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A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the molecular and neuronal mechanisms are not fully understood. Here, we reported that impaired neuronal macroautophagy in the prelimbic cortical (PrL) subregion of the mPFC paralleled the occurrence of anxiety-like behaviors in rats with chronic spared nerve injury (SNI). Intriguingly, such macroautophagy impairment was mainly observed in a FOS/c-Fos+ neuronal subpopulation in the PrL. Chemogenetic inactivation of this comorbid anxiety-related neuronal ensemble relieved pain-induced anxiety-like behaviors. Rescuing macroautophagy impairment in this neuronal ensemble relieved chronic pain-associated anxiety and mechanical allodynia and restored synaptic homeostasis at the molecular level. By contrast, artificial disruption of macroautophagy induced early-onset co-morbid anxiety in neuropathic rats, but not general anxiety in normal rats. Taken together, our work identifies causal linkage between PrL neuronal macroautophagy dysfunction and comorbid anxiety in neuropathic pain and provides novel insights into the role of PrL by differentiating its contribution in pain-induced comorbid anxiety from its modulation over general anxiety-like behaviors. Abbreviation: AAV: adeno-associated viruses; ACC: anterior cingulate cortex; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CNO: clozapine-N-oxide; CQ: chloroquine; DIA: data independent acquisition; DIO: double floxed inverse orf; DLG4/PSD-95: discs large MAGUK scaffold protein 4; Dox: doxycycline; GABA: γ-aminobutyric acid; GFP: green fluorescent protein; GO: gene ontology; Gi: inhibitory guanine nucleotide-binding proteins; HsCHRM4/M4D: human cholinergic receptor muscarinic 4; HsSYN: human synapsin; KEGG: Kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; LC3-II: PE conjugated microtubule-associated protein 1 light chain3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mPFC: medial prefrontal cortex; P2A: 2A self-cleaving peptide; PPI: protein-protein interaction networks; PrL: prelimbic cortex; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; rtTA: reverse tetracycline-transactivator; SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; SHANK3: SH3 and multiple ankyrin repeat domains 3; SLC1A1/EAAC1: solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, systemXag), member 1; SNAP23: synaptosomal-associated protein 23; SNI:spared nerve injury; SQSTM1/p62: sequestosome 1; SYT3: synaptotagmin 3; TRE: tetracycline-responsive element; TRE3G: third-generation tetracycline-responsive element. [ABSTRACT FROM AUTHOR]

Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host's antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-β promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection. Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-β:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2 [ABSTRACT FROM AUTHOR]

To determine the validity of bioelectrical impedance analysis (BIA) in quantifying fat-free mass (FFM) compared to air-displacement plethysmography (ADP) in patients with a motor neurone disease (MND). FFM of 140 patients diagnosed with MND was determined by ADP using the BodPod (i.e. the gold standard), and by BIA using the whole-body Bodystat. FFM values were translated to predicted resting energy expenditure (REE); the actual REE was measured using indirect calorimetry, resulting in a metabolic index. Validity of the BIA compared to the ADP was assessed using Bland-Altman analysis and Pearson's r. To assess the clinical relevance of differences, we evaluated changes in metabolic index and in individualized protein demand. Despite the high correlation between ADP and BIA (r = 0.93), averaged across patients, the assessed mean fat-free mass was 51.7 kg (± 0.9) using ADP and 54.2 kg (± 1.0) using BIA. Hence, BIA overestimated fat-free mass by 2.5 kg (95% CI 1.8–3.2, p < 0.001). Clinically, an increased metabolic index would be more often underdiagnosed in patients with MND using BIA (31.4% according to BIA versus 44.2% according to ADP, p = 0.048). A clinically relevant overestimation of ≥ 15 g in protein demand was observed for 4 (2.9%) patients using BIA. BIA systematically overestimates FFM in patients with MND. Although the differences are limited with ADP, underscoring the utility of BIA for research, overestimation of fat-free mass may have consequences for clinical decision-making, especially when interest lies in determining the metabolic index. [ABSTRACT FROM AUTHOR]

The prognosis of multiple myeloma (MM) continues to improve. Recent progress in therapies, using immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), and anti-CD38 monoclonal antibodies, has greatly improved patients' outcomes. Despite these advancements, relapses still happen often, and patients can become resistant to the usual treatments. Newer treatments, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies (BsAbs) targeting B-cell maturation antigen (BCMA), have resulted in excellent outcomes in patients with limited treatment options. G protein – coupled receptor, class C group 5 member D (GPRC5D) is considered a very promising target with early results from clinical trials showing high response rates in patients with relapsed or refractory multiple myeloma. This review covers the efficacy and safety of CAR-T and BsAbs targeting GPRC5D in MM, focusing on talquetamab – the inaugural FDA-approved BsAb targeting GPRC5D. Talquetamab has exhibited promising response rates alongside a distinctive side effect profile. Additionally, ongoing trials examining talquetamab in combination with agents like daratumumab and teclistamab are discussed. We offer insights into the potential utilization of various GPRC5D-based therapies in the treatment paradigm for MM, either independently or in combination with established therapies. [ABSTRACT FROM AUTHOR]

Phytophthora capsici is a major oomycete-based pathogen causing enormous damage to the productivity of peppers and other major crops from different families worldwide by causing fruit rot, root rot and foliar blight diseases. This study utilized molecular signatures present within ITS1-5.8S rRNA-ITS2 and Ras-related GTP binding protein (Ypt1) genes to evaluate haplotype variability and phylogenetic relationships among P. capsici isolates with significant epidemiological implications. Twelve different evolutionary lineages specific to particular hosts and/or geographical regions and differed by SNPs, and 30 haplotypes with maximum diversity in India and Pakistan, were identified by sequence analysis of the ITS regions. It includes country-specific lineages Pc-I and Pc-II in India with multiple host ranges; capsicum-specific lineages Pc-IV, Pc-V and Pc-VIII from Pakistan; papaya-specific lineages Pc-VI from China, and watermelon-specific lineages Pc-VII and Pc-XI from USA. Many lineages showed cross-border infections, such as Pc-III and Pc-X in India and Pakistan, Cocoa-specific lineage Pc-IX in Brazil and Cote d'Ivoire, and Capsicum-specific multi-continental lineage Pc-XII. Similarly, 11 different SNP lineages and 32 haplotypes with maximum diversity in China were identified for the Ypt1 gene. These findings would be very useful for epidemiological surveillance of P. capsici strains worldwide, understanding population and evolutionary dynamics, developing lineage- and haplotype-specific diagnostic assays and disease-resistant varieties. [ABSTRACT FROM AUTHOR]

Rational protein engineering has often focused on increasing the thermal stability of proteins by incorporating stabilising mutations. Introducing fluorinated amino acids into proteins has been used to enhance protein stability. Free energy simulations of the immunoglobulin binding domain of streptococcal protein G in both the folded and unfolded states were performed to understand the detailed mechanism of the effect of fluorinated aromatic and aliphatic amino acid side chains on the stability of Leu → Hfl (hexafluoroleucine) and Phe → Pff (pentafluorophenylalanine) mutations at the solvent-exposed position of the globular protein. Our simulated free energy differences (ΔΔG) for the Leu → Hfl and Phe → Pff mutations are −0.43 ± 0.14 kcal/mol and −0.45 ± 0.20 kcal/mol, respectively. These calculated free energy differences are in excellent agreement with the corresponding reported experimental values. Surprisingly, a significant contribution to the increased stability of the Hfl-containing fluorinated protein comes from van der Waals interactions and, in the case of Pff, electrostatic interactions. The simulated quantitative agreement with experimental values validates the free energy simulation method to elucidate the effect of fluorination on protein stability. The implications of the simulation results for our understanding of the effect of fluorination on protein stability are discussed. [ABSTRACT FROM AUTHOR]

