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Translocational regulation of proinsulin biosynthesis in pancreatic β-cells is unknown, although several studies have reported an important accessory role for the Translocon-Associated Protein complex to assist preproinsulin delivery into the endoplasmic reticulum via the heterotrimeric Sec61 translocon (comprising α, β, and γ subunits). The actual protein-conducting channel is the α-subunit encoded either by Sec61A1 or its paralog Sec61A2. Although the underlying channel selectivity for preproinsulin translocation is unknown, almost all studies of Sec61α to date have focused on Sec61α1. There is currently no evidence to suggest that this gene product plays a major role in proinsulin production, whereas genome-wide association studies indicate linkage of Sec61A2 with diabetes. Here, we report that evolutionary differences in mouse preproinsulin signal peptides affect proinsulin biosynthesis. Moreover, we find that, although some preproinsulin translocation can proceed through Sec61α1, Sec61α2 has a greater impact on proinsulin biosynthesis in pancreatic β-cells. Remarkably, Sec61α2 translocon deficiency exerts a significant inhibitory effect on the biosynthesis of preproinsulin itself, including a disproportionate increase of full-length nascent chain unreleased from ribosomes. This study not only reveals novel translocational regulation of proinsulin biosynthesis but also provides a rationale for genetic evidence suggesting an important role of Sec61α2 in maintaining blood glucose homeostasis. Article Highlights: Preproinsulin uses its signal peptide to engage the Sec61 translocon, but, surprisingly, existing data show no appreciable insulin biosynthesis or diabetes phenotypes linked to the Sec61A1 gene. We wanted to know whether Sec61α2 might contribute to insulin biosynthesis, because genome-wide association studies link Sec61A2 (the unstudied paralog of Sec61A1) to type 2 diabetes. We found a major drop in proinsulin biosynthesis in β-cells deficient for Sec61α2 (much more than for Sec61α1), and, further, this reflexively inhibits preproinsulin translation. Our data suggest that the genetic linkage of Sec61A2 to type 2 diabetes may be explained by a defect in insulin biosynthesis. [ABSTRACT FROM AUTHOR]

In the investigation of heterotrimeric G protein‐mediated signal transduction in planta, their roles in the transmittance of low K+ stimuli remain to be elucidated. Here, we found that the primary root growth of wild‐type Arabidopsis was gradually inhibited with the decrease of external K+ concentrations, while the primary root of the mutants for G protein β subunit AGB1 and γ subunits AGG1, AGG2 and AGG3 could still grow under low K+ conditions (LK). Exogenous NAA application attenuated primary root elongation in agb1 and agg1/2/3 but promoted the growth in wild‐type seedlings under LK stress. Using ProDR5:GFP, ProPIN1:PIN1‐GFP and ProPIN2:PIN2‐GFP reporter lines, a diminishment in auxin concentration at the radicle apex and a reduction in PIN1and PIN2 efflux carrier abundance were observed in wild‐type roots under LK, a phenomenon not recorded in the agb1 and agg1/2/3. Further proteolytic and transcriptional assessments revealed an enhanced degradation of PIN1 and a suppressed expression of PIN2 in the wild‐type background under LK, contrasting with the stability observed in the agb1 and agg1/2/3 mutants. Our results indicate that the G protein β and γ subunits play pivotal roles in suppressing of Arabidopsis root growth under LK by modulating auxin redistribution via alterations in PIN1 degradation and PIN2 biosynthesis. Summary statement: We demonstrate that Gβγ dimers modulate auxin distribution at the root tip via the auxin efflux transporters PIN1/PIN2, consequently enhancing the adaptive response of plant roots to low K+ conditions. This underscores the critical role of G protein β and γ subunits in the morphogenetic processes of root architecture under low K+ stress. [ABSTRACT FROM AUTHOR]

Pertussis toxin (PT) is a virulent factor produced by Bordetella pertussis, the causative agent of whooping cough. PT exerts its pathogenic effects by ADP-ribosylating heterotrimeric G proteins, disrupting cellular signaling pathways. Here, we investigate the potential of two antiarrhythmic drugs, amiodarone and dronedarone, in mitigating PT-induced cellular intoxication. After binding to cells, PT is endocytosed, transported from the Golgi to the endoplasmic reticulum where the enzyme subunit PTS1 is released from the transport subunit of PT. PTS1 is translocated into the cytosol where it ADP-ribosylates inhibitory α-subunit of G-protein coupled receptors (Gαi). We showed that amiodarone and dronedarone protected CHO cells and human A549 cells from PT-intoxication by analyzing the ADP-ribosylation status of Gαi. Amiodarone had no effect on PT binding to cells or in vitro enzyme activity of PTS1 but reduced the signal of PTS1 in the cell suggesting that amiodarone interferes with intracellular transport of PTS1. Moreover, dronedarone mitigated the PT-mediated effect on cAMP signaling in a cell-based bioassay. Taken together, our findings underscore the inhibitory effects of amiodarone and dronedarone on PT-induced cellular intoxication, providing valuable insights into drug repurposing for infectious disease management. [ABSTRACT FROM AUTHOR]

Key points The striking increase of uterine blood flow during pregnancy is essential for normal fetal development as well as for cardiovascular well‐being of the mother. Yet, the underlying mechanisms of pregnancy‐mediated vasodilatation of the uterine artery are not fully understood. In this study, we test the hypothesis that Rad, a monomeric G protein, is a novel regulatory mechanism in inhibiting CaV1.2 channel currents in uterine artery haemodynamic adaptation to pregnancy in a sheep model. We found that pregnancy significantly upregulates Rad expression and decreases CaV1.2 channel currents in uterine arterial smooth muscle cells. Rad knockdown ex vivo and in vivo increases CaV1.2 activity and channel window currents by reducing steady‐state inactivation in uterine arteries of pregnant sheep, recapitulating the phenotype of uterine arteries in non‐pregnant animals. Moreover, Rad knockdown in vivo in pregnant sheep enhances myogenic tone and phenylephrine‐induced vasoconstriction of uterine arteries. Whereas knockdown of Rad has no effect on mesenteric arterial CaV1.2 channel activity and mean arterial blood pressure, it significantly increases uterine vascular resistance and decreases uterine artery blood flow. Our study reveals a novel cause‐and‐effect mechanism of Rad in pregnancy‐induced suppression of CaV1.2 channel activity in uterine arteries to facilitate increased uterine blood flow, providing new insights into fundamental mechanisms of uterine haemodynamic adaptation to pregnancy. Pregnancy suppresses CaV1.2 channel currents in uterine arterial smooth muscle cells. Rad, a monomeric G protein, is upregulated in uterine arteries of pregnant sheep. Rad knockdown ex vivo or in vivo increases CaV1.2 channel currents in uterine arteries from pregnant ewes. In vivo knockdown of Rad elevates uterine vascular resistance and decreases uterine blood flow in pregnant sheep. The study reveals a novel mechanism of Rad in pregnancy‐induced suppression of CaV1.2 channel activity in uterine arterial haemodynamic adaptation to pregnancy. [ABSTRACT FROM AUTHOR]

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). LRRK2 protein contains two enzymatic domains: a GTPase (Roc-COR) and a kinase domain. Disease-causing mutations are found in both domains. Now, studies have focused largely on LRRK2 kinase activity, while attention to its GTPase function is limited. LRRK2 is a guanine nucleotide-binding protein, but the mechanism of direct regulation of its GTPase activity remains unclear and its physiological GEF is not known. Here, we identified CalDAG-GEFI (CDGI) as a physiological GEF for LRRK2. CDGI interacts with LRRK2 and increases its GDP to GTP exchange activity. CDGI modulates LRRK2 cellular functions and LRRK2-induced neurodegeneration in both LRRK2 Drosophila and mouse models. Together, this study identified the physiological GEF for LRRK2 and provides strong evidence that LRRK2 GTPase is regulated by GAPs and GEFs. The LRRK2 GTPase, GAP, or GEF activities have the potential to serve as therapeutic targets, which is distinct from the direct LRRK2 kinase inhibition. [ABSTRACT FROM AUTHOR]

