Giardia duodenalis is one of the most common enteric protozoan parasites in vertebrates, such as humans, domestic and wild animals, causing giardiasis. To the best of our knowledge, little is known about the genetic diversity of G. duodenalis assemblages. This study aimed to identify genetic diversity of G. duodenalis assemblages in Iranian stray dogs.glutamate dehydrogenase gene.A total of 450 fecal samples were collected from 2015 to 2016 from stray dogs of Northwest Iran. All specimens were observed microscopically following concentration and flotation techniques. Subsequently, DNA samples were extracted, amplified, and sequenced targeting the G. duodenalis in infected dogs was estimated at 1.6%, based on microscopic and molecular diagnoses. Sequencing and phylogenetic analyses indicated a high level of genetic diversity of assemblage C .The overall prevalence of G. duodenalis assemblage C haplotypes is unequivocally circulates among stray dogs in Northwest Iran.The pairwise sequence distances between the identified isolates of assemblage C showed an intradiversity of 0.3%-1.3% and identity of 98.7%-100%. Current findings indicate that a significant genetic diversity of  Giardia duodenalis  is one of the zoonotic protozoan flagellate unicellular parasites that infect a wide range of vertebrates, such as humans and dogs. Giardiasis occurs with ingestion of the cysts, either through contaminated water, food, and materials or through host-to-host contact [G. duodenalis from different host species are morphologically indistinguishable. However, some molecular methods using semi-conserved genes include glutamate dehydrogenase (gdh) [small subunit ribosomal locus (18S rRNA) [elongation factor-1 (ef-1) [triose phosphate isomerase (tpi) [G. duodenalis (A-H) have been reported [The relationship between humans and dogs was discovered historically 15,000 years ago . It esti contact ,4. Genot8S rRNA) , elongat1 (ef-1) , and trise (tpi) ,9. Basedreported .G. duodenalis; however, potentially zoonotic assemblages found in humans (assemblages A and B) may have also been isolated from dogs [Assemblages A and B isolates are potentially zoonotic and have been reported in a broad range of hosts, including humans, dogs, cats, livestock, and wildlife ,11. In crom dogs ,19.G. duodenalis from those that have zoonotic potential, using a molecular approach, such as PCR-based procedures [In terms of a public health perspective, it is imperative to distinguish specific host assemblages (assemblages A and B) from ocedures .G. duodenalis assemblages in the canine community, particularly in Zanjan of Northwest Iran.According to the previous studies, it demonstrated the prevalence of different infections with zoonotic genotypes in different geographical areas; 5.5% in Australia, 61% in Thailand, and 80.5% in Belgium . There aG. duodenalis assemblages isolated from infected stray dogs to integrate the baseline data on canine genetic diversity in this part of Iran.This phylomolecular study was aimed to determine the infection rate and heterogeneity features of The study proposal was reviewed and approved by the Institutional Research Ethics Committee of Zanjan University of Medical Sciences (Reference number A-12-153-8).The study was conducted on stray dogs in the city of Zanjan , which is located in Northwest Iran. Agriculture and animal husbandry are the most important occupations of the inhabitants of this province. There were 2,366,411 farm animals in the province . The stugdh gene. G. duodenalis cysts were isolated from fecal samples using a sucrose gradient centrifugation technique [A total of 450 fecal samples from stray dogs were collected from selected areas of Zanjan Province, such as streets and farms in rural areas of different parts of Zanjan city. Fecal samples were placed in labeled sterile “Ziploc” bags and immediately transported to the research center of the Zanjan University of Medical Sciences, on the same day of sample collection. All samples were examined by microscopy and later analyzed by the polymerase chain reaction (PCR) targeting the echnique . The pregdh gene was amplified with primers reported by Read et al. [DNA was extracted from 200 mg of each fecal sample using the AccuPrep Stool DNA extraction kit . The manufacturer’s protocol was used with the following modifications: 600 μL of digestion buffer  was mixed with samples. Glass pearls (0.45-0.52 mM in diameter) were added to the mixtures, and the samples were vortexed for 10 min. Subsequently, these