single cell-RNAseq (scRNA-seq) of liver ILC2s, lung ILC2s and hepatocytes, GATA3 transcription complex, ATAC-seq, GATA3-ChIP-seq, and Metabolomics of primary hepatocytes. Results: IL-33 strongly induced IL-13 production by liver ILC2s, lowered fasting blood glucose, and suppressed pyruvate-induced glucose elevation; these effects were not observed in NSG mice lacking immune cells including ILC2s. A hypoglycemic effect of IL-33 was observed in liver ILC2-transplanted NSG mice, but not in mice transplanted with IL-13-deficient ILC2s, suggesting the effect is IL-13-dependent. scRNA-seq results showed that liver ILC2s showed higher IL-13 expression than lung ILC2s, and IL-33 treatment suppressed the expression of gluconeogenic enzymes in the G6pc-high hepatocyte population. The interaction between liver ILC2s and G6pc-high hepatocytes via IL-13/IL-13 receptors was also demonstrated; co-culture of ILC2s with primary cultured hepatocytes resulted in decreased expression of hepatocyte gluconeogenic enzymes and accumulation of multiple gluconeogenic substrates. In addition, IL-13 neutralizing antibody treatment attenuated the effect of the decreased expression of gluconeogenic enzymes in hepatocytes. Transcription factor GATA3 complex analysis and GATA3-ChIPseq suggested binding of GATA3 of the AP-1 family (JunB) in liver ILC2s. Overexpression, knockdown, and scRNA-seq results suggest that AP1 may act in a GATA3-repressive manner in liver ILC2s. Discussion The functional characteristics of liver ILC2s, hepatocyte heterogeneity and subcluster were demonstrated by scRNA-seq, suggesting that liver ILC2s may directly suppress hepatocyte gluconeogenesis via IL-13. This may play a novel role in the homeostasis of hepatic glucose metabolism. Presentation: 6/1/2024

6523 Kisspeptin Administration Does Not Induce Anxiety in Humans

Abstract Disclosure: E.G. Mills: None. L. Thurston: None. L. Yang: None. S. Suladze: None. T. Hunjan: None. M. Phylactou: None. B. Patel: None. S.A. Clarke: None. A.J. Shah: None. C. Izzi-Engbeaya: None. J. Tsoutsouki: None. P. Bech: None. N. Ertl: None. L. Demetriou: None. M.B. Wall: None. D. Goldmeier: None. A. Abbara: None. A.N. Comninos: None. W.S. Dhillo: None. Background: The neuropeptide kisspeptin is a critical endogenous activator of the reproductive system. Due to its key role in regulating reproductive and behavioural processes, there has been escalating interest in targeting kisspeptin-pathways to treat reproductive and psychosexual disorders. However, conflicting evidence from pre-clinical animal models proposes that kisspeptin can have an anxiolytic, anxiogenic or have no effects on anxiety. Given the rapid development of kisspeptin-based therapeutics, it is important to clarify kisspeptin’s effects on anxiety in humans. Herein, we report the largest study examining the effects of kisspeptin on psychometric measures of anxiety and circulating cortisol levels in humans. Methods: Ninety-three eugonadal participants (n=29 healthy men, n=32 men with low sexual desire, n=32 women with low sexual desire) completed a double-blind, randomised, placebo-controlled, crossover study. Participants attended twice: once for intravenous infusion of kisspeptin-54 (1nmol/kg/h) over 75mins and for a rate-matched placebo. Blood sampling took place at 15-minute intervals from -30 to 75mins to measure circulating kisspeptin, LH, testosterone and cortisol [in men] and oestradiol [in women]. The validated ‘State-Trait Anxiety Inventory (STAI) Y2-Trait’ was completed prior to the infusions to exclude abnormal anxiety traits, with all scores within normal limits. Participants also completed the ‘STAI Y1-State’ before and prior to the end of the infusions to assess for any dynamic effects of the infusions on anxiety. Results: Ninety-three participants (mean age ±SD 30.9±8.7yrs, BMI 24.0±3.7kg/m2) completed the study. Intravenous kisspeptin significantly increased serum LH to similar levels previously described using this administration protocol, confirming that the dose was biologically active (P<0.001). Importantly, state anxiety was unaltered by kisspeptin, compared to placebo (mean difference in ‘STAI Y1-State’ scores during the infusions: kisspeptin -0.03±8.11, placebo 0.77±8.08, P=0.43). Moreover, kisspeptin had no effect on circulating cortisol compared to placebo during the 75minute study period (P=0.92). As expected, kisspeptin had no significant effects on downstream sex-steroid levels during the 75minute study period, thereby excluding these as confounders. Discussion: This is the largest study demonstrating that a biologically active dose of kisspeptin to healthy volunteers and patients with psychosexual disorders does not affect psychometric measures of anxiety and associated circulating cortisol levels. Given that animal studies have yielded conflicting results, this provides important clinical data and reassurance that kisspeptin administration in humans does not induce anxiety and so informs the rapid development of kisspeptin-based therapeutics for reproductive and psychosexual disorders. Presentation: 6/3/2024