Background: Heterotrimeric G proteins are crucial signaling molecules involved in cell signaling, plant development, and stress response. However, the genome-wide identification and analysis of G proteins in castor (Ricinus communis L.) have not been researched. Results: In this study, RcG-protein genes were identified using a sequence alignment method and analyzed by bioinformatics and expression analysis in response to salt stress. The results showed that a total of 9 G-protein family members were identified in the castor genome, which were classified into three subgroups, with the majority of RcG-proteins showing homology to soybean G-protein members. The promoter regions of all RcG-protein genes contained antioxidant response elements and ABA-responsive elements. Go enrichment analysis displayed that RcG-protein genes were involved in the G protein-coupled receptor signaling pathway, regulation of root development, and response to the bacterium. Real-time PCR showed varying responses of all RcG-protein genes to salt stress. RcGB1 was notably expressed in both roots and leaves under salt treatment, suggesting that it may be an essential gene associated with salt tolerance in the castor. Conclusions: This study offers a theoretical framework for exploring G-protein function and presents potential genetic assets for improving crop resilience through genetic enhancement. [ABSTRACT FROM AUTHOR]

Vision and magnetoreception in navigating songbirds are strongly connected as recent findings link a light dependent radical-pair mechanism in cryptochrome proteins to signalling pathways in cone photoreceptor cells. A previous yeast-two-hybrid screening approach identified six putative candidate proteins showing binding to cryptochrome type 4a. So far, only the interaction of the cone specific G-protein transducin α-subunit was investigated in more detail. In the present study, we compare the binding features of the G-protein α-subunit with those of another candidate from the yeast-two-hybrid screen, cellular retinol binding protein. Purified recombinant European robin retinol binding protein bound retinol with high affinity, displaying an EC50 of less than 5 nM, thereby demonstrating its functional state. We applied surface plasmon resonance and a Förster resonance transfer analysis to test for interactions between retinol binding protein and cryptochrome 4a. In the absence of retinol, we observed no robust binding events, which contrasts the strong interaction we observed between cryptochrome 4a and the G-protein α-subunit. We conclude that retinol binding protein is unlikely to be involved in the primary magnetosensory signalling cascade. [ABSTRACT FROM AUTHOR]

Shear stress sensing represents a vital mode of mechanosensation. 1 Previous efforts have mainly focused on characterizing how various cell types—for example, vascular endothelial cells—sense shear stress arising from fluid flow within the animal body. 1,2 How animals sense shear stress derived from their external environment, however, is not well understood. Here, using C. elegans as a model, we show that external fluid flow triggers behavioral responses in C. elegans , facilitating their navigation of the environment during swimming. Such behavioral responses primarily result from shear stress generated by fluid flow. The sensory neurons AWC, ASH, and ASER are the major shear stress-sensitive neurons, among which AWC shows the most robust response to shear stress and is required for shear stress-induced behavior. Mechanistically, shear stress signals are transduced by G protein signaling in AWC, with cGMP as the second messenger, culminating in the opening of cGMP-sensitive cyclic nucleotide-gated (CNG) channels and neuronal excitation. These studies demonstrate that C. elegans senses and responds to shear stress and characterize the underlying neural and molecular mechanisms. Our work helps establish C. elegans as a genetic model for studying shear stress sensing. • C. elegans senses and responds to shear stress generated by fluid flow • AWC, ASH, and ASER neurons are the major shear stress-sensitive neurons in C. elegans • Shear stress transduction in AWC is mediated by G protein signaling • Shear stress sensors in AWC are likely GPCRs How animals sense shear stress derived from their external environment is not well understood. Zhang et al. report that C. elegans senses and responds to shear stress generated by fluid flow in the environment and does so through G protein signaling. These results help to establish C. elegans as a genetic model for studying shear stress sensing. [ABSTRACT FROM AUTHOR]

Altered hepatic glucose fluxes are critical during the pathogenesis of type 2 diabetes. G protein-coupled receptors represent important regulators of hepatic glucose production. Recent studies have shown that hepatocytes express GPCRs that can couple to G12/13, a subfamily of heterotrimeric G proteins that has attracted relatively little attention in the past. Here we show, by analyzing several mutant mouse strains, that selective activation of hepatocyte G12/13 signaling leads to pronounced hyperglycemia and that this effect involves the stimulation of the ROCK1-JNK signaling cascade. Using both mouse and human hepatocytes, we also show that activation of endogenous sphingosine-1-phosphate type 1 receptors strongly promotes glucose release in a G12/13-dependent fashion. Studies with human liver samples indicate that hepatic GNA12 (encoding Gα12) expression levels positively correlate with indices of insulin resistance and impaired glucose homeostasis, consistent with a potential pathophysiological role of enhanced hepatic G12/13 signaling. Altered hepatic glucose fluxes play a key role in the pathogenesis of type 2 diabetes. Here, Pittala et al. show that activation of G12/13 signaling in hepatocytes enhances hepatic glucose production, leading to greatly increased blood glucose levels. [ABSTRACT FROM AUTHOR]

Glucose homeostasis is a complex process regulated by multiple organs and hormones, with insulin playing a central role. Recent evidence underscores the role of small GTP-binding proteins, particularly Rac1, in regulating insulin secretion and glucose uptake. However, the role of Rac1-regulatory proteins in these processes remains largely unexplored. In this study, we investigated the role of β2-chimaerin, a Rac1-specific GTPase-activating protein (GAP), in glucose homeostasis using whole-body β2-chimaerin knockout mice. Our data revealed that β2-chimaerin deficiency results in improved glucose tolerance and enhanced insulin sensitivity in mice. These metabolic effects were associated with increased insulin-induced AKT phosphorylation in the liver and activation of downstream pathways that regulate gluconeogenesis and glycogen synthesis. We show that insulin activates Rac1 in the liver. However, β2-chimaerin deletion did not significantly alter Rac1 activation in this organ, suggesting that β2-chimaerin regulates insulin signaling via a Rac1-independent mechanism. These findings expand our understanding of Rac1 regulation in glucose metabolism, and identify β2-chimaerin as a novel modulator of hepatic insulin signaling, with potential implications for the development of insulin resistance and diabetes. [ABSTRACT FROM AUTHOR]

GTP-binding proteins are essential molecular switches that regulate a wide range of cellular processes. Their function relies on the specific recognition and binding of guanine within their binding pockets. This study aims to elucidate the molecular determinants underlying this recognition. A large-scale data mining of the Protein Data Bank yielded 298 GTP-binding protein complexes, which provided a structural foundation for a systematic analysis of the intermolecular interactions that are responsible for the molecular recognition of guanine in proteins. It was found that multiple modes of non-bonded interactions including hydrogen bonding, cation–π interactions, and π–π stacking interactions are employed by GTP-binding proteins for binding. Subsequently, the strengths of non-bonded interaction energies between guanine and its surrounding protein residues were quantified by means of the double-hybrid DFT method B2PLYP-D3/cc-pVDZ. Hydrogen bonds, particularly those involving the N2 and O6 atoms of guanine, confer specificity to guanine recognition. Cation–π interactions between the guanine ring and basic residues (Lys and Arg) provide significant electrostatic stabilization. π–π stacking interactions with aromatic residues (Phe, Tyr, and Trp) further contribute to the overall binding affinity. This synergistic interplay of multiple interaction modes enables GTP-binding proteins to achieve high specificity and stability in guanine recognition, ultimately underpinning their crucial roles in cellular signaling and regulation. Notably, the NKXD motif, while historically considered crucial for guanine binding in GTP-binding proteins, is not universally required. Our study revealed significant variability in hydrogen bonding patterns, with many proteins lacking the NKXD motif but still effectively binding guanine through alternative arrangements of interacting residues. [ABSTRACT FROM AUTHOR]