To identify the potential anabolic properties of a dairy-plant protein blend as compared to single plant-based and single dairy protein, the postprandial amino acid (AA) response of pea protein, milk protein, micellar casein, and a casein–pea protein blend was investigated in healthy older adults (age 72.3 ± 3.4 years, BMI 25.3 ± 2.9 kg/m2). Plasma AA levels were measured, before and up to 5 h after ingestion of each 20 g protein. Blending casein–pea in a 60/40 mixture resulted in improved plasma AA availability, i.e. area under the curve (AUC) and peak height, of total (essential) AA and of key AAs methionine and leucine compared to pea only, while preserving the higher availability of arginine. The casein/pea blend clearly showed an AA response that was in between that of its single constituents, indicating that blending could be a solution to improve a lower quality (plant) protein, which could be of relevance for older adults. [ABSTRACT FROM AUTHOR]

Constitutively active mutations in the Gαi2 and GαoA subunits of heterotrimeric G proteins have been found in various human cancers, including breast cancer, but their precise roles in tumor formation, progression, and metastasis remain poorly understood. This study focused on GαoAR243H and Gαi2R179C mutants in breast cancer. These mutants alone were insufficient to initiate mammary tumor formation in mice. However, when introduced into transgenic mouse models of breast cancer induced by Neu expression or PTEN loss, the Gαi2R179C mutant notably enhanced spontaneous lung metastasis, without affecting primary tumor initiation and growth. Ectopic expression of the GαoAR243H and Gαi2R179C mutants in tumor cells promoted cell migration in vitro and dissemination into multiple organs in vivo by activating the c-Src signaling pathway. These mutants activate c-Src through direct interaction, involving specific residues in the switch domains II of Gαi subunits, which only partially overlap with those involved in inhibiting adenylyl cyclases. This study uncovers a critical role of Gαi/o signaling in accelerating breast cancer metastasis through the c-Src pathway. These findings hold clinical significance as they may pave the way for personalized therapies targeting c-Src to inhibit breast cancer metastasis in patients with active Gαi/o mutations or elevated Gαi/o signaling. [ABSTRACT FROM AUTHOR]

Impaired insulin secretion due to pancreatic β-cell injury is an important cause of type 2 diabetes (T2D). Regulators of guanine nucleotide binding protein (G protein) signaling proteins played a key role in regulating insulin sensitivity in vivo. To explore the role of RGS7 on palmitic acid-induced pancreatic β-cell injury, pancreatic β-cells Beta-TC-6 and Min6 were treated with palmitic acid (PA) to similar type 2 diabetes (T2D) injury in vitro. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT), 5-ethynyl-2′-deoxyuridine (EdU), and flow cytometry were used to analyze cell viability, proliferation, and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) kits were used to analyze the changes of inflammation-related cytokines. The expression of gene and protein was measured by quantitative real-time PCR (qRT-PCR) and western blot. PA modeling induced apoptosis, increased levels of inflammation-related cytokines, and suppressed cell viability and proliferation of pancreatic β-cells. RGS7 silence markedly alleviated the cell injury induced by PA. RGS7 overexpression further aggravated apoptosis and inflammatory response in PA-induced pancreatic β-cells and inhibited cell viability and proliferation. It is worth noting that RGS7 activated the chemokine signaling pathway. Silence of the key gene of the chemokine signaling pathway could eliminate the negative effect of RGS7 on PA-induced pancreatic β-cells. RGS7 silence protects pancreatic β-cells from PA-induced injury by inactivating the chemokine signaling pathway. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes the N-ϵ-(γ-glutamyl) lysine bonds between side chains of glutamine and lysine residues resulting in proteolytically resistant cross-links. Increased TG2 activity and levels are involved in the pathophysiology of various diseases, including liver injury, cystic fibrosis, celiac sprue, metastatic cancers, and several neurodegenerative conditions. Inhibiting TG2 activity is considered a potential strategy to combat these diseases. Although guanine nucleotide (GTP) could inhibit TG2, its inhibitory activity decreased with increased calcium concentration. Search for GTP analogs that could strongly bind and inhibit TG2 activity is intense. This study screened the PubChem database for about two thousand GTP-like compounds for TG2. Using docking and molecular dynamics simulations we identified three compounds (C4959, C4215, and C9560) that could selectively interact with TG2. These three compounds have less affinity for several other intracellular and extracellular GTP-binding proteins suggesting selectivity for TG2. Interestingly, C9560 showed stronger interactions and better binding energy with TG2 than C4959 and C4215, suggesting that C9560 can form a more stable complex with TG2. Our study indicates that C9560, a GTP analog, could be exploited as a promising candidate to inhibit TG2-mediated fibrotic conditions. [ABSTRACT FROM AUTHOR]

Constant exposure of human gingival fibroblasts (HGFs) to oral pathogens trigger selective immune responses. Recently, the activation of immune response to cyclic dinucleotides (CDNs) via STING has come to the forefront. Reports show that other proteins outside the STING-TBK1-IRF3 axis respond to CDNs but a global view of impacted proteome in diverse cells is lacking. HGFs are constantly exposed to bacterial-derived cyclic-di-adenosine monophosphate (c-di-AMP) and cyclic-di-guanosine monophosphate (c-di-GMP). To understand the response of HGFs to bacterial-derived CDNs, we carried out a global proteomics analysis of HGFs treated with c-di-AMP or c-di-GMP. The expression levels of several proteins modulated by CDNs were examined. Interferon signaling proteins such as Ubiquitin-like protein ISG15 (ISG15), Interferon-induced GTP-binding protein Mx1 (MX1), Interferon-induced protein with tetratricopeptide repeats (IFIT) 1 (IFIT1), and (IFIT3) were significantly upregulated. Interestingly, other pathways not fully characterized to be regulated by CDNs, such as necroptosis signaling, iron homeostasis signaling, protein ubiquitination, EIF2 signaling, sumoylation and nucleotide excision repair pathways were also modulated by the bacterial-derived CDNs. This study has added to the increasing appreciation that beyond the regulation of cytokine production via STING, cyclic dinucleotides also broadly affect many critical processes in human cells. [ABSTRACT FROM AUTHOR]

The objective of this work was to optimize the hydrolysis conditions of hybrid sturgeon (Huso dauricus × Acipenser schrenckii) protein using flavourzyme and compound proteinase, and to evaluate the antioxidant activity of hydrolysates. The lyophilized flavourzyme hydrolysate (FH) and compound proteinase hydrolysate (CPH) with degree of hydrolysis of 18.16% and 19.35% under optimal conditions had 85.36% and 87.24% protein content, 89.19 and 96.97 g/100 g protein total amino acid content, 92.86% and 95.12% protein solubility, and 902.47 nm and 1025.76 nm particle size, respectively. The CPH had the highest DPPH and ABTS radical scavenging activities, which were 71.04 and 66.84% with IC50 at 2.85 and 3.37 mg/mL, respectively; while the FH had higher metal-chelating activity, which was 58.37% with IC50 at 24.81 mg/mL. The obtained hydrolysates have excellent antioxidant properties and could be used as food additives and natural substitutes for synthetic antioxidants compounds, particularly in food and pharmaceuticals. [ABSTRACT FROM AUTHOR]