samples were subjected to seven cycles of freeze/thawing using liquid nitrogen and boiling water to disrupt the cyst walls. At this stage, 20 μL of proteinase K  and 40 μL of 2% sodium dodecyl sulfate were added to each sample and placed in a water bath at 60°C overnight. Finally, the parasite DNA was extracted according to the protocol recommended by the manufacturer. The concentration of extracted DNAs was measured by NanoDrop . The extracted DNAs were stored at −20°C until analysis. A DNA fragment (432 bp) from the gdh gene in both directions using the GDHeF/GDHiF primers. The ambiguity sites of sequences were edited according to the reference sequence (RefSeq) using the Sequencher Tm v.4.1.4 software based on IUPAC codes . To assess the genetic diversity, the DnaSP software was used according to the analysis of molecular variance (AMOVA) to calculate the diversity of haplotypes (Hd); nucleotide diversity (π); and number of haplotypes (Hn). A sequence distance matrix pairwise was computed using the DNASTAR MegAlign program to show the percent identity (%) and intraspecies diversity of G. duodenalis assemblages between geographical sequences of the gdh gene. To authenticate the genetic relationships between identified G. duodenalis assemblages, a phylogenetic tree was generated by the MEGA 5.05 software  based on the maximum likelihood algorithm and the Kimura 2-parameter model. The distance scale was estimated at 0.02. G. ardeae  was addressed as an outgroup branch. Multiple alignments between the amino acid and nucleotide sequences of G. duodenalis were performed based on Clustal W method .Amplicons from positive fecal samples were sequenced  targeting the gdh gene were identified in 7 (1.6%) samples consistent with G. duodenalis. Sequence analysis revealed that seven positive samples belonged to assemblage C. In the consensus position of 314 bp in the assemblage, 12 variable (polymorphic) sites were also detected  (Giardia assemblage C showed an intraspecies diversity of 0.3%-1.3% and identity of 98.7%-100% (Microscopic examination and PCR amplification (432 bp fragment) of the detected . The num; 0.802) . The pai.7%-100% .Indel) mutations were found in G. duodenalis assemblage C. In comparison to globally RefSeqs, the multiple amino acid alignments of G. duodenalis assemblage C (Zan1*-Zan7*) indicated the occurrence of two non-synonymous substitutions in codons 72, 85, and 100, where Seine (S) replaced a leucine (L), asparagine (N) replaced a S, and L replaced a proline (P), respectively  is placed in its specific clade adjacent to assemblage D clade  samples to their humans in the same spatial and temporal settings [On the other hand, it should be noted that the preferred option to conclusively determine the zoonotic transmission of settings .G. duodenalis isolates from stray dogs in the city of Zanjan, northwest of Iran. In our study, G. duodenalis was detected 1.6% of fecal samples with microscopic examination and PCR method. Other studies in Iran have reported a low prevalence of G. duodenalis infections using microscopic examination, such as 0.07% of the prevalence found in 98 stray dogs and 0.09% of the infection rate with G. duodenalis of 100 samples studied [G. duodenalis using PCR analysis [G. duodenalis infection in the canine population. Silva et al. [G. duodenalis infection [et al. [Giardia found in 359 samples [G. duodenalis infection, such as canine population  and the use of diagnostic methods  [The current study was conducted to determine the genotype diversity of  studied ,35. Our analysis . Howevera et al.  showed tnfection , and lat [et al.  also rep samples . Taken i or PCR) .G. duodenalis in dogs’ feces compared to microscopy [Giardia in our canine communities. However, recent molecular tools, such as a PCR-based diagnostic system, and sequencing with housekeeping genes, such as 18S rRNA, bg, gdh, ef-1, and tpi, are the most frequently used loci for the differentiation of Giardia at genotype levels [From this study, the detection method was considered the main factor affecting the prevalence rates , althougcroscopy . Surprise levels ,41.gdh loci has been used to differentiate the common assemblages of G. duodenalis and genetic diversity, the gdh locus proved to be stable for the isolates identified in different host species and geographic locations [G. duodenalis showed the presence of C genotype. It is noteworthy to mention that all genotypes were canine adapted and were not found isolates from other zoonotic assemblages. Our