Background: There is increasing interest in developing FPR2 agonists (compound 43, ACT‐389949 and BMS‐986235) as potential pro‐resolving therapeutics, with ACT‐389949 and BMS‐986235 having entered phase I clinical development. FPR2 activation leads to diverse downstream outputs. ACT‐389949 was observed to cause rapid tachyphylaxis, while BMS‐986235 and compound 43 induced cardioprotective effects in preclinical models. We aim to characterise the differences in ligand‐receptor engagement and downstream signalling and trafficking bias profile. Experimental Approach: Concentration‐response curves to G protein dissociation, β‐arrestin recruitment, receptor trafficking and second messenger signalling were generated using FPR2 ligands (BMS‐986235, ACT‐389949, compound 43 and WKYMVm), in HEK293A cells. Log(τ/KA) was obtained from the operational model for bias analysis using WKYMVm as a reference ligand. Docking of FPR2 ligands into the active FPR2 cryoEM structure (PDBID: 7T6S) was performed using ICM pro software. Key Results: Bias analysis revealed that WKYMVm and ACT‐389949 shared a very similar bias profile. In comparison, BMS‐986235 and compound 43 displayed approximately 5‐ to 50‐fold bias away from β‐arrestin recruitment and trafficking pathways, while being 35‐ to 60‐fold biased towards cAMP inhibition and pERK1/2. Molecular docking predicted key amino acid interactions at the FPR2 shared between WKYMVm and ACT‐389949, but not with BMS‐986235 and compound 43. Conclusion and Implications: In vitro characterisation demonstrated that WKYMVm and ACT‐389949 differ from BMS‐986235 and compound 43 in their signalling and protein coupling profile. This observation may be explained by differences in the ligand‐receptor interactions. In vitro characterisation provided significant insights into identifying the desired bias profile for FPR2‐based pharmacotherapy. [ABSTRACT FROM AUTHOR]

Unlike animals, plants are sessile organisms that cannot migrate to more favorable conditions and must constantly adapt to a variety of biotic and abiotic stresses. Therefore, plants exhibit developmental plasticity to cope, which is probably based on the underlying trade-off mechanism that allocates energy expenditure between growth and stress responses to achieve appropriate growth and development under different environmental conditions. Plant heterotrimeric G protein signaling plays a crucial role in the trade-off involved in the regulation of normal growth and stress adaptation. This review examines the composition and signaling processes of heterotrimeric G proteins in plants, detailing how they balance growth and adaptive responses in plant immunity and thermomorphogenesis through recent advances in the field. Understanding the trade-offs associated with plant G protein signaling will have significant implications for agricultural innovation, particularly in the development of crops with improved resilience and minimal growth penalties under environmental stress. [ABSTRACT FROM AUTHOR]

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments. [ABSTRACT FROM AUTHOR]

Eukaryotes have two paralogous developmentally regulated GTP-binding (DRG) proteins: DRG1 and DRG2, both of which have a conserved binding partner called DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively. DFRPs are important for the function of DRGs and interact with their respective DRG via a conserved region called the DFRP domain. Despite being highly similar, DRG1 and DRG2 have strict binding specificity for their respective DFRP. Using AlphaFold generated structure models of the human DRG/DFRP complexes, we have biochemically characterized their interactions and identified interface residues involved in determining specificity. This analysis revealed that as few as five mutations in DRG1 can switch binding from DFRP1 to DFRP2. Moreover, while DFRP1 binding confers increased stability and GTPase activity to DRG1, DFRP2 binding only supports increased stability. Overall, this work provides new insight into the structural determinants responsible for the binding specificities of the DRG/DFRP complexes. [Display omitted] • DRG1 and DRG2 are paralogs that have different specificities for DFRP-binding partners • Interface residues in DRG1 facilitate the specific binding to DFRP1 • Mutation of DRG1 interface residues can switch binding specificity from DFRP1 to DFRP2 • DFRP1 binding but not DFRP2 binding can stimulate DRG1 GTPase activity Based on AlphaFold models and sequence analyses, Westrip et al. identified binding specificity determining residues in the developmentally regulated GTP-binding protein DRG1. Mutation of these residues can switch DRG1 binding from the DRG family regulatory protein 1 (DFRP1) toward DFRP2, but only DRFP1 binding enhanced the GTPase activity of DRG1. [ABSTRACT FROM AUTHOR]

While it is known that temperature sensors trigger calcium (Ca2+) signaling to confer cold tolerance in cells, less is known about sensors that couple with other secondary messengers. Here, we identify a cold sensor complex of CHILLING-TOLERANCE DIVERGENCE 6 (COLD6) and osmotin-like 1 (OSM1), which triggers 2′,3′-cyclic adenosine monophosphate (2′,3′-cAMP) production to enhance cold tolerance in rice. COLD6, which is encoded by a major quantitative trait locus (QTL) gene, interacts with the rice G protein α subunit (RGA1) at the plasma membrane under normal conditions. Upon exposure to chilling, cold-induced OSM1 binds to COLD6, kicking out RGA1 from interaction. This triggers an elevation of 2′,3′-cAMP levels for enhancing chilling tolerance. Genetic data show that COLD6 negatively regulates cold tolerance and functionally depends on OSM1 in chilling stress. COLD6 alleles were selected during rice domestication. Knockout and natural variation of COLD6 in hybrid rice enhanced chilling tolerance, hinting design potential for breeding. This highlighted a module triggering 2′,3′-cAMP to improve chilling tolerance in crops. [Display omitted] • A major QTL, COLD6 , negatively regulates cold tolerance in rice • COLD6 is associated with environmental adaptation during rice domestication • COLD6 interacts with osmotin-like 1 (OSM1) protein for cold tolerance • COLD6-OSM1 module triggers 2′,3′-cAMP signaling, enhancing cold tolerance in rice While roles for Ca2+ signaling in cold tolerance are known, less is known about sensors coupled with other secondary messengers. Luo et al. identify a chilling sensor complex COLD6, encoded by a major QTL gene, and OSM1, which triggers 2′,3′-cAMP signaling to enhance cold tolerance in rice. [ABSTRACT FROM AUTHOR]

Transient receptor potential canonical (TRPC)5 channel is a non-selective cation channel that plays a significant role in membrane depolarization and calcium influx. TRPC5 not only forms homotetramers itself but also heterotetramers with TRPC1. However, accurately testing and confirming these heterotetrameric channels at specific ratios has proven challenging. Therefore, creating heteromeric concatemers of TRPC5 and TRPC1 with a fixed stoichiometry of 1:1 becomes essential. This study aims to meticulously identify and reaffirm the properties of TRPC5 homomers and heteromers with a 1:1 fixed stoichiometry to determine the optimal ratio for the TRPC1/5 heterotetramer. The overall characteristics were consistent with those of the previous studies, but several specific features were different. The TRPC1–TRPC5 concatemer is activated by Englerin A and GiQL, whereas carbachol alone does not trigger its activation. Additionally, GqQL significantly inhibited the current when co-expressed with the concatemer. Interestingly, carbachol can activate the TRPC1–TRPC5 concatemer in the presence of internal GTPγS, highlighting the influence of intracellular signaling conditions on its activation. Meanwhile, the TRPC5–TRPC5 concatemer is responsive to both carbachol and Englerin A. In conclusion, we provide evidence that the TRPC1–TRPC5 heteromeric concatemer with fixed stoichiometry need specific conditions to respond to carbachol, whereas the TRPC5–TRPC5 homomeric concatemer responds physiologically to carbachol. Additional research may be necessary to ascertain the optimal stoichiometry for the TRPC1–TRPC5 concatemer to enhance its electrophysiological properties. [ABSTRACT FROM AUTHOR]

Aims: High non-fasting triglycerides (TG) concentration is linked to the development of atherosclerosis, and physical activity is commonly recommended to reduce postprandial TG concentration and cardiovascular diseases. Previous studies have demonstrated that acute whole-body (walking and running) or lower-body (leg cycling) aerobic exercise reduces postprandial TG. However, it is unclear whether upper-body exercise (i.e. arm-cranking) with sufficient energy expenditure lowers postprandial TG. Therefore, this study aimed to evaluate the effects of energy-matched upper- and lower-body exercises on postprandial TG concentrations the next day in healthy young men. Method and Materials: Fifteen healthy young men (age 22.5 ± 1.7 years, height 173.8 ± 5.7 cm, body mass 68.2 ± 8.5 kg, peak oxygen uptake 48.0 ± 5.5 mL/min/kg and physically active) participated in a three-arm crossover trials: 1) arm-cranking, 2) leg-cycling exercise at 70% of mode-specific peak oxygen uptake to induce a net energy expenditure of 1,255 kJ, or 3) rested between 16:00 and 17:00 h on day 1 and consumed two standardised meals for breakfast (10:00 h) and lunch (13:00 h) on day 2. The mean macronutrient content of the breakfast was 44.9 ± 5.6 g fat, 104.8 ± 13.0 g carbohydrate, and 29.4 ± 3.6 g protein, which provided 3.95 ± 0.49 MJ energy (43% fat, 45% carbohydrate, and 12% protein), and that of the lunch was 45.2 ± 5.6 g fat, 106.7 ± 13.2 g carbohydrate, and 33.9 ± 4.2 g protein, which provided 4.06 ± 0.50 MJ energy (42% fat, 44% carbohydrate, and 14% protein). Results: Time-averaged postprandial serum TG concentrations over 8 h differed among trials (main effect of trial p < 0.001) and were lower in the upper- and lower-body exercise trials than in the control trial (1.46 ± 0.54 vs. 1.50 ± 0.69 vs. 1.79 ± 0.83 mmol/L, respectively). The incremental TG area under the curve (AUC) (main effect of trial, p = 0.012) was 39% and 37% higher in the control trial than in the upper- and lower-body exercise trials (p = 0.025 and p = 0.033, respectively). There were no significant differences in incremental TG AUC between the upper- and lower-body exercise trials. Conclusion: An acute bout of energy-matched upper- and lower-body exercises similarly lowered postprandial TG concentrations the following day in healthy young men. Trial registration number: UMIN000045449. Date of registration: 10 September 2021. [ABSTRACT FROM AUTHOR]