1. The following study explored the expression and preliminary function of the RGS3 gene. The spatial and temporal expression patterns of the RGS3 gene were analysed in the ovarian stroma of Shendan No. 6 Green shell hens and Hy-line Brown hens at four time points (6, 28, 40, and 52 weeks old), as well as in various organs and follicles of Hy-line Brown hens. 2. Based on the genomic and protein sequences of RGS3 in NCBI database, phylogenetic trees were constructed using MEGA-X. The protein interaction network was analysed using STRING. According to the results of protein–protein interaction network and pathways, the mRNA expression levels of RGS3 and three interaction proteins were explored by qRT-PCR in vitro. 3. Spatio-temporal expression data revealed that RGS3 mRNA was expressed in all the organs tested, being highest in the hypothalamus. In different follicles, RGS3 mRNA was highly expressed in post-ovulatory follicles, followed by ovarian stroma and large white follicles. The expression levels of RGS3 mRNA in the ovarian stroma were significantly higher in Shendan No. 6 Green shell hens than that in the Hy-line Brown hens at all egg-laying stages. 4. The phylogenetic tree results showed that ducks, geese and chickens had higher homology based on the genomic and protein sequence of RGS3. Moreover, chicken RGS3 interacted with GSK3B, RAF1 and BRAF based on STRING prediction. In vitro follicle stimulating hormone (FSH) treatment showed that mRNA expression levels of RGS3 and those of its predicted interacting proteins BRAF and GSK3B decreased with increasing FSH concentration. The results suggested that RGS3 responds to FSH and may play an important role in the regulation of follicular development in chicken. [ABSTRACT FROM AUTHOR]

Landfill leachate, due to its recalcitrant nature and toxicity, poses a serious environmental threat, which requires the implementation of effective treatment processes. In this work, a full-scale treatment system consisting of two Sequencing Batch Reactors (SBRs) was used for the processing of landfill leachate of intermediate to mature age (BOD/COD ratio of 0.16). Biosystem operation resulted in BOD5, COD and TKN removal efficiencies of 81%, 39% and 76%, respectively, whereas the low residual NO3--N concentration in the effluent (4.01 ± 0.10 mg/L) was indicative of the efficient denitrification process. Assessment of hydrolytic potential of activated sludge revealed high endocellular and extracellular lipase activities, which reached values up to 206 and 141 U/g protein respectively, possibly as the consequence of plastics degradation during maturation process. Implementation of Illumina sequencing indicated the predominance of Alphaproteobacteria, accompanied by members of Bacteroidetes, Betaproteobacteria and Chloroflexi. Paracoccus was the predominant genus identified, followed by representatives of the genera Bellilinea, Flavobacterium, Thauera and Truepera. Nitrosomonas was the major ammonia-oxidizing bacterium (AOB), while nitrite oxidation was mainly achieved by the uncultured nitrite-oxidizing bacterium (NOB) Candidatus Nitrotoga. [ABSTRACT FROM AUTHOR]

The association of AMP-activated protein kinase (AMPK) with membranes plays a critical role in the regulation of AMPK activation and function. Protein lipid modification, including palmitoylation, myristoylation, and farnesyation, constitutes a crucial mechanism in the regulation of protein dynamic interactions with membranes. Among the three subunits of the AMPK heterotrimeric complex, the structural subunit AMPKβ is myristoylated and the catalytic subunit AMPKa is palmitoylated. Here, we report the characterization of AMPKa palmitoylation. We found that AMKPa was palmitoylated at Cys209 and Cys543, and this was required for AMPK activation and subcellular membrane compartmentalization. To understand the regulation of AMPKa palmitoylation, we have identified DHHC17 as a candidate palmitoyltransferase for AMPKa and found that DHHC17, by palmitoylating AMPKa, modulated AMPK membrane association and activation in response to energy stress. To determine the role of DHHC17 in cell function, we generated DHHC17 liver-specific knockout mice and found that inactivation of DHHC17 in the mouse liver impaired AMPK activation and hepatic autophagy and caused a type 2 diabetes-like syndrome. Overall, our studies demonstrate that AMPKa palmitoylation plays a critical role in AMPK activation and that DHHC17, through its modulation of AMPK signaling, constitutes a new regulator of hepatic metabolism. [ABSTRACT FROM AUTHOR]

The β-adrenoceptor blockers may have anti-oxidant properties or induce β-arrestin recruitment beyond classical desensitization of receptor/G protein coupling, offering potential therapeutic benefits. Here, we investigated the effects of carvedilol, metoprolol and propranolol in an animal model of cisplatin-induced nephrotoxicity. Rats received the β-blockers (3 or 12 mg/kg/day) with or without cisplatin, and kidney function was investigated using renal scintigraphy, histopathology, and serum variables. Metoprolol and propranolol as well as low-dose carvedilol did not alter kidney function, per se. Meanwhile, high-dose carvedilol reduced renal accumulation of Technetium-99m (99mTc)–labeled dimercaptosuccinic acid (99mTc-DMSA) without significant effect on other variables. Furthermore, low-dose carvedilol prevented cisplatin-induced reduction of tracer uptake, but high-dose of this drug aggravated the situation. In this regard, both low and high -doses of carvedilol significantly inhibited cisplatin effects on kidney histology, BUN and creatinine levels. Also, high-dose propranolol inhibited cisplatin adverse effects on radiotracer uptake, histological manifestations, BUN and creatinine levels, while metoprolol failed to cause a notable effect. Taken together, carvedilol and high-dose propranolol may offer potential benefits in cisplatin nephrotoxicity. [ABSTRACT FROM AUTHOR]

GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation. [ABSTRACT FROM AUTHOR]

We recently identified a CD63-interacting protein to understand the role of CD63 in virion production of the human immunodeficiency virus type 1, and we have found that Rab3a forms a complex with CD63. In this study, we analysed the effect of Rab3a on virion production of the murine leukaemia virus (MLV), which is another member of the retrovirus family. We found that Rab3a silencing induced lysosomal degradation of the MLV Gag protein, and recovery of the Rab3a expression restored the level of the Gag protein through a complex formation of MLV Gag and Rab3a, indicating that Rab3a is required for MLV Gag protein expression. In contrast, CD63 silencing decreased the infectivity of released virions but had no effect on virion production, indicating that CD63 facilitates the infectivity of released MLV particles. Although Rab3a induced CD63 degradation in uninfected cells, the complex of MLV Gag and Rab3a suppressed the Rab3a-mediated CD63 degradation in MLV-infected cells. Finally, we found that the MLV Gag protein interacts with Rab3a to stabilize its own protein and CD63 that facilitates the infectivity of released MLV particles. Considering the involvement of Rab3a in lysosome trafficking to the plasma membrane, it may also induce cell surface transport of the MLV Gag protein. [ABSTRACT FROM AUTHOR]