results are in line with Abe et al. [gdh loci that showed canine-specific genotypes, assemblage D [Giardia genotypes and assemblages D and C in dog-specific strains of G. intestinalis has been reported [et al. [gdh coding genes and showed only the host-adapted assemblages C and D  [et al. [bg, gdh, and tpi genes demonstrated that genetic assemblages C and D of G. duodenalis were present among dogs in China [Since the ocations ,16,42,43e et al.  study thmblage D . The prereported . Ouza et [et al.  conductectively) . Zhang e [et al.  in a stuin China . Based oin China ,47.18SrDNA, bg, and tpi, reported different zoonotic assemblages. A study in China conducted by Zheng et al. [et al. [gdh and β-giardin, a number of inter-assemblage mixed infections, including A+B, A+D, and A+B+D, have also been reported. Genotyping and further subtyping have relied primarily on the sequence analysis on fragments of single-copy gene targets, such as tpi, b-giardin (bg), or gdh. However, this approach is now being reexamined because mixed infections may not always be distinguished by analyzing a single-locus and conflicting classification between loci [Giardia genotyping of stray dogs in this area. Although no zoonotic assemblages have been reported in the present research, study in canine and human populations using genotyping based on multilocus sequences and other valid molecular tools to obtain comprehensive information about G. intestinalis is strongly recommended.In contrast to our results, other studies with one or multiloci, such as g et al.  demonstrg et al. . In a stg et al. . Adell-A [et al.  also sho [et al. . Resultseen loci . This fiG. duodenalis assemblage C haplotypes is unequivocally circulating among stray dogs in Northwest Iran. This is therefore highly recommended for future studies with a larger sample size conducted in different parts of Iran to identify the genetic diversity of G. duodenalis in stray dogs and the association with human pathogenic strains of this protozoan parasite.Our finding indicates, for the first time, that the significant genetic diversity of AF, AN, and AH conceived and designed the experiments. AS, AF, and MHK contributed to the analysis and interpretation of data. MHK wrote the manuscript, while AK, ASi, VN, and RN assisted in writing and revision of the manuscript. All authors have read and approved the final manuscript.


In a signaling network, not only the functions of molecules are important but when  and where  those functions are exerted and orchestrated is what defines the signaling output. To temporally and spatially modulate signaling events, cells generate specialized functional domains with variable lifetime and size that concentrate signaling molecules, enhancing their transduction potential. The plasma membrane is a key in this regulation, as it constitutes a primary signaling hub that integrates signals within and across the membrane. Here, we examine some of the mechanisms that cells exhibit to spatiotemporally regulate signal transduction, focusing on the early events of T cell activation from triggering of T cell receptor to formation and maturation of the immunological synapse. Complex organisms have evolved intricate molecular networks in which a single molecule can potentially participate in multiple pathways, performing signaling and/or structural roles. In such cellular networks, the functionality of molecules is defined by their structure, their potential interactors, and by their cellular context, that is, by their localization and time of action, which maximizes the potential capability of each molecule. Segregating signals spatiotemporally to control “when” and “how much” a stimulus is communicated is key for specificity and modulation, essential features of cellular signal transduction. Extracellular signals are transduced into the cell through the plasma membrane. On one hand, the plasma membrane acts as primary cell barrier, conferring self-containment and physical support to the cell. On the other hand, it constitutes a three-dimensional sensory and signaling platform that integrates signals in the lateral and axial planes , aiding 3, leading to recruitment of several proteins involved in rearrangement of the actin cytoskeleton, essential for cell polarization and formation of the IS.T cells perfectly exemplify the spatiotemporal regulation of signaling. Despite the diversity in the molecular interactions between T cells and antigen presenting cells (APC) ,3, some This sequence of events