This study aimed to assess the effect of Persian gum (PG) and microbial transglutaminase enzyme (MTGase) on some physicochemical and microstructural characteristics of low‐fat ultrafiltrated Iranian white cheese during 60 days of cold storage. To manufacture the low‐fat cheese samples, PG (0%, 0.25%, and 0.5%) and MTGase (0, 0.5 and 1 U/g of protein) were used and cheeses (low‐ and full‐fat) without PG and MTGase were considered as controls. The obtained results revealed that the treated samples with PG had a higher titratable acidity, moisture content, and moisture‐to‐protein ratio (M:P), but a lower protein content as compared to the low‐fat control (p ≤ 0.05). Furthermore, the cheese samples treated with the higher level of MTGase (i.e., 1 U/g of protein) had a higher protein and fat content (p ≤ 0.05). Methyl palmitate (C16:0) and methyl oleate (C18:1) were found to be the most abundant fatty acids in the tested cheeses. Additionally, scanning electron micrographs revealed that addition of PG and MTGase improved the microstructure of the samples, so that the sample containing 0.5 U enzyme and 0.5% gum was more similar to its full‐fat counterpart and obtained the highest overall acceptance score among the cheeses. Therefore, our findings revealed that with incorporation of PG and MTGase, a low‐fat ultrafiltrated white cheese could be produced with an appropriate functional, physicochemical, and microstructural characteristics comparable to the full‐fat product. [ABSTRACT FROM AUTHOR]

The Ras-related GTP-binding protein D (RRAGD) gene plays a crucial role in cellular processes. Recently, RRAGD variants found in patients have been implicated in a novel disorder with kidney tubulopathy and dilated cardiomyopathy. Currently, the consequences of RRAGD variants at the organismal level are unknown. Therefore, this study investigated the impact of RRAGD variants on cardiac function using a zebrafish embryo model. Furthermore, the potential usage of rapamycin, an mTOR inhibitor, as a therapy was assessed in this model. Zebrafish embryos were injected with RRAGD p.S76L and p.P119R cRNA and the resulting heart phenotypes were studied. Our findings reveal that overexpression of RRAGD mutants resulted in decreased ventricular fractional shortening, ejection fraction, and pericardial swelling. In RRAGD S76L-injected embryos, lower survival and heartbeat were observed, whereas survival was unaffected in RRAGD P119R embryos. These observations were reversible following therapy with the mTOR inhibitor rapamycin. Moreover, no effects on electrolyte homeostasis were observed. Together, these findings indicate a crucial role of RRAGD in cardiac function. In the future, the molecular mechanisms by which RRAGD variants result in cardiac dysfunction and if the effects of rapamycin are specific for RRAGD-dependent cardiomyopathy should be studied in clinical studies. NEW & NOTEWORTHY: The resultant heart-associated phenotypes in the zebrafish embryos of this study serve as a valuable experimental model for this rare cardiomyopathy. Moreover, the potential therapeutic property of rapamycin in cardiac dysfunctions was highlighted, making this study a pivotal step toward prospective clinical applications. [ABSTRACT FROM AUTHOR]

Adhesion G protein-coupled receptors are unique molecules. They are able to transmit classical signals via G protein activation as well as mediate functions solely through their extracellular N termini, completely independently of the seven transmembrane helices domain and the C terminus. This dual mode of action is highly unusual for G protein-coupled receptors and allows for a plethora of possible cellular consequences. However, the physiological implications and molecular details of this N terminus-mediated signaling are poorly understood. Here, we show that several distinct seven transmembrane helices domain-independent/ trans functions of the adhesion G protein-coupled receptor latrophilin homolog latrophilin-1 in the nematode Caenorhabditis elegans together regulate reproduction: sperm guidance, ovulation, and germ cell apoptosis. In these contexts, the receptor elicits its functions in a noncell autonomous manner. The functions might be realized through alternative splicing of the receptor specifically generating N terminus-only variants. Thus, our findings shed light on the versatility of seven transmembrane helices domain-independent/N terminus-only/ trans functions of adhesion G protein-coupled receptor and discuss possible molecular details. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are sensors for the G protein complex to sense changes in environmental factors and molecular switches for G protein complex signal transduction. In this study, the homologous gene of GPCR-like proteins was identified from maize and named as ZmCOLD1. Subcellular analysis showed that the ZmCOLD1 protein is localized to the cell membrane and endoplasmic reticulum. A CRISPR/Cas9 knock-out line of ZmCOLD1 was further created and its plant height was significantly lower than the wild-type maize at both the seedling and adult stages. Histological analysis showed that the increased cell number but significantly smaller cell size may result in dwarfing of zmcold1, indicating that the ZmCOLD1 gene could regulate plant height development by affecting the cell division process. Additionally, ZmCOLD1 was verified to interact with the maize Gα subunit, ZmCT2, though the central hydrophilic loop domain by in vivo and in vitro methods. Abscisic acid (ABA) sensitivity analysis by seed germination assays exhibited that zmcold1 were hypersensitive to ABA, indicating its important roles in ABA signaling. Finally, transcriptome analysis was performed to investigate the transcriptional change in zmcold1 mutant. Overall, ZmCOLD1 functions as a GPCR-like protein and an important regulator to plant height. [ABSTRACT FROM AUTHOR]

Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). The neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6fl/fl; DAT-iCreER mice to determine the role of RGS6 in DA neurons on EtOH consumption, reward, and relapse behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse ventral tegmental area (VTA), and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6−/− mice. Strikingly, RGS6 loss from DA neurons before or after EtOH behavioral reward is established significantly reduced (~ 50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies identify DA neurons as the locus of RGS6 action in promoting EtOH consumption, preference, reward, and relapse. RGS6 is unique among R7 RGS proteins in promoting rather than suppressing behavioral responses to drugs of abuse and to modulate EtOH behavioral reward. This is a result of RGS6's pre-synaptic actions that we hypothesize promote VTA DA transmission by suppressing GPCR-Gαi/o-DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH. [ABSTRACT FROM AUTHOR]

Therapies that target the serotonin 2A receptor (5-HT2AR) are promising. However, probes are needed to better understand the role of 5-HT2AR. Here, we design and synthesize a photoswitch and photoswitchable 5-HT2AR ligand based on highly potent agonist TCB-2 and arylazopyrazole, which also boasts photoswitchable G protein vs. β-arrestin pathway bias. [ABSTRACT FROM AUTHOR]

Introduction: Major depressive disorder (MDD), characterized by severe neuropsychiatric symptoms and significant cognitive deficits, continues to present both etiological and therapeutic challenges. However, the specific underlying mechanisms and therapeutic targets remain unclear. Methods: We analyzed human postmortem dorsolateral prefrontal cortex (dlPFC) samples from MDD patients using datasets GSE53987 and GSE54568, identifying three key genes: AGA, FBXO38, and RGS5. To model depressive-like behavior, we employed chronic social defeat stress (CSDS) and subsequently measured the expression of AGA, FBXO38, and RGS5 in the dlPFC using qPCR and Western blot analysis following CSDS exposure. Results: CSDS significantly induced depressive-like behavior, and both the protein and transcriptional expression levels of AGA, FBXO38, and RGS5 in the dlPFC of mice were markedly reduced a0fter stress, consistent with findings from datasets GSE53987 and GSE54568. Conclusion: Our research suggests that AGA, FBXO38, and RGS5 are potential biomarkers for MDD and could serve as valuable targets for MDD risk prediction. [ABSTRACT FROM AUTHOR]