Cell Mediated Reactions Create TGF-β Delivery Limitations in Engineered Cartilage

During native cartilage development, endogenous TGF-β activity is tightly regulated by cell-mediated chemical reactions in the extracellular milieu (e.g., matrix and receptor binding), providing spatiotemporal control in a manner that is localized and short acting. These regulatory paradigms appear to be at odds with TGF-β delivery needs in tissue engineering (TE) where administered TGF-β is required to transport long distances or reside in tissues for extended durations. In this study, we perform a novel examination of the influence of cell-mediated reactions on the spatiotemporal distribution of administered TGF-β in cartilage TE applications. Reaction rates of TGF-β binding to cell-deposited ECM and TGF-β internalization by cell receptors are experimentally characterized in bovine chondrocyte-seeded tissue constructs. TGF-β binding to the construct ECM exhibits non-linear Brunauer–Emmett–Teller (BET) adsorption behavior, indicating that as many as seven TGF-β molecules can aggregate at a binding site. Cell-mediated TGF-β internalization rates exhibit a biphasic trend, following a Michaelis-Menten relation (Vmax=2.4 molecules cell-1 s-1, Km=1.7 ng mL-1) at low ligand doses (≤130ng/mL), but exhibit an unanticipated non-saturating power trend at higher doses (≥130ng/mL). Computational models are developed to illustrate the influence of these reactions on TGF-β spatiotemporal delivery profiles for conventional TGF-β administration platforms. For TGF-β delivery via supplementation in culture medium, these reactions give rise to pronounced steady state TGF-β spatial gradients; TGF-β concentration decays by ∼90% at a depth of only 500 μm from the media-exposed surface. For TGF-β delivery via heparin-conjugated affinity scaffolds, cell mediated internalization reactions significantly reduce the TGF-β scaffold retention time (160 to 360-fold reduction) relative to acellular heparin scaffolds. This work establishes the significant limitations that cell-mediated chemical reactions engender for TGF-β delivery and highlights the need for novel delivery platforms that account for these reactions to achieve optimal TGF-β exposure profiles. Statement of SignificanceDuring native cartilage development, endogenous TGF-β activity is tightly regulated by cell-mediated chemical reactions in the extracellular milieu (e.g., matrix and receptor binding), providing spatiotemporal control in a manner that is localized and short acting. However, the effect of these reactions on the delivery of exogenous TGF-β to engineered cartilage tissues remains not well understood. In this study, we demonstrate that cell-mediated reactions significantly restrict the delivery of TGF-β to cells in engineered cartilage tissue constructs. For delivery via media supplementation, reactions significantly limit TGF-β penetration into constructs. For delivery via scaffold loading, reactions significantly limit TGF-β residence time in constructs. Overall, these results illustrate the impact of cell-mediated chemical reaction on TGF-β delivery profiles and support the importance of accounting for these reactions when designing TGF-β delivery platforms for promoting cartilage regeneration.

A Novel Porcine Model of Thoracic Aortic Aneurysms Reveals Increased Endogenous Matrix Metalloproteinase Activity

A Novel Porcine Model of Thoracic Aortic Aneurysms Reveals Increased Endogenous Matrix Metalloproteinase Activity

Context transcription factors establish cooperative environments and mediate enhancer communication

Many enhancers control gene expression by assembling regulatory factor clusters, also referred to as condensates. This process is vital for facilitating enhancer communication and establishing cellular identity. However, how DNA sequence and transcription factor (TF) binding instruct the formation of high regulatory factor environments remains poorly understood. Here we developed a new approach leveraging enhancer-centric chromatin accessibility quantitative trait loci (caQTLs) to nominate regulatory factor clusters genome-wide. By analyzing TF-binding signatures within the context of caQTLs and comparing episomal versus endogenous enhancer activities, we discovered a class of regulators, ‘context-only’ TFs, that amplify the activity of cell type-specific caQTL-binding TFs, that is, ‘context-initiator’ TFs. Similar to super-enhancers, enhancers enriched for context-only TF-binding sites display high coactivator binding and sensitivity to bromodomain-inhibiting molecules. We further show that binding sites for context-only and context-initiator TFs underlie enhancer coordination, providing a mechanistic rationale for how a loose TF syntax confers regulatory specificity.