Cell migration, a crucial step in numerous biological processes, is tightly regulated in space and time. Cells employ Rho GTPases, primarily Rho, Rac, and Cdc42, to regulate their motility. Like other small G proteins, Rho GTPases function as biomolecular switches in regulating cell migration by operating between GDP bound 'OFF' and GTP bound 'ON' states. Guanine nucleotide exchange factors (GEFs) catalyse the shuttling of GTPases from OFF to ON state. G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that are involved in many signalling phenomena including cell survival and cell migration events. In this review, we summarize signalling mechanisms, involving GPCRs, leading to the activation of RhoGEFs. GPCRs exhibit diverse GEF activation modes that include the interaction of heterotrimeric G protein subunits with different domains of GEFs, phosphorylation, protein–protein interaction, protein–lipid interaction, and/or a combination of these processes. [ABSTRACT FROM AUTHOR]

In all living cells, the ribosome translates the genetic information carried by messenger RNAs (mRNAs) into proteins. The process of ribosome recycling, a key step during protein synthesis that ensures ribosomal subunits remain available for new rounds of translation, has been largely overlooked. Despite being essential to the survival of the cell, several mechanistic aspects of ribosome recycling remain unclear. In eubacteria and mitochondria, recycling of the ribosome into subunits requires the concerted action of the ribosome recycling factor (RRF) and elongation factor G (EF-G). Recently, the conserved protein HflX was identified in bacteria as an alternative factor that recycles the ribosome under stress growth conditions. The homologue of HflX, the GTP-binding protein 6 (GTPBP6), has a dual role in mitochondrial translation by facilitating ribosome recycling and biogenesis. In this review, mechanisms of ribosome recycling in eubacteria and mitochondria are described based on structural studies of ribosome complexes. [ABSTRACT FROM AUTHOR]

A magnetic separation-based immunosensor for deoxynivalenol assay, coupled with a nano-affinity cleaning up for instrumental confirmation, was developed. Maltose-binding protein with mimic epitope of DON was coupled to CdSe/ZnS quantum dot nanobeads for fluorescent reporter. Graphene oxide was linked with magnetic nanoparticles and protein G for immunomagnetic adsorbent. The magnetic separation of reporter could be inhibited by DON so the fluorescent remained in the solution indicates a good quantitative correlation to DON concentration. The DON separated by immune adsorbent can be cleaned up for instrumental confirmation. The optimized immunosensor showed results as follows: the linear range was in 0.3–3000 ng mL−1. The limits of detection (S/N = 3) were 0.50, 1.0, and 3.0 ng mL−1 in different backgrounds. The recoveries for DON-spiked samples range from 78.7% to 88.5%. Both results of UPLC-MS/MS based on affinity column purification and coupled with nano-affinity cleaning up showed a good agreement with the current immunosensor. [ABSTRACT FROM AUTHOR]

Regulators of G protein signaling (RGSs) are proteins that negatively regulate G protein signal transduction. In this study, seven putative RGSs were characterized in the nematode-trapping (NT) fungus, Arthrobotrys oligospora. Deleting Rgs genes significantly increased intracellular cAMP levels, and caused defects in mycelia growth, stress resistance, conidiation, trap formation, and nematocidal activity. In particular, the ΔAoFlbA mutant was unable to produce conidia and traps. Transcriptomic analysis showed that amino acid metabolic and biosynthetic processes were significantly enriched in the ΔAoFlbA mutant compared to WT. Interestingly, Gas1 family genes are significantly expanded in A. oligospora and other NT fungi that produce adhesive traps, and are differentially expressed during trap formation in A. oligospora. Disruption of two Gas1 genes resulted in defective conidiation, trap formation, and pathogenicity. Our results indicate that RGSs play pleiotropic roles in regulating A. oligospora mycelial growth, development, and pathogenicity. Further, AoFlbA is a prominent member and required for conidiation and trap formation, possibly by regulating amino acid metabolism and biosynthesis. Our results provide a basis for elucidating the signaling mechanism of vegetative growth, lifestyle transition, and pathogenicity in NT fungi. [ABSTRACT FROM AUTHOR]

Septins, a conserved family of GTP-binding proteins, are widely recognized as an essential cytoskeletal component, playing important roles in a variety of biological processes, including division, polarity, and membrane remodeling, in different eukaryotes. Although the roles played by septins were identified in the model organism Saccharomyces cerevisiae, their importance in other fungi, especially pathogenic fungi, have recently been determined. In this review, we summarize the functions of septins in pathogenic fungi in the cell cycle, autophagy, endocytosis and invasion host-microbe interactions that were reported in the last two years in the field of septin cell biology. These new discoveries may be expanded to investigate the functions of septin proteins in fungal pathogenesis and may be of wide interest to the readers of Microbiology and Molecular Pathology. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of chronic administration of caffeine on the anatomical characteristics of taste buds, the expression level of taste receptor protein in mice, and preference for a palatable solution. We found that following a 21-day administration of caffeine, mice showed increased behavioural responses to sweet stimuli (acesulfame-K solution). Mirroring this behavioural change, chronic caffeine treatment evidently decreased the maximal cross-sectional area and height of the longitudinal axis of fungiform taste buds, the number of taste cells per fungiform taste bud, and the expression of G protein α-gustducin, while the expression of the sweet taste receptors T1R2 and T1R3 was reversed. Our findings demonstrate that chronic administration of caffeine has an impact on taste sensitivity and changes in taste bud features, which may contribute to the alteration of taste behaviour. [ABSTRACT FROM AUTHOR]

The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5ʹ end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in Leishmania species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in L. infantum. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in Trypanosoma brucei, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the Leishmania-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in Leishmania (W45) did not substantially impair the identified interactions. Finally, the crystal structure of Leishmania EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its Trypanosoma orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation. [ABSTRACT FROM AUTHOR]

Background: The effects of ingesting varying essential amino acid (EAA)/protein-containing food formats on protein kinetics during energy deficit are undetermined. Therefore, recommendations for EAA/protein food formats necessary to optimize both whole-body protein balance and muscle protein synthesis (MPS) during energy deficit are unknown. We measured protein kinetics after consuming iso-nitrogenous amounts of free-form essential amino acid-enriched whey (EAA + W; 34.7 g protein, 24 g EAA sourced from whey and free-form EAA), whey (WHEY; 34.7 g protein, 18.7 g EAA), or a mixed-macronutrient meal (MEAL; 34.7 g protein, 11.4 g EAA) after exercise during short-term energy deficit. Methods: Ten adults (mean ± SD; 21 ± 4 y; 25.7 ± 1.7 kg/m2) completed a randomized, double-blind crossover study consisting of three, 5 d energy-deficit periods (− 30 ± 3% of total energy requirements), separated by 14 d. Whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and in response to combination exercise consisting of load carriage treadmill walking, deadlifts, and box step-ups at the end of each energy deficit using L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Treatments were ingested immediately post-exercise. Mixed-muscle protein synthesis (mixed-MPS) was measured during exercise through recovery. Results: Change (Δ postabsorptive + exercise to postprandial + recovery [mean treatment difference (95%CI)]) in whole-body (g/180 min) PS was 15.8 (9.8, 21.9; P = 0.001) and 19.4 (14.8, 24.0; P = 0.001) greater for EAA + W than WHEY and MEAL, respectively, with no difference between WHEY and MEAL. ΔPB was − 6.3 (− 11.5, − 1.18; P = 0.02) greater for EAA + W than WHEY and − 7.7 (− 11.9, − 3.6; P = 0.002) greater for MEAL than WHEY, with no difference between EAA + W and MEAL. ΔNET was 22.1 (20.5, 23.8; P = 0.001) and 18.0 (16.5, 19.5; P = 0.00) greater for EAA + W than WHEY and MEAL, respectively, while ΔNET was 4.2 (2.7, 5.6; P = 0.001) greater for MEAL than WHEY. Mixed-MPS did not differ between treatments. Conclusions: While mixed-MPS was similar across treatments, combining free-form EAA with whey promotes greater whole-body net protein balance during energy deficit compared to iso-nitrogenous amounts of whey or a mixed-macronutrient meal. Trial registration: ClinicalTrials.gov, Identifier no. NCT04004715. Retrospectively registered 28 June 2019, first enrollment 6 June 2019 [ABSTRACT FROM AUTHOR]