G protein subunit Gamma 5 (GNG5) has been found to be involved in regulating glioma progression. However, its function and mechanism in glioblastoma (GBM) progression need to be further elucidated. GBM cell proliferation, apoptosis, invasion and stemness were assessed by cell counting kit 8 assay, EdU assay, flow cytometry, transwell assay and sphere formation assay. The mRNA and protein levels of GNG5 and Yin Yang 1 (YY1) were determined by quantitative real-time PCR and western blot (WB). Detection of the glucose consumption, lactate production and ATP/ADP ratios were used to assess cell glycolysis. Besides, Wnt/β-catenin pathway-related protein levels were examined by WB. Mice xenograft model was also constructed to explore GNG5 roles in vivo. GNG5 was highly expressed in GBM, and its silencing inhibited GBM cell proliferation, invasion, stemness and glycolysis, while promoted apoptosis. Transcription factor YY1 could bind to the GNG5 promoter region and induce its expression. GNG5 overexpression reversed the inhibitory effects of YY1 silencing on GBM cell growth, invasion, stemness and glycolysis. YY1/GNG5 axis could activate the Wnt/β-catenin pathway, and Wnt/β-catenin pathway agonists SKL2001 could revert the effects of GNG5 silencing on GBM cell progression. Furthermore, GNG5 facilitated GBM tumor growth by mediating the Wnt/β-catenin pathway. YY1-mediated GNG5 promoted GBM progression through the Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

The small G protein Arf1 has been identified as playing a selective role in supporting cancer stem cells (CSCs), making it an attractive target for cancer therapy. However, the current Arf1 inhibitors have limited translational potential due to their high toxicity and low specificity. In this study, two new potent small‐molecule inhibitors of Arf1, identified as DU101 and DU102, for cancer therapy are introduced. Preclinical tumor models demonstrate that these inhibitors triggered a cascade of aging in CSCs and enhance anti‐tumor immunity in mouse cancer and PDX models. Through single‐cell sequencing, the remodeling of the tumor immune microenvironment induced by these new Arf1 inhibitors is analyzed and an increase in tumor‐associated CD8+ CD4+ double‐positive T (DPT) cells is identified. These DPT cells exhibit superior features of active CD8 single‐positive T cells and a higher percentage of TCF1+PD‐1+, characteristic of stem‐like T cells. The frequency of tumor‐infiltrating stem‐like DPT cells correlates with better disease‐free survival (DFS) in cancer patients, indicating that these inhibitors may offer a novel cancer immunotherapy strategy by converting the cold tumor immune microenvironment into a hot one, thus expanding the potential for immunotherapy in cancer patients. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications Fentanyls and nitazenes are μ‐opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we determined the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues, compared to morphine and DAMGO.In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ receptors to assess Gi protein activation and β‐arrestin 2 recruitment.The apparent rate of agonist dissociation from the μ receptor varied: morphine, DAMGO, alfentanil and fentanyl dissociated rapidly, whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow apparent rate of dissociation was not because of G protein receptor kinase‐mediated arrestin recruitment as its apparent rate of dissociation was not increased by inhibition of G protein‐coupled receptor kinases (GRKs) with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments.With fentanyls and nitazenes that slowly dissociate from the μ receptor, antagonism by naloxone is pseudo‐competitive. In overdoses involving fentanyls and nitazenes, higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose. [ABSTRACT FROM AUTHOR]

The treatment of carbendazim-contaminated effluents is a challenge because of its complex composition and toxicity. A promising solution lies in biodegradation and the fungus Actinomucor elegans LBM 290 shows significant potential in this regard. Thus, the aim of this study was to biodegrade MBC by A. elegans LBM 290 in a liquid medium addressing the changes in the fungal morphology and protein production. The fungus A. elegans LBM 290 efficiently remove the fungicide carbendazim, with 86.6% removal within 8 days. This degradation is a combination of biodegradation (24.54%) and adsorption (62.08%). Exposure to carbendazim negatively affected the fungus, causing a decrease in biomass and morphological changes. Proteomic analysis revealed the fungal response to carbendazim stress through increased production of Cu–Zn superoxide dismutase, an antioxidant enzyme that combats oxidative stress, and the presence of a G protein subunit, suggesting participation in stress signaling pathways. These findings contribute to understanding the strategies of A. elegans LBM 290 to cope with carbendazim exposure in aquatic environments. [ABSTRACT FROM AUTHOR]

Objectives Methods Results Conclusions Patients with polypoidal choroidal vasculopathy (PCV) exhibit variability in response to anti‐VEGF therapy. This study aimed to analyse the aqueous humour proteomic profiles of PCV patients and provide preliminary insights for the identification of biomarkers associated with anti‐VEGF drug responsiveness.PCV patients who were treatment‐naïve or untreated for more than 3 months were prospectively recruited from two hospitals in Beijing and Tianjin. Based on the relative changes in central macular thickness (ΔCMT/baseline‐CMT) before and after anti‐VEGF treatment, the PCV patients were divided into a good response (GR) group (≤−25%) and a poor response (PR) group (>−25%). Aqueous humour proteomics was performed by the Data‐independent Acquisition‐Mass Spectrometry (DIA‐MS) method, and differentially expressed proteins (DEPs) analysis between the different PCV groups and the control group was conducted. Key DEPs were selected for preliminary validation in the aqueous humour using the Luminex method retrospectively.A total of 31 PCV patients (31 eyes) were included, 13 in the GR group and 18 in the PR group. A total of 414 DEPs were identified, including 36 significantly upregulated proteins, such as G protein regulatory factor 10 (RGS10), podocin (PODN) and epidermal growth factor (EGF), and 32 downregulated proteins, including RAB11FIP4 (Rab11 family‐interacting protein 4), α‐synuclein (SNCA), haemoglobin subunit δ (HBD) and interleukin 6 (IL6). Compared to the cataract control group (10 eyes), 134 proteins were significantly upregulated, and 72 were downregulated. KEGG pathway enrichment analysis revealed that the GR and PR groups differ in terms of cell communication, and cell signal transduction. Protein–protein interaction analysis revealed interactions between EGF and various DEPs. Validation of aqueous humour proteins using the Luminex method revealed that changes in the levels of EGF were associated with the anti‐VEGF treatment response in PCV patients.PCV patients with good or poor anti‐VEGF responses exhibit distinct aqueous humour proteomic profiles. Aqueous EGF may serve as a biomarker for the ‘precise treatment’ of PCV. [ABSTRACT FROM AUTHOR]

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF. [ABSTRACT FROM AUTHOR]

Endothelin type B receptor (ETBR) plays a crucial role in regulating blood pressure and humoral homeostasis, making it an important therapeutic target for related diseases. ETBR activation by the endogenous peptide hormones endothelin (ET)−1–3 stimulates several signaling pathways, including Gs, Gi/o, Gq/11, G12/13, and β-arrestin. Although the conserved NPxxY motif in transmembrane helix 7 (TM7) is important during GPCR activation, ETBR possesses the lesser known NPxxL motif. In this study, we present the cryo-EM structure of the ETBR–Gi complex, complemented by MD simulations and functional studies. These investigations reveal an unusual movement of TM7 to the intracellular side during ETBR activation and the essential roles of the diverse NPxxL motif in stabilizing the active conformation of ETBR and organizing the assembly of the binding pocket for the α5 helix of Gi protein. These findings enhance our understanding of the interactions between GPCRs and G proteins, thereby advancing the development of therapeutic strategies. Tani et al. present the cryo-EM structure of the endothelin type B receptor (ETBR) in complex with the Gi protein, emphasizing the movements of transmembrane helix 7 during ETBR activation. The study underscores the crucial role of the NPxxL motif in stabilizing the receptor's active state and organizing the Gi protein binding site, enhancing our understanding of GPCR activation and aiding therapeutic development. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Knockdown of THOC1 reduces the proliferation of hepatocellular carcinoma and increases the sensitivity to cisplatin." The author identified errors in two images in the original article, but these errors do not affect the overall results, discussion, or conclusion of the article. The corrected figures are provided in the correction notice. The publisher remains neutral with regard to jurisdictional claims and institutional affiliations. [Extracted from the article]