A digital CRISPR-dCas9-based gene remodeling biocomputer programmed by dietary compounds in mammals

CRISPR-dCas9 (dead Cas9 protein) technology, combined with chemical molecules and light-triggered genetic switches, offers customizable control over gene perturbation. However, these simple ON/OFF switches cannot precisely determine the sophisticated perturbation process. Here, we developed a resveratrol and protocatechuic acid-programmed CRISPR-mediated gene remodeling biocomputer (REPACRISPR) for conditional endogenous transcriptional regulation of genes invitro and invivo. Two REPACRISPR variants, REPACRISPRi and REPACRISPRa, were designed for the logic control of gene inhibition and activation, respectively. We successfully demonstrated the digital computations of single or multiplexed endogenous gene transcription by using REPACRISPRa. We also established mathematical models to predict the dose-responsive transcriptional levels of a target endogenous gene controlled by REPACRISPRa. Moreover, high levels of endogenous gene activation in mice mediated by the AND logic gate demonstrated computational control of CRISPR-dCas9-based epigenome remodeling in mice. This CRISPR-based biocomputer expands the synthetic biology toolbox and can potentially advance gene-based precision medicine. A record of this paper's transparent peer review process is included in the supplemental information.

Clinical and Humanistic Burden of Non-inhibitor HaemophiliaA in Five European Countries: Insights from the CHESSII Study.

HaemophiliaA (HA) is a congenital bleeding disorder caused by a deficiency/absence of factorVIII (FVIII) and characterised by frequent, acute and prolonged spontaneous or traumatic bleeding events, often leading to haemophilic arthropathy and progressive joint deterioration. HA severity is characterized by endogenous FVIII activity: mild (> 5-40%), moderate (1-5%), or severe (< 1%). HA poses a substantial clinical and socioeconomic burden on people with HA (PWHA), their caregivers, and society. This analysis evaluates clinical and patient-centric outcomes of a cohort of individuals with non-inhibitor HA sampled from France, Germany, Italy, Spain, and the UK in the 'Cost of Haemophilia in Europe: A Socioeconomic Survey II' (CHESSII) study. CHESSII was a cross-sectional burden-of-illness study collecting clinical and socioeconomic data on adult (≥ 18years) individuals with haemophiliaA or B of any severity with or without inhibitors from eight European countries. Descriptive analyses were conducted examining physician-reported demographics, clinical and health resource utilisation information. PWHA-reported health-related quality of life (HRQoL) using the EQ-5D-5L and Work Productivity and Activity Impairment (WPAI) were also examined. Outcomes were stratified by HA severity and reported at country level. Demographics and clinical characteristics of the cohort (N = 880) were generally consistent across countries. Individuals with severe HA experienced more frequent bleeding events and joint disease despite broad use of factor replacement therapy long-term prophylaxis. A minority of those with mild or moderate HA also experienced such challenges. HRQoL and workforce participation diminished, and chronic pain increased, with increasing HA severity. This analysis provides up-to-date insights on the impact of HA across five European countries. Increasing HA severity was generally associated with worse clinical outcomes, HRQoL and workforce participation. These findings suggest a place for continued evidence-based tailored treatment and clinical management approaches in addressing the residual burden of HA.

Novel control strategy employing anaerobic/aerobic/anoxic/aerobic/anoxic mode to enhance endogenous denitrification and anammox for municipal wastewater treatment

Anaerobic/Aerobic/Anoxic (AnOA) process utilizes endogenous denitrification to remove nitrogen. However, low endogenous denitrification activity critically restricts its application owing to insufficient carbon sources. In this study, a novel control strategy employing anaerobic/aerobic/anoxic/aerobic/anoxic (AOAOA) mode was introduced to treat low Carbon/Nitrogen (C/N) ratio municipal wastewater over 262 days. The concentration of total inorganic nitrogen (TIN) was only 3.9 ± 2.0 mg/L in the effluent, with a high nitrogen removal efficiency (NRE) of 94.3 %. The relative abundance of Candidatus Competibacter increased from 1.2 % to 2.3 %, ensuring an efficient endogenous denitrification process. Additionally, Candidatus Brocadia enriched from 0.02 % to 0.6 %, contributing to 63.1 % nitrogen removal during the anoxic stage in Phase Ⅲ. This study presents a promising approach for enhancing endogenous denitrification and anammox in the AnOA process, contributing to sustainable wastewater treatment.