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Rasd1, also known as Dexras1 or AGS1, is rapidly induced by dexamethasone (Dex). While prior data indicates that Rasd1 is highly expressed in the pituitary and that the gene may function in regulation of corticotroph activity, its exact cellular localization in this tissue has not been delineated. Nor has it been determined which endocrine pituitary cell type(s) are responsive to Dex-induced expression of Rasd1. We hypothesized that Rasd1 is primarily localized in corticotrophs and furthermore, that its expression in these cells would be upregulated in response to exogenous Dex administration. Rasd1 expression in each pituitary cell type both under basal conditions and 1-hour post Dex treatment were examined in adult male mice. While a proportion of all endocrine pituitary cell types expressed Rasd1, a majority of corticotrophs and thyrotrophs expressed Rasd1 under basal condition. In vehicle treated animals, approximately 50–60% of corticotrophs and thyrotrophs cells expressed Rasd1 while the gene was detected in only 15–30% of lactotrophs, somatotrophs, and gonadotrophs. In Dex treated animals, Rasd1 expression was significantly increased in corticotrophs, somatotrophs, lactotrophs, and gonadotrophs but not thyrotrophs. In Dex treated animals, Rasd1 was detected in 80–95% of gonadotrophs and corticotrophs. In contrast, Dex treatment increased Rasd1 expression to a lesser extent (55–60%) in somatotrophs and lactotrophs. Corticotrophs of the pars intermedia, which lack glucocorticoid receptors, failed to display increased Rasd1 expression in Dex treated animals. Rasd1 is highly expressed in corticotrophs under basal conditions and is further increased after Dex treatment, further supporting its role in glucocorticoid negative feedback. In addition, the presence and Dex-induced expression of Rasd1 in endocrine pituitary cell types, other than corticotrophs, may implicate Rasd1 in novel pituitary functions. [ABSTRACT FROM AUTHOR]

RNAs are highly regulated at the post-transcriptional level in neurodegenerative diseases and just a few mutations can significantly affect the fate of neuronal cells. To date, the impact of G-quadruplex (G4) regulation in neurodegenerative diseases like Parkinson's disease (PD) has not been analysed. In this study, in silico potential G4s located in deregulated genes related to the nervous system were initially identified and were found to be significantly enriched. Several G4 sequences found in the 5ʹ untranslated regions (5ʹUTR) of mRNAs associated with Parkinson's disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5ʹUTRs of these candidates upstream of a luciferase reporter system led to the demonstration that the G4s of both Parkin RBR E3 Ubiquitin Protein Ligase (PRKN) and Vacuolar Protein Sorting-Associated Protein 35 (VPS35) significantly repressed the translation of both genes in SH-SY5Y cells. Subsequently, a strategy of using label-free RNA affinity purification assays with either of these two G4 sequences as bait isolated the Guanine Nucleotide-Binding Protein-Like 1 (GNL1). The latter was shown to have a higher affinity for the G4 sequences than for their mutated version. This study sheds light on new RNA G-quadruplexes located in genes dysregulated in Parkinson disease and a new G4-binding protein, GNL1. [ABSTRACT FROM AUTHOR]

The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization. [ABSTRACT FROM AUTHOR]

There is a need for acceptable gluten-free foods that provide greater protein. Rice is used in gluten-free products and new higher protein rice varieties now exist. Stores surveyed for products with rice flour as the main ingredient showed 36% snacks and 13% breads, with baked snacks 1.3 g protein/serving. Bread was chosen as the focus for testing higher protein rice flour (HPR). HPR were compared to commercial rice flours (CW) for pasting properties, protein, fat, and fiber content. Gluten-free bread was prepared and tested for color and texture. A consumer sensory study rated appearance, aroma, texture, and taste of the breads. Brown and white HPR were 10.1% and 9.25% protein. There were no differences in acceptance of breads made with any flour. Health benefit information for HPR breads increased purchase intent. Rice flour with greater protein can be used in gluten-free rice flour baked goods without affecting properties. [ABSTRACT FROM AUTHOR]

Small GTPases (e.g., Rac1) play key roles in glucose-stimulated insulin secretion (GSIS) in the β-cell. We investigated regulation by RhoGDIβ of glucose-induced activation of Rac1 and insulin secretion. RhoGDIβ is expressed in INS-1 832/13 cells, rodent and human islets. siRNA-mediated knockdown of RhoGDIβ in INS-1 832/13 cells significantly attenuated glucose-induced Rac1 activation without affecting its translocation and membrane association. Further, suppression of RhoGDIβ expression exerted minimal effects on GSIS at the height of inhibition of Rac1 activation, suggesting divergent effects of RhoGDIβ on Rac1 activation and insulin secretion in the glucose-stimulated β-cell. We provide the first evidence for the expression of RhoGDIβ in rodent and human β-cells, and its differential regulatory roles of this protein in G protein activation and GSIS. Abbreviations: Arf6: ADP ribosylation factor; Cdc42: Cell Division Cycle; GAP: GTPase-activating protein; GDI: GDP dissociation inhibitor; GDIα: GDP dissociation inhibitorα; GDIβ: GDP dissociation inhibitorβ; GEF: Guanine nucleotide exchange factor; GSIS: Glucose-stimulated insulin secretion; Rac1: Ras-Related C3 Botulinum Toxin Substrate 1 [ABSTRACT FROM AUTHOR]

According to structural and functional similarities, small GTP-binding proteins can be divided into five distinct families, in which, Ras-related nuclear protein (Ran) GTPases regulates proteins and RNA across nuclear envelope. To excavate differentially expressed proteins in the pistil after pollination, proteomics was recruited. One protein spot was identified as BoRAN3, was down-regulated expressed in the pistil. Phylogenetic tree assay found RAN3 in Brassica oleracea, Eutrema salsugineum, Brassica napus and Brassica rapa grouped into one clade. Compared BoRAN3 expression levels in reproductive organs found it was expressed in all five organs, specifically highly in the pistil. qRT-PCR assays found that BoRAN3 was up-regulated expsed in the early pollination process. Sub-cellular assay demonstrated that BoRAN3 located in the nucleus. GUS assay found that BoRAN3 was expressed in the cotyledon, root, stoma, lateral root primordia, petal, filigree and both the front and the bottom of pistil. Taken together, our results demonstrated that BoRAN3 was highly expressed in the pistil and up-regulated expressed in the pistil during early pollination process. Based on those results, we speculated that the up-regulated expressed of BoRAN3 in the pistil during early pollination process hints that it may related to pollen germination and pollen tube development. [ABSTRACT FROM AUTHOR]