In vivo , the temperature inside preovulatory follicles of cows is approximately 1 °C lower than rectal temperature. However, standard bovine oocyte in vitro maturation (IVM) protocols use 38.5 °C based on rectal temperature. This study evaluated the effect of reducing IVM temperature to 37.5 °C on the proteomic profile of oocytes compared to the routine 38.5 °C. Nuclear maturation rate and cumulus cell (CC) expansion (30 COCs per group, 21 replicates) were assessed by observing the first polar body and using a subjective scoring method (0–4). Total nitrite concentrations in the culture medium were measured using the Griess method. Differential proteomics was performed using LC-MS/MS on pooled oocyte samples (500 matured oocytes per group, three replicates), followed by gene ontology enrichment, protein-protein interaction, and putative miRNA target analyses. No significant differences were observed between the groups in nuclear maturation, CC expansion, or nitrite concentration (P > 0.05). A total of 806 proteins were identified, with 7 up-regulated and 12 down-regulated in the treatment group compared to the control. Additionally, 12 proteins were unique to the control group, and 8 were unique to the treatment group. IVM at 37.5 °C resulted in the upregulation of proteins involved in protein folding and GTP binding, and the downregulation of enzymes with oxidoreductase activity and proteins involved in cytoskeletal fiber formation. Furthermore, 43 bovine miRNAs potentially regulating these genes (DES, HMOX2, KRT75, FARSA, IDH2, CARHSP1) were identified. We conclude that IVM of bovine oocytes at 37.5 °C induces significant proteomic changes without impacting nuclear maturation, cumulus cell expansion, or nitrite concentration in the IVM medium. • Temperature reduction during IVM altered the proteomic profile but did not affect nuclear maturation. • The total nitrite concentration in the IVM medium of COCs was similar between groups cultured at 38.5 °C and 37.5 °C • Proteomic analysis identified 12 unique proteins in the control group and 8 unique proteins in the treatment group • IVM at 37.5 °C resulted in the upregulation of proteins involved in protein folding and GTP binding. • 43 bovine miRNAs were found to potentially regulate key genes affected by the temperature change during IVM of bovine oocytes. [ABSTRACT FROM AUTHOR]

Aims: High non-fasting triglycerides (TG) concentration is linked to the development of atherosclerosis, and physical activity is commonly recommended to reduce postprandial TG concentration and cardiovascular diseases. Previous studies have demonstrated that acute whole-body (walking and running) or lower-body (leg cycling) aerobic exercise reduces postprandial TG. However, it is unclear whether upper-body exercise (i.e. arm-cranking) with sufficient energy expenditure lowers postprandial TG. Therefore, this study aimed to evaluate the effects of energy-matched upper- and lowerbody exercises on postprandial TG concentrations the next day in healthy young men. Method and Materials: Fifteen healthy young men (age 22.5 ± 1.7 years, height 173.8 ± 5.7 cm, body mass 68.2 ± 8.5 kg, peak oxygen uptake 48.0 ± 5.5 mL/min/kg and physically active) participated in a three-arm crossover trials: 1) arm-cranking, 2) leg-cycling exercise at 70% of mode-specific peak oxygen uptake to induce a net energy expenditure of 1,255 kJ, or 3) rested between 16:00 and 17:00 h on day 1 and consumed two standardised meals for breakfast (10:00 h) and lunch (13:00 h) on day 2. The mean macronutrient content of the breakfast was 44.9 ± 5.6 g fat, 104.8 ± 13.0 g carbohydrate, and 29.4 ± 3.6 g protein, which provided 3.95 ± 0.49 MJ energy (43% fat, 45% carbohydrate, and 12% protein), and that of the lunch was 45.2 ± 5.6 g fat, 106.7 ± 13.2 g carbohydrate, and 33.9 ± 4.2 g protein, which provided 4.06 ± 0.50 MJ energy (42% fat, 44% carbohydrate, and 14% protein). Results: Time-averaged postprandial serum TG concentrations over 8 h differed among trials (main effect of trial p < 0.001) and were lower in the upper- and lower-body exercise trials than in the control trial (1.46 ± 0.54 vs. 1.50 ± 0.69 vs. 1.79 ± 0.83 mmol/L, respectively). The incremental TG area under the curve (AUC) (main effect of trial, p = 0.012) was 39% and 37% higher in the control trial than in the upper- and lower-body exercise trials (p = 0.025 and p = 0.033, respectively). There were no significant differences in incremental TG AUC between the upper- and lower-body exercise trials. Conclusion: An acute bout of energy-matched upper- and lower-body exercises similarly lowered postprandial TG concentrations the following day in healthy young men. Trial registration number: UMIN000045449. Date of registration: 10 September 2021. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the correlation between Rab10 (GTP binding protein RAB10), TLR4 (Toll-like receptor 4), and NF-κB (nuclear factor kappa-B) levels and therapeutic effects in peripheral blood of patients with breast cancer after surgery. The study included 160 patients with stage I-III breast cancer who underwent surgical treatment at our hospital's Department of Breast Surgery and Oncology between January 2021 and June 2021. ELISA was used to assess Rab10, TLR4, and NF-κB levels in peripheral blood. Based on their levels of Rab10, TLR4, and NF-κB in peripheral blood, participants were categorized into two groups: the low marker expression group (72 participants with relatively low expression of Rab10, TLR4, and NF-κB: Rab10<2.0ng/ml; TLR4<2.75ng/ml; NF-κB<3.5ng/ml) and the high marker expression group (88 participants with relatively high expression: Rab10 ≥ 2.0 ng/ml; TLR4 ≥ 2.75ng/ml; NF-κB ≥ 3.5ng/ml). All participants provided informed consent to participate the study. The baseline data of the two groups of patients, the presence or absence of lymph node metastasis and recurrence within 3 years after surgery, as well as the survival status within 3 years after surgery (including median overall survival and median progression-free survival) were statistically analyzed. The expressions of Rab10, TLR4, and NF-κB in the peripheral blood of patients were detected through enzyme-linked immunosorbent assay (ELISA). Kendall's tau-b correlation analysis was conducted to examine the relationship between the expressions of Rab10, TLR4, and NF-κB and the therapeutic effects outcomes. The levels of Rab10, TLR4, and NF - κ B in peripheral blood of the high marker expression group were higher than those of the low marker expression group (Rab10: 1.87 ± 0.18 vs. 3.15 ± 0.24 ng/ml; TLR4: 2.17 ± 0.20 vs. 3.26 ± 0.25 ng/ml); NF-κB: 2.68 ± 0.27 vs. 4.63 ± 0.30 ng/ml; P < 0.05). Analyzing the relationship between patient staging and Rab10, TLR4, and NF - κ B expression, the number of patients in high marker expression group III-IV increased compared to the low marker expression group (54.55% vs. 36.12%; P < 0.05), while the number of patients in high marker expression group I-II decreased compared to the low marker expression group (45.45% vs. 63.88%; P < 0.05). It was found that the number of patients with no recurrence or metastasis in the high marker expression group decreased compared to the low marker expression group (56.81% vs. 73.61%; P < 0.05), while the number of patients with recurrence or metastasis in the high marker expression group increased compared to the low marker expression group (43.19% vs. 26.39%; P < 0.05). The median overall survival and median progression free survival in the high marker expression group were shorter than those in the low marker expression group (median overall survival: 21.45 ± 2.68 months vs. 28.38 ± 3.44 months; median progression free survival: 15.25 ± 2.37 vs. 20.72 ± 2.58 months; P < 0.05). Kendall's tau-b correlation indicated a positive correlation between the expressions of Rab10, TLR4, and NF-κB and a poor therapeutic effects (P < 0.05), suggesting that elevated levels of Rab10, TLR4, and NF-κB may lead to a worsened therapeutic effects. There is a significant correlation between the presence of Rab10, TLR4, and NF-κB in the peripheral blood of breast cancer patients. Elevated levels of Rab10, TLR4, and NF-κB are linked to an increased risk of recurrence, metastasis, reduced overall survival, and progression-free survival. [ABSTRACT FROM AUTHOR]