The role of starch digestion in the brewing of gluten-free beers

Celiac disease affects an estimated 1% of the global population, and symptoms are triggered by the consumption of gluten in sensitized individuals. Other gluten sensitivities exist but are poorly characterized or often under-diagnosed, leading a number of people to pursue a gluten-free or gluten-reduced diet even in the absence of a diagnosis. Gluten is a complex suite of proteins from the prolamin fraction of storage proteins in barley, wheat, and rye, meaning that foods or beverages that are produced from these cereals will also contain gluten. Beer is one such beverage that is traditionally produced from malted barley and is thus incompatible with a gluten-free lifestyle. The simplest option to produce a gluten-free beer would seem to be to use alternative, gluten-free, grains such as millet, sorghum, teff, rice, corn or other starchy materials. However, barley has thousands of years of development as a brewing agent behind it and these alternative ingredients cannot be incorporated into the brewing process without substantial alterations. Particularly, these ingredients generally have much lower endogenous starch degrading activity and much higher starch gelatinization temperatures as compared to barley which makes generating sufficient fermentable sugars for yeast fermentation a challenge. In this review we will further elucidate these challenges as well as the solutions that are being developed to bring beer to the gluten-free masses.

Antioxidant Features of Humic Products by ABTS Assay.

Oxidative stress plays a pivotal role in driving immunosenescence by disrupting cellular homeostasis and impairing immune function. Humic substances exhibit scavenging activity against reactive oxygen species (ROS), inhibit ROS generation via metal chelation, and modulate endogenous antioxidant enzyme activity. Additionally, humic substances display anti-inflammatory effects, further supporting cellular redox balance. Given their antioxidant activity, humic substances hold promise as natural compounds for mitigating oxidative stress-associated immunosenescence. Here we describe the evaluation of antioxidant capacities of humic products by ABTS spectrophotometric assay.

TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes.

TLR3 is expressed in human skin and keratinocytes, and given its varied role in skin inflammation, development, and regeneration, we sought to determine the cellular response in normal human keratinocytes to TLR3 activation. We investigated this mechanism by treating primary human keratinocytes with both UVB, an endogenous and physiologic TLR3 activator, and poly(I:C), a synthetic and selective TLR3 ligand. TLR3 activation with either UVB or poly(I:C) altered keratinocyte morphology, coinciding with the key features of epithelial-to-mesenchymal transition: increased epithelial-to-mesenchymal transition gene expression, enhanced migration, and increased invasion properties. These results confirm and extend previous studies demonstrating that in addition to its classical role in the innate immune response, TLR3 signaling also regulates stem cell-like properties and developmental programs.

The Modulatory Effect of Exercise on the Endocannabinoid Signaling Pathway in the Epileptic Rats

Background: Regular moderate exercise and endogenous cannabinoid activity have independently been shown to alleviate seizure (SE) attacks. Objectives: This study aimed to investigate the effect of physical activity on the expression levels of cannabinoid CB1 and CB2 receptors in the brains Methods: Male Wistar rats were divided into five groups: Sham, SE, physical activity (PA), PA + SE, and PA before SE. Epileptic SEs were induced by administering pentylenetetrazol (PTZ; intraperitoneally, 35 mg/kg) every other day for four weeks in the SE, PA + SE, and PA before SE groups. Animals in the PA, PA + SE, and PA before SE groups participated in treadmill running (30 minutes per day, five days a week). The mean number of cortical and hippocampal (CA1, CA3) CB1 and CB2 receptors was assessed using immunohistochemistry. Results: The study data revealed a significant reduction of CB1 and CB2 receptors in the CA1, CA3, and cortex of the SE group compared to the sham group. A significant increase in CB1 receptors was observed in the PA and PA before SE groups compared to the SE group in both cortical and hippocampal areas. Physical activity significantly increased hippocampal and cortical CB2 receptor distribution in the PA, PA + SE, and PA before SE groups compared to the SE group. Conclusions: These findings suggest that exercise modulates the expression of hippocampal and cortical cannabinoid receptors in epileptic rats, highlighting the involvement of the endocannabinoid pathway in the anti-epileptic effects of exercise.