Septins, a conserved family of GTP-binding proteins, are widely recognized as an essential cytoskeletal component, playing important roles in a variety of biological processes, including division, polarity, and membrane remodeling, in different eukaryotes. Although the roles played by septins were identified in the model organism Saccharomyces cerevisiae, their importance in other fungi, especially pathogenic fungi, have recently been determined. In this review, we summarize the functions of septins in pathogenic fungi in the cell cycle, autophagy, endocytosis and invasion host-microbe interactions that were reported in the last two years in the field of septin cell biology. These new discoveries may be expanded to investigate the functions of septin proteins in fungal pathogenesis and may be of wide interest to the readers of Microbiology and Molecular Pathology. [ABSTRACT FROM AUTHOR]

Regulators of G protein signaling (RGSs) are proteins that negatively regulate G protein signal transduction. In this study, seven putative RGSs were characterized in the nematode-trapping (NT) fungus, Arthrobotrys oligospora. Deleting Rgs genes significantly increased intracellular cAMP levels, and caused defects in mycelia growth, stress resistance, conidiation, trap formation, and nematocidal activity. In particular, the ΔAoFlbA mutant was unable to produce conidia and traps. Transcriptomic analysis showed that amino acid metabolic and biosynthetic processes were significantly enriched in the ΔAoFlbA mutant compared to WT. Interestingly, Gas1 family genes are significantly expanded in A. oligospora and other NT fungi that produce adhesive traps, and are differentially expressed during trap formation in A. oligospora. Disruption of two Gas1 genes resulted in defective conidiation, trap formation, and pathogenicity. Our results indicate that RGSs play pleiotropic roles in regulating A. oligospora mycelial growth, development, and pathogenicity. Further, AoFlbA is a prominent member and required for conidiation and trap formation, possibly by regulating amino acid metabolism and biosynthesis. Our results provide a basis for elucidating the signaling mechanism of vegetative growth, lifestyle transition, and pathogenicity in NT fungi. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a hypertensive disorder of uncertain etiology that is the leading cause of maternal and fetal morbidity or mortality. The dysregulation of microRNAs (miRNAs) has been highlighted as a potential factor involved in the development of PE. Therefore, our study investigated a novel miRNA, miRNA 183 (miR- 183), and its underlying association with PE. Expression of miR-183, forkhead box P1 (FOXP1), and G protein subunit gamma 7 (GNG7) in placental tissues of patients with PE was determined. Gain- and loss-of-function experiments were conducted to explore modulatory effects of miR-183, FOXP1, and GNG7 on the viability, invasion, and angiogenesis of trophoblast cells in PE. Finally, we undertook in vivo studies to explore effects of FOXP1 in the PE model. The results revealed suppressed expression of FOXP1 and significant elevations in miR-183 and GNG7 expression in placental tissues of PE patients. FOXP1 was observed to promote proliferation, invasion, and angiogenesis in human chorionic trophoblastic cells. miR-183 resulted in depletion of FOXP1 expression, while FOXP1 was capable of restraining GNG7 expression and promoting the mTOR pathway. The findings confirmed the effects of FOXP1 on PE. In conclusion, miR-183 exhibits an inhibitory role in PE through suppression of FOXP1 and upregulation of GNG7. [ABSTRACT FROM AUTHOR]

Sulfonation is an important high affinity elimination pathway for phenolic compounds. In this study sulfonation of 7-hydroxycoumarin and 13 its derivatives were evaluated in liver cytosols of human and six animal species. 7-hydroxycoumarin and its derivatives are strongly fluorescent, and their sulfate conjugates are nonfluorescent at excitation 405 nm and emission 460 nm. A convenient fluorescence based kinetic assay of sulfonation was established. The sulfonation rate of most of the 7-hydroxycoumarin derivatives was low in liver cytosol of human and pig, whereas it was high with most compounds in dog and intermediate in rat, mouse, rabbit, and sheep. Sulfonation of the 7-hydroxycoumarin derivatives followed Michaelis-Menten kinetics with Km values of 0.1–12 µM, Vmax of 0.005–1.7 µmol/(min * g protein) and intrinsic clearance (Vmax/Km) of 0.004–1.9 L/(min * g cytosolic protein). Fluorescence based measurement of sulfonation of 7-hydroxycoumarin derivatives provides a sensitive and convenient high-throughput assay to determine sulfonation rate in different species and tissues and can be applied to evaluate sulfonation kinetics and inhibition. [ABSTRACT FROM AUTHOR]

The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract. [ABSTRACT FROM AUTHOR]

Hesperidin, a citrus flavonoid, can exert numerous beneficial effects on human health. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract. In the present study, we investigated potential effects of hesperidin on pacemaker potential of ICC in murine small intestine and GI motility. A whole-cell patch-clamp configuration was used to record pacemaker potential in ICC, and GI motility was investigated in vivo by recording gastric emptying (GE) and intestinal transit rate (ITR). Hesperidin depolarized pacemaker potentials of ICC in a dose-dependent manner. Pre-treatment with methoctramine or 4-DAMP did not inhibit hesperidin-induced pacemaker potential depolarization. Neither a 5-HT3 receptor antagonist (Y25130) nor a 5-HT7 receptor antagonist (SB269970) reduced the effect of hesperidin on ICC pacemaker potential, whereas the 5-HT4 receptor antagonist RS39604 was found to inhibit this effect. In the presence of GDP–β–S, hesperidin-induced pacemaker potential depolarization was inhibited. Moreover, in the presence of U73122 and calphostin C, hesperidin did not depolarize pacemaker potentials. Furthermore, hesperidin accelerated GE and ITR in vivo. These results imply that hesperidin depolarized ICC pacemaker potential via 5-HT4 receptors, G protein, and PLC/PKC dependent pathways and that it increased GI motility. Therefore, hesperidin may be a promising novel drug to regulate GI motility. [ABSTRACT FROM AUTHOR]