Understanding the kinetics of LSD in receptors and subsequent induced signaling is crucial for comprehending both the psychoactive and therapeutic effects of LSD. Despite extensive research on LSD's interactions with serotonin 2A and 2B receptors, its behavior on other targets, including dopamine receptors, has remained elusive. Here, we present cryo-EM structures of LSD/PF6142-bound dopamine D 1 receptor (DRD1)-legobody complexes, accompanied by a β-arrestin-mimicking nanobody, NBA3, shedding light on the determinants of G protein coupling versus β-arrestin coupling. Structural analysis unveils a distinctive binding mode of LSD in DRD1, particularly with the ergoline moiety oriented toward TM4. Kinetic investigations uncover an exceptionally rapid dissociation rate of LSD in DRD1, attributed to the flexibility of extracellular loop 2 (ECL2). Moreover, G protein can stabilize ECL2 conformation, leading to a significant slowdown in ligand's dissociation rate. These findings establish a solid foundation for further exploration of G protein-coupled receptor (GPCR) dynamics and their relevance to signal transduction. [Display omitted] • Structures of DRD1-legobody complexes aided by a β-arrestin-mimicking nanobody NBA3 • Unique binding poses of LSD at DRD1 • Attractive versus repulsive interactions of β-arrestin and G protein with GPCR • Stability of G protein to the GPCR's ECL2 in the extracellular domain LSD-bound DRD1-legobody complex structure expands the understanding of LSD binding poses across different GPCRs and provides a robust foundation for investigating the kinetics of LSD and its implications for signal transduction and the resultant profound effects. [ABSTRACT FROM AUTHOR]

Transient receptor potential canonical (TRPC)5 channel is a non-selective cation channel that plays a significant role in membrane depolarization and calcium influx. TRPC5 not only forms homotetramers itself but also heterotetramers with TRPC1. However, accurately testing and confirming these heterotetrameric channels at specific ratios has proven challenging. Therefore, creating heteromeric concatemers of TRPC5 and TRPC1 with a fixed stoichiometry of 1:1 becomes essential. This study aims to meticulously identify and reaffirm the properties of TRPC5 homomers and heteromers with a 1:1 fixed stoichiometry to determine the optimal ratio for the TRPC1/5 heterotetramer. The overall characteristics were consistent with those of the previous studies, but several specific features were different. The TRPC1-TRPC5 concatemer is activated by Englerin A and GiQL, whereas carbachol alone does not trigger its activation. Additionally, GqQL significantly inhibited the current when co-expressed with the concatemer. Interestingly, carbachol can activate the TRPC1-TRPC5 concatemer in the presence of internal GTPγS, highlighting the influence of intracellular signaling conditions on its activation. Meanwhile, the TRPC5-TRPC5 concatemer is responsive to both carbachol and Englerin A. In conclusion, we provide evidence that the TRPC1-TRPC5 heteromeric concatemer with fixed stoichiometry need specific conditions to respond to carbachol, whereas the TRPC5-TRPC5 homomeric concatemer responds physiologically to carbachol. Additional research may be necessary to ascertain the optimal stoichiometry for the TRPC1-TRPC5 concatemer to enhance its electrophysiological properties. [ABSTRACT FROM AUTHOR]

Background: Asthma is a heterogeneous inflammatory disease with two main clinical endotypes: type 2 (T2) high and low asthma. The plasticity and autophagy in dendritic cells (DCs) influence T helper (Th)2 or Th17 differentiation to regulate asthma endotypes. Enhanced autophagy in DCs fosters Th2 differentiation in allergic environments, while reduced autophagy favors Th17 cell differentiation in sensitized and infected environments. Autophagy regulation in DCs involves interaction with various pathways like G protein‐coupled receptor (GPCR), mammalian target of rapamycin (mTOR), or phosphoinositide 3‐kinase (PI3K) pathway. However, specific molecules within DCs influencing asthma endotypes remain unclear. Methods: Gene expression data series (GSE) 64896, 6858, 2276, and 55247 were obtained from gene expression omnibus (GEO) database. Differentially expressed genes (DEGs) between CD103+ and CD11b+ DCs after induction by ovalbumin (OVA) and lipopolysaccharide (LPS) were analyzed using GEO2R. DEGs were examined through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein–protein interaction (PPI) analyses. The hub gene network was construct with STRING database and Cytoscape. Autophagy differences in DCs and the selected hub gene in GSE6858, GSE2276, and GSE55247 were evaluated using student t tests. Results: Our analysis identified 635 upregulated and 360 downregulated genes in CD11b+ DCs, compared to CD103+ DCs. These DEGs were associated with "PI3K‐AKT signaling pathway," "Ras signaling pathway," and so forth. Thirty‐five hub genes were identified, in which G protein subunit gamma transduction protein 2 (Gngt2) in CD11b+ DCs exhibited a relatively specific increase in expression associated with autophagy defects under the induction environment similar to T2 low asthma model. No significant difference was found in lung Gngt2 expression between T2 high asthma model and control group. Conclusion: Our analysis suggested Gngt2 acted as an adapter molecule that inhibited autophagy, promoting Th17‐mediated airway inflammation via the GPCR pathway in a T2 low asthma mice model. Targeting this pathway provides new asthma treatment strategies in preclinical research. [ABSTRACT FROM AUTHOR]

Halorhodospira (Hlr.) halochloris is a triply extremophilic phototrophic purple sulfur bacterium, as it is thermophilic, alkaliphilic, and extremely halophilic. The light‐harvesting‐reaction center (LH1–RC) core complex of this bacterium displays an LH1‐Qy transition at 1,016 nm, which is the lowest‐energy wavelength absorption among all known phototrophs. Here we report the cryo‐EM structure of the LH1–RC at 2.42 Å resolution. The LH1 complex forms a tricyclic ring structure composed of 16 αβγ‐polypeptides and one αβ‐heterodimer around the RC. From the cryo‐EM density map, two previously unrecognized integral membrane proteins, referred to as protein G and protein Q, were identified. Both of these proteins are single transmembrane‐spanning helices located between the LH1 ring and the RC L‐subunit and are absent from the LH1–RC complexes of all other purple bacteria of which the structures have been determined so far. Besides bacteriochlorophyll b molecules (B1020) located on the periplasmic side of the Hlr. halochloris membrane, there are also two arrays of bacteriochlorophyll b molecules (B800 and B820) located on the cytoplasmic side. Only a single copy of a carotenoid (lycopene) was resolved in the Hlr. halochloris LH1–α3β3 and this was positioned within the complex. The potential quinone channel should be the space between the LH1–α3β3 that accommodates the single lycopene but does not contain a γ‐polypeptide, B800 and B820. Our results provide a structural explanation for the unusual Qy red shift and carotenoid absorption in the Hlr. halochloris spectrum and reveal new insights into photosynthetic mechanisms employed by a species that thrives under the harshest conditions of any phototrophic microorganism known. [ABSTRACT FROM AUTHOR]

Purpose: Chewing duration can affect food particle size, gastric processing, and postprandial glycemia, but these effects have not been investigated with exercise. This study examined how the chewing duration of a food bar impacts glycemic and metabolic responses, gastrointestinal (GI) symptoms, psychological affect, and performance during endurance running. Methods: This randomized, unblinded, crossover study had 15 males (35.2 ± 7.4 years, VO2peak: 56.1 ± 5.2 ml/kg/min) attend three laboratory visits. Visit 1 required a VO2peak test, 10 min familiarization run at 60% VO2peak, and familiarization time-to-exhaustion (TTE) test (10 min at 90% VO2peak, followed by TTE at 100% VO2peak). Visits 2 and 3 consisted of a 60 min run at 60% VO2peak, followed by TTE testing. Participants were fed 45 g of a bar (180 kcal, 4 g fat, 33 g carbohydrate, 3 g protein, 1 g fiber) in 9 g servings 30 min before running, and 27 g of bar in 9 g servings at three timepoints during the 60 min run. Participants consumed the servings in 20 (20CHEW) or 40 (40CHEW) masticatory cycles, at 1 chew/second. Outcomes included blood glucose, substrate use, GI symptoms, perceived exertion (RPE), overall feeling, and TTE. Results: Post-prandial blood glucose, GI symptoms, and RPE increased over time, but there were no significant between-condition or condition-by-time effects. TTE showed no significant between-condition effect (20CHEW: 288 ± 133 s; 40CHEW: 335 ± 299 s; p = 0.240). Overall feeling demonstrated a time-by-condition effect (p = 0.006), suggesting possible better maintenance over time with 40CHEW. Conclusion: Cumulatively, the results suggest that extended chewing minimally impacts physiology, perceptions, and performance during 60 min moderate-intensity running. [ABSTRACT FROM AUTHOR]