Development of a Dual Reporter System to Simultaneously Visualize Ca2+ Signals and AMPK Activity.

In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.

Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity.

Engineering histone acylation states can inform mechanistic epigenetics and catalyze therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the programmable deposition of acetylation and longer-chain acylations. We show that targeting an engineered lysine crotonyltransferase results in weak levels of endogenous enhancer activation yet retains potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while substantially reducing cytotoxicity, enabling improved viral delivery. We leveraged these capabilities to perform single-cell CRISPR activation screening and map enhancers to the genes they regulate in situ. We also discover acylation-specific interactions and find that recruitment of p300, regardless of catalytic activity, to prime editing sites can improve editing efficiency. These new programmable epigenome editing tools and insights expand our ability to understand the mechanistic role of lysine acylation in epigenetic and cellular processes and perform functional genomic screens.

Visualizing PIEZO1 Localization and Activity in hiPSC-Derived Single Cells and Organoids with HaloTag Technology.

PIEZO1 is critical to numerous physiological processes, transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging, our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore, the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca 2+ -sensitive HaloTag ligands, achieving temporal resolution approaching that of patch clamp electrophysiology. Finally, we used lightsheet imaging of hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue organoids. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and targeted therapeutic development.

Chemogenetic Activation of RFRP Neurons Reduces LH Pulse Frequency in Female but not Male Mice.

The neuropeptide RFRP-3 (RFamide-related peptide-3) is thought to play a role in the negative regulation of fertility. However, the exogenous administration of RFRP-3 yields varying results depending on the dose and route of administration, sex of the subject, and many other variables. Manipulation of in vivo neuronal activity using DREADDs (designer receptor exclusively activated by designer drugs) technology enables investigation of cell type-specific neuronal activation in a manner that better reflects endogenous neuronal activity. To test the effects of RFRP neuronal activation on pulsatile luteinizing hormone (LH) secretion. We generated mice expressing the stimulatory hM3Dq designer receptor exclusively in RFRP cells using 2 different Cre-loxP-mediated approaches: (1) we bred mice to express hM3Dq in all Rfrp-Cre-expressing cells, including some that transiently expressed Rfrp-Cre neonatally (RFRP × hM3Dq mice), and (2) we stereotaxically injected Cre-dependent hM3Dq into the dorsomedial nucleus of RFRP-Cre mice to drive hM3Dq expression exclusively in a subpopulation of adult Rfrp-Cre neurons (RFRP-AAV-hM3Dq mice). We then investigated the effects of acute hM3Dq activation on LH pulse frequency in RFRP × hM3Dq mice, RFRP-AAV-hM3Dq mice, and their respective controls. In both female RFRP × hM3Dq and RFRP-AAV-hM3Dq mice, chemogenetic activation of Cre-driven hM3Dq led to a significant 35% to 50% reduction in LH pulse frequency compared with controls, while no differences in pulse amplitude or mean LH concentration were observed. In marked contrast, RFRP activation did not cause any changes to LH pulse dynamics in male mice. These data show for the first time that activation of neurons that have expressed Rfrp, or of a subset of adult RFRP neurons, can independently suppress LH pulsatility in female, but not male mice.

Saliciylic Acid is also Effective Along with Abscisic Acid and Gibberellic Acid in the Orchid Post-Pollination Process

The aim of this study is to determine the endogenous hormone activities of abscisic acid (ABA), Jasmonic acid (JA) and Salicylic acid (SA) in compatible (Intraspecific = ISP) and incompatible pollination (Intergeneric = IGP) in stigmatic ovaries during orchid post-pollination process. It was designed to be Himantoglossum robertianum in ISP experiments and Himantoglossum robertianum and Orchis italica in IGP experiments. Polynariums taken from O. italica have been applied with needles to the flower stigmas of H. robertianum. Ovaries with stigma from both pollination types were taken separately for each day for a total of 10 days and quantitative endogenous hormone analyzes have been performed by LC-MS/MS. After all, SA hormone has been detected for the first time in both ISP and IGP, but only ISP values were significant. The statistical analysis revealed that while SA is significant in only ISP, ABA values were found to be highly significant in both ISP and IGP. ISP9 also showed high significance. Additionally, ABA values were generally higher than SA values, especially in IGP experiments. The results also emphasized the importance of ovarian endogenous hormone activities for the first time. JA was not detected in the analysis.