The adverse effects of sleep disorders on male fertility are of increased concern. In this study, a rat model of chronic sleep restriction (CSR) was established using the modified multiplatform method. The effects of CSR on the fertility of male rats were evaluated first based on sexual behavior. Serum hormones, including testosterone (T), prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and sperm parameters (concentration, viability, motility, deformation rate) were measured, and testicular histology was analysed by hematoxylin and eosin staining. The transcriptional differences between CSR rats and control rats were detected by RNA sequencing (RNA-Seq), and DNA methylation was then detected by bisulfite sequencing. After the differentialy expressed genes of CSR rats were sequenced and screened, representative up- and down-regulated genes were randomly sampled to verify the sequencing results by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Finally, functional annotations were completed, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomic (KEGG) pathway analyses. The results showed that the sexual behavior of CSR rats did not change when compared with control group rats. The sperm concentration, viability and motility of the CSR rats decreased significantly, while the sperm malformation rate increased significantly. In the KEGG pathway analysis database, some specific differentially expressed genes were screened, which are involved in metabolic pathways, inflammation-related pathways, the renin-angiotensin system, as well as others. However, the aforesaid differentially expressed genes in the testes were not related to their DNA methylation status. CSR could significantly reduce the fertility of male rats, and one of its mechanisms occurs by altering gene expression in the testes, which is not related to their state of DNA methylation. The results of this study suggest that CSR could cause male infertility by significantly altering the testicular transcriptome. Abbreviations: CSR: chronic sleep restriction; SD: sleep deprivation; RNA-Seq: RNA sequencing; NGS: next generation sequencing; qRT-PCR: real-time quantitative reverse transcription polymerase chain reaction; KEGG: Kyoto encyclopedia of genes and genomic; NO: nitric oxide; INOS: Inducible nitric oxide synthase; Il6: interleukin-6; Tnf: tumour necrosis factor alpha; Hsd11b1: hydroxysteroid 11-beta dehydrogenase 1; Dnmt3a: DNA methyltransferase 3Ax; PSD: paradoxic sleep deprivation; DNMTs: DNA methyltransferases family; REM: rapid eye movement sleep; PGD: preimplantation genetic diagnosis; PGS: preimplantation genetic screening; ECS: expanded carrier screening; T: testosterone; FSH: follicle stimulating hormone; LH: luteinizing hormone; PRL: prolactin; BC group: Blank Control group; MC group: Model Control group; Hist1h2ba: histone cluster 1 H2ba; Lgr4: leucine-rich repeat-containing G protein-coupled receptor 4; Atrn: attractin ; Ogg1: 8-oxoguanine DNA glycosylase; SNVs: single nucleotide variants ; HPG axis: hypothalamic-pituitary-adrenal axis; Star protein: steroid acute regulatory protein; Dmac2l: distal membrane arm assembly complex 2 like; Esr1: estrogen receptor 1; MAPK pathways: mitogen-activated protein kinase pathways; Sos2: SOS Ras/Rho guanine nucleotide exchange factor 2; Jak2: Janus kinase 2; Pik3cb: phosphatidylinositol-4,5-bisphosphate 3-kinase, and catalytic subunit beta; Kras: KRAS proto-oncogene and GTPase; RRBS: reduced representation bisulfite sequencing; DEGs: differently expressed genes; SPF: Specific Pathogen Free; HE: hematoxylin & eosin; DMR: differentially methylated region; GO Analysis: Gene Ontology analysis; SINE: short interspersed nuclear elements; LINE: long interspersed nuclear elements; LTR: long terminal repeats [ABSTRACT FROM AUTHOR]

Purpose: Recent studies have shown that growth-associated protein-43 (GAP-43) may influence the mitotic-spindle orientation of Madin-Darby Canine Kidney (MDCK) cells through interacting with G proteins in vitro. However, whether GAP-43 interacts with the G proteins under the influence of mitotic spindle positioning related to the orientation of cell division during neurogenesis remains unclear. In order to explore the molecular mechanism in vivo, the GAP-43 transgenic mice were produced and the angles of cell division in the ventricular zone (VZ) during neurogenesis (embryonic period between 13.5 and 17.5 days) were measured in both transgenic mice and wild type mice by spindle angle analysis. Materials and methods: The interaction of GAP-43 and Gαi was detected by co-immunoprecipitation (co-IP), whereas the localization of GAP-43 was determined by immunofluorescence. Results: The results obtained using co-IP and immunofluorescence showed that GAP-43 is localized on the cell membrane and interacts with Gαi. This interaction dramatically induced a significant increase in the proportion of horizontally and intermediately dividing cells during the embryonic period of 13.5 days in the transgenic mouse brain, as observed by spindle angle analysis. Conclusions: It can be concluded that GAP-43 is involved in the orientation of cell division by interacting with Gαi, and that this may be an important mechanism for neurogenesis in the mammalian brain. [ABSTRACT FROM AUTHOR]

GTP binding protein overexpressed in skeletal muscle (GEM) is an important gene with many functions, such as regulating the rearrangement of cytoskeleton and the activity of voltage-dependent calcium channel, and GEM was regarded as a candidate gene for obesity. However, little investigation has been carried out to explore whether GEM affected the intramuscular fat (IMF) deposition of goat. To explore the role of GEM gene in goat, this gene was cloned and its tissue and temporal expression profile were detected. Effect of GEM on adipogenesis was examined by losing function of GEM in vitro. Thereafter, several lipid metabolism-related genes were examined, including CCAAT/enhancing-binding protein α (C/EBPα), CCAAT/enhancing-binding protein β (C/EBPβ), lipoprotein lipase (LPL), preadipocyte factor 1 (Pref-1), peroxisome proliferator activated receptor γ (PPARγ) and sterol regulatory element binding protein 1 (SREBP1). We found that the goat GEM gene consisted of 936 bp, which encoded a protein of 311 amino acids. The expression of GEM was higher in spleen, lung and large intestine and it appeared sharp in the interim stage of differentiation. Furthermore, GEM knockdown blocked adipogenesis and the expression of C/EBPα, C/EBPβ, LPL, PPARγ and SREBP1. These results indicated that GEM might promote lipid accumulation and adipogenesis. [ABSTRACT FROM AUTHOR]

Plum peel is the by-product of plum processing and has all the functional properties of the plum fruit. The aim of this study was to investigate chemical composition of black plum peel and to use it as a functional ingredient to produce Masghati. The components of black plum peel in 100g were 3.40 g protein, 0.66 g fat, 13.73 g total sugars, and 2.50 g crude fiber. Plum peel is a good source of Ca, P, Zn, and ascorbic acid (201.29, 88.60, 0.97, and 25 mg/100 g raw materials, respectively). The amount of antioxidant and phenolic compounds in black plum peel was significant (88.59% and 105.91 mg/g GA, respectively). Heavy metals contained Hg, Cd, and Pb and chlorine and organophosphorus pesticides were very low and unrecognizable in black plum peel. Gelatin (1.5, 1.8, and 2.3%) and black plum peel puree (14, 27, and 41%) used for Masghati formulations and chemical properties, sensory evaluations, and texture profile analysis (TPA) of Halva Masghati samples were investigated. The results of texture evaluation showed that hardness and chewiness increased by increasing gelatin and puree, while adhesiveness decreased (P < .05). Cohesiveness increased by addition of the gelatin (P < .05). Sensory evaluations showed that total acceptance increased by black plum peel puree, while decreased by gelatin concentration (P < .05). Masghati including 1.8% gelatin and 41% puree had the lowest amount of hardness, adhesiveness, chewiness, and suitable color characteristics, was determined as the best formulation among the other investigated samples. [ABSTRACT FROM AUTHOR]

Atherosclerosis is the chronic inflammatory disease, and inflammation-elicited endothelial activation is an early event in the development of atherosclerosis. The P2Y11 receptor is a purinergic receptor and a member of the P2 family of G coupled protein which has been shown to modulate vascular function. Progress in the study of purine receptors has been tremendous and these receptors have become pharmacological targets for various diseases. In this study, we show that the P2Y11R antagonist NF157 can mitigate oxidized LDL (ox-LDL)-induced endothelial inflammation. Our study demonstrates that P2Y11R is expressed to a fair degree in human aortic endothelial cells and is induced by treatment with ox-LDL. Blockage of P2Y11R by its selective antagonist NF157 ameliorates ox-LDL-induced adhesion of THP-1 monocytes to endothelial cells. NF157 inhibits ox-LDL-induced expression of adhesion molecules including E-selectin and VCAM-1. NF157 also suppresses ox-LDL-associated ROS production and induction of the NADPH oxidase subunit NOX-4. Moreover, NF157 has an inhibitory effect on the production of major cytokines including IL-6 and TNF-α. Mechanistically, we show that NF157 mitigates ox-LDL-induced phosphorylation of MAPK kinase p38 and NF-κB activation. Our findings indicate that blockage of P2Y11R signalling by its antagonist NF157 may protect endothelial cells from ox-LDL-induced endothelial inflammation. Therefore, NF157 may have therapeutic implications in the modulation of atherosclerosis-associated inflammation. [ABSTRACT FROM AUTHOR]

Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes. [ABSTRACT FROM AUTHOR]

The characteristics and functional properties of gelatin obtained from Chinese giant salamander (Andrias davidianus) skin were investigated. After alkaline and acetic acid pre-treatment, yield of the gelatin by hot water extraction (60°, 8 h) was 17.63 g/100 g (wet basis), and the gelatin contained 89.46 g/100 g protein, 0.3 g/100 g fat, and 0.41 g/100 g ash (dry basis). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results of the gelatin showed clear band pattern for collagen subunits,β- and α-chains. The gelatin contained 18.85% imino acids, 28.62% hydrophobic amino acids, and 19.68% essential amino acids, which is slightly lower than porcine skin gelatin (PSG). The gelatin exhibited thermo-reversible properties, with gel strength of 192.50 ± 11.47 g, rupture distance of 10.17 ± 0.20 mm, gelling temperature of 4.98°, and melting temperature of 18.04°, Emulsifying activity index (EAI) of the gelatin diminished with increasing concentrations from 10 to 40 mg/mL (p <.05), whereas the emulsifying stability index (ESI) improved significantly (p <.05). Higher concentrations (30 and 40 mg/mL) could significantly enhance the gelatin's foam expansion (FE) and foam stability (FS) (p <.05). Water absorption ability and oil absorption ability of the gelatin were 7.1 g water/g gelatin and 9.8 g oil/g gelatin, respectively. These results imply that the gelatin with good interfacial properties could potentially be used as a novel ingredient in food emulsion systems. Abbreviations: CGSS, Chinese giant salamander skin; CSG, Chinese giant salamander skin gelatin; PSG, porcine skin gelatin; EAI, emulsion activity index; ESI, emulsion stability index; FE, foam expansion; FS, foam stability; WAC, water absorption capacity; OAC, oil absorption capacity. [ABSTRACT FROM AUTHOR]

Sea cucumbers belonging to the Holothuroidea class are a group of worm-like and soft-bodied echinoderms that live in nearly all marine environments. Mediterranean Holothuria tubulosa is an economic sea cucumber species found in Turkish seas. This study aimed to present the first report of detailed amino acid profile and investigate the biochemical and nutritional characteristics of H. tubulosa seasonally collected from one of the densest natural beds, located at Çanakkale Strait (Southern Coast of Marmara Sea), Turkey. According to our results, the highest protein, lipid, and ash contents (%) were found to be 10.2, 1.9, and 5.1, respectively. The highest percentages of saturated, monounsaturated, and polyunsaturated fatty acids were 42.9, 28.3, and 37.8, respectively. Total amino acids (mg/g crude protein) ranged between 867.2 and 974.0 among all seasons. All essential amino acids were found in body wall tissues and ranged between 7.1 and 5.9. Mineral composition was found to be desirable at the recommended and threshold levels. In general, the proximate composition of H. tubulosa was directly affected by seasons (p <.05); however, amino acid, fatty acid, and mineral compositions were found to be similar among all seasons, except summer (p >.05). [ABSTRACT FROM AUTHOR]

Chemotaxis, which is chemoattractant-guided directional cell migration, plays major roles in recruitment of neutrophils, the metastasis of cancer cells, and the development of the model organism Dictyostelium discoideum. These cells share remarkable similarities in the signaling pathways by which they control chemotaxis. They all use a G protein-coupled receptor (GPCR)-mediated signal transduction pathway to sense the chemotactic gradient to guide cell migration. Diverse chemokines activate Rac through conserved GPCR signaling pathways. ELMO proteins are an evolutionarily conserved, essential component of the ELMO/Dock complex, which functions as a guanine nucleotide exchange factor (GEF) for small G protein Rac activation. The linkages between the GPCR-initiated gradient sensing compass and the Rac-mediated migrating machinery have long been missing. Here, we summarize recent findings on ELMO proteins that directly interact with G protein and transduce GPCR signaling to control the reorganization of actin-based cytoskeleton through regulating Rac activation during chemotaxis, first in D. discoideum and then in mammalian cancer cells. This represents an evolutionarily conserved signaling shortcut from GPCR to the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

The processing of fish in general entails quantities of by-products, such as exoskeleton and cephalothorax of shrimp, which may reach 70%. Protein recovered from by-products has been investigated in recent years for its ability to offer more nutritious foods and improved functional properties. The objective of the present study was the use of the pH shifting process to recover proteins from solubilization and precipitation that are present in the by-products (heads and shells) of white shrimp (Litopenaeus vannamei). The proximal composition of the by-products presented 58.5% protein (d.b). The isoelectric point used to precipitate the protein was defined at pH 4. The protein concentrate presented 73.1% of protein. The yield of the protein concentrate mass resulted in 47.8% (w.b). The greatest solubility found for the protein concentrate was 51.3% at pH 8. The oil and water retention capacity resulted in values of 8.5 and 2.5 mL/g of protein, respectively, and the highest emulsifying capacity was found at pH 8. The by-products and concentrate amino acids profiles confirmed the presence of 16 amino acids. In general, the shrimp protein concentrate obtained showed the potential to be included as an ingredient in food formulations. [ABSTRACT FROM AUTHOR]

Arf GTPase-activating proteins (Arf GAPs) were first identified as regulators of the small GTP-binding proteins ADP-ribosylation factors (Arfs). The Arf GAPs are a large family of proteins in metazoans, outnumbering the Arfs that they regulate. The members of the Arf GAP family have complex domain structures and some have been implicated in particular cellular functions, such as cell migration, or with particular pathologies, such as tumor invasion and metastasis. The specific effects of Arfs sometimes depend on the Arf GAP involved in their regulation. These observations have led to speculation that the Arf GAPs themselves may affect cellular activities in capacities beyond the regulation of Arfs. Recently, 2 Arf GAPs, ASAP1 and AGAP1, have been found to bind directly to and influence the activity of myosins and kinesins, motor proteins associated with filamentous actin and microtubules, respectively. The Arf GAP-motor protein interaction is critical for cellular behaviors involving the actin cytoskeleton and microtubules, such as cell migration and other cell movements. Arfs, then, may function with molecular motors through Arf GAPs to regulate microtubule and actin remodeling. [ABSTRACT FROM AUTHOR]

Cold-stored fruits of the loquat (Eriobotrya japonica Lindl.) plant suffer from strong lignification, which leads to a hard texture and a crude mouthfeel of the pulp. However, the signalling mechanisms of loquat fruits under cold storage remain unclear. In this study, the heterotrimeric G protein α subunit (Gα) gene (EjLGA1) from loquat was isolated, and in silico analysis was performed. The levels of EjLGA1 expression in cold-stored loquat fruits with different pretreatments were also investigated. The largest open-reading frame of EjLGA1 is 1173 bp, encoding a polypeptide of 390 amino acids. The nucleotide and amino acid sequences of the Gα subunit are highly conserved among loquat and 48 other seed plants, indicating that the loquat Gα subunit likely has similar functions in other plants. A reliable model of the loquat Gα subunit was constructed, which revealed the structure of the subunit. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed that EjLGA1 is highly expressed under cold storage, which suggests the significance of EjLGA1 function in loquat fruits in response to cold stress. However, ethephon and 1-methylcyclopropene (1-MCP) pretreatments did not regulate EjLGA1expression. These results provide new insight into the signalling mechanism in cold-stored loquat fruits. [ABSTRACT FROM AUTHOR]