This article explores the role of the Rad protein in the regulation of L-type calcium channels (LTCCs) in heart muscle cells. LTCCs are crucial for heart contraction, and when they are not properly regulated, it can lead to heart problems. The Rad protein, a type of small GTP-binding protein, has been found to inhibit LTCC activity. The article investigates how Rad interacts with LTCCs and its potential for treating heart failure. The authors discovered that mice without the Rad protein had increased calcium and electrical activity in their heart muscle cells. They propose that targeting the interaction between Rad and the Cav1.2 calcium channel could be a potential therapy, but more research is needed to understand this interaction. The authors also stress the importance of considering different subpopulations of the Cav1.2 calcium channel. Overall, the article highlights the complexity of cellular signaling and the need for careful experimental design to fully comprehend the molecular mechanisms involved. [Extracted from the article]

Background and Purpose: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on‐target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago‐PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. Experimental Approach: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β‐arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2‐AG, and AMB‐FUBINACA. Key Results: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago‐PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2‐AG but not AEA or AMB‐FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA‐induced signalling in all pathways tested; however, no notable potentiation of 2‐AG or AMB‐FUBINACA was observed. Conclusion and Implications: Ago‐PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2‐AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including β‐arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo. [ABSTRACT FROM AUTHOR]

In cattle, bovine respiratory syncytial virus (BRSV) is associated with secondary bacterial infections; however, the mechanisms of the interaction between BRSV and bacteria are unclear. Trueperella pyogenes (T. pyogenes) causes pneumonia in cattle and is involved in secondary infections following viral infections. In this study, we evaluated the effect of BRSV infection on the adhesion of T. pyogenes to BRSV-infected cells. BRSV infection significantly enhanced the adhesion of T. pyogenes to cells in a multiplicity of infection- and time-dependent manner. The BRSV-mediated change in the adhesion of T. pyogenes was widely observed in various cell types and bacterial strains. The results from the gentamicin protection assay showed that BRSV infection did not affect the intracellular invasion ability of T. pyogenes. Furthermore, adhesion assays conducted using BRSV G protein-expressing cells and anti-BRSV G antibodies revealed that the increased adhesion of T. pyogenes to cells was mediated by the G protein of BRSV. In addition, immunofluorescence assay revealed the colocalization of BRSV G protein and T. pyogenes. Thus, BRSV infection can potentially lead to bovine respiratory disease complex by promoting the adhesion of T. pyogenes to the infected cells. [ABSTRACT FROM AUTHOR]

Developmental defects of enamel (DDE) is a dental disease with a high prevalence and no effective means of prevention. One of the major causes of DDE is infection, but the pathogenesis is still unclear. Melatonin is known for its anti-inflammatory and mineralization-promoting activities. However, the effects of melatonin on inflammation-induced DDE remain unknown. Here, we investigated the pathogenesis and potential therapeutic targets of inflammation-induced DDE. First, the effect of lipopolysaccharide-induced inflammation in pregnant mice on the enamel mineralization of the offspring was detected by 3D X-ray microscope analysis, immunohistochemical assays, and quantitative real-time polymerase chain reaction (qRT-PCR). Then, the ameloblastic differentiation ability of ameloblast lineage cells (ALCs) in macrophage conditioned medium (CM) was detected. Subsequently, ameloblastic mineralization after melatonin administration was studied both in vivo and in vitro. The underlying mechanism of melatonin was investigated by RNA sequencing and small interfering RNA transfection. Enamel mineralization was decreased in the inflammatory environment both in vivo and in vitro. Furthermore, melatonin treatment ameliorated these defects. RNA sequencing analysis revealed that regulator of G protein signaling 2 (Rgs2) was downregulated in the inflammation group, whereas it was upregulated after the addition of melatonin. Further studies showed that Rgs2 knockdown resulted in decreased ameloblastic mineralization in ALCs. After Rgs2 knockdown of ALCs in M1-CM with melatonin, the effect of melatonin-mediated attenuation of DDE was greatly reduced. Our results demonstrate that melatonin ameliorates inflammation-induced DDE by upregulating RGS2, suggesting that RGS2 is a potential therapeutic target for inflammation-induced DDE. [ABSTRACT FROM AUTHOR]

The majority of tumor comprehensive genomic profiling (CGP) currently does not include a matched normal control. The use of a tumor-only CGP approach needs the development of a strategy to refine germline pathogenic/likely pathogenic variants (gP/LPVs) calls, so as to limit the performance of unnecessary germline reflex tests and instead successfully identify patients who are carriers of likely gP/LPVs. Guidelines have been developed for the identification of gP/LPVs in BRCA1/2 genes on the basis of tumor-only CGP results and for the evaluation of the appropriateness of performing germline reflex BRCA1/2 testing. In this study, an algorithm to assist decision-making for germline reflex testing of BRCA1/2 variants following tumor-only CGP is proposed. [ABSTRACT FROM AUTHOR]

Pituitary gigantism is a rare manifestation of chronic growth hormone (GH) excess that begins before closure of the growth plates. Nearly half of patients with pituitary gigantism have an identifiable genetic cause. X-linked acrogigantism (X-LAG; 10% of pituitary gigantism) typically begins during infancy and can lead to the tallest individuals described. In the 10 years since its discovery, about 40 patients have been identified. Patients with X-LAG usually develop mixed GH and prolactin macroadenomas with occasional hyperplasia that secrete copious amounts of GH, and frequently prolactin. Circulating GH-releasing hormone is also elevated in a proportion of patients. X-LAG is caused by constitutive or sporadic mosaic duplications at chromosome Xq26.3 that disrupt the normal chromatin architecture of a topologically associating domain (TAD) around the orphan G-protein–coupled receptor, GPR101. This leads to the formation of a neo-TAD in which GPR101 overexpression is driven by ectopic enhancers ("TADopathy"). X-LAG has been seen in 3 families due to transmission of the duplication from affected mothers to sons. GPR101 is a constitutively active receptor with an unknown natural ligand that signals via multiple G proteins and protein kinases A and C to promote GH/prolactin hypersecretion. Treatment of X-LAG is challenging due to the young patient population and resistance to somatostatin analogs; the GH receptor antagonist pegvisomant is often an effective option. GH, insulin-like growth factor 1, and prolactin hypersecretion and physical overgrowth can be controlled before definitive adult gigantism occurs, often at the cost of permanent hypopituitarism. [ABSTRACT FROM AUTHOR]

Spirulina (Arthospira platensis) is known for its rich content of natural compounds like phycocyanin, chlorophylls, carotenoids, and high protein levels, making it a nutrient-dense food. Over the past decade, research has aimed to optimize the extraction, separation, and purification of these valuable metabolites, focusing on technologies such as high-pressure processing, ultrasound-assisted extraction, and microwave-assisted extraction as well as enzymatic treatments, chromatographic precipitation, and membrane separation. In this study, various extraction methods (conventional vs. ultrasound-assisted), solvents (water vs. phosphate buffer), solvent-to-biomass ratios (1:5 vs. 1:10), and ultrafiltration (PES membrane of MWCO 3 kDa, 2 bar) were evaluated. The quantities of total protein, phycocyanin (PC), chlorophyll a (Cla), and total carotenoids (TCC) were measured. The results showed that ultrasound-assisted extraction (UAE) with phosphate buffer at a 1:10 ratio yielded a metabolite-rich retentate (MRR) with 37.0 ± 1.9 mg/g of PC, 617 ± 15 mg/g of protein, 0.4 ± 0.2 mg/g of Cla, and 0.15 ± 0.14 mg/g of TCC. Water extraction in the concentration process achieved the highest concentrations in MRR, with approximately 76% PC, 92% total protein, 62% Cla, and 41% TCC. These findings highlight the effective extraction and concentration processes to obtain a metabolite-rich retentate from Spirulina biomass, reducing the volume tenfold and showing potential as a functional ingredient for the food, cosmetic, and pharmaceutical industries. [ABSTRACT FROM AUTHOR]