occurs at different timescales. The type of stimuli and sensor receiving and transducing the signal  ,21, the Here, we aim to examine the mechanisms by which T cells modulate and segregate, signaling spatiotemporally, during early T cell activation and formation of the IS.The very first stage of T cell activation is highly influenced by mechanical signals. Signaling events mediated by mechanical cues are rather fast, being able to communicate information in the order of nano- to milliseconds ,21,26. MThe role of the actin cytoskeleton in mechanotransduction is well known, and the importance of actin during T cell activation has been thoroughly reviewed ,33,34. Y2+ signaling in resting cells [2+ channel, the transient receptor potential cation channel subfamily V member 2 (TRPV2), showed lower response to membrane stretching when silenced in Jurkat (human lymphoma CD4+ cells) [Other mechanotransducers in T cells are mechanosensitive ion channels. These channels transduce changes in membrane tension and membrane stretching by altering their conformation and subsequently adjusting the transit of ions b. Thus, ng cells . Another+ cells) . These s2+ signaling and IL-2 secretion, increasing the sensitivity of T cell recognition by two orders of magnitude compared to low affinity antigens [The TCR is the main player in the initial steps that lead to T cell activation. The TCR has been traditionally studied from the perspective of ligand-receptor triggering, but in the last decade several groups have investigated its mechanosensitive potential. Integration of mechanical forces is only one aspect of TCR triggering, so we encourage the reader to consider two review articles that perfectly summarize the activation models proposed to date, and aim to combine the described triggering mechanisms into a unified model ,48. The antigens . As obseantigens ,56,57.2+ influx, phosphorylation of proteins, and production of cytokines by just one of the aforementioned mechanisms  or by jL lysine ,60. ThouL lysine ). Thus, After initial triggering, cells modulate signaling by controlling the strength and duration of the transduction events. Signal strength seems to correlate in some T cell types (CD4+) with the duration of the interaction of T cell and APC , which, The activation threshold varies for each cell, stage of the immune response (naïve vs. activated T cells), and affinity and avidity for the antigen presented. T cells have developed a system of positive and negative co-stimulatory receptors to modulate their responsiveness to antigen, which is the case of CD28 (positive) and CTLA-4 (negative) that can regulate the threshold needed for activation ,65 Figuc. LCK waBrockmeyer et al. described how TCR activation leads to at least three distinctive phosphorylation kinetic profiles or signaling waves . In a fiLCK is a key player during T cell activation. Despite having been studied in the context of early activation, how it localizes and phosphorylates remain unclear. During the initial stages of activation, a first signaling wave by active LCK phosphorylates CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) upon TCR triggering c. After Similar to TCR and LCK, LAT exists as two different pools in T cells: One at the plasma membrane and one intracellular. LAT is internalized in a clathrin-dependent manner c, going These few examples stress the relevance of how key molecules are delivered to the plasma membrane and activated over time during T cell activation. Although endocytosis of exhausted  and non-exhausted signaling molecules could initially contribute to downregulation of signaling, growing evidence suggests that active signaling occurs in endosomes of T cells ,88. ThisCompartmentalization aids sorting of molecules to perform different tasks. Cellular compartments include organelles or, at a much smaller scale, specialized functional domains. These domains can be found in the cytoplasm as, for instance, self-contained lipid droplets, but they have been mostly depicted in the plasma membrane. Membrane domains can range in size from microns  to nanometers, as in the case of highly ordered lipid domains or “lipid rafts”. Most of these specialized domains share a relatively high concentration of receptors, adhesion proteins, and other signaling molecules, as well as a particular lipid composition; but their lifetime might differ, leading to more transient or stable domains. Membrane domains require a certain level of stability to keep their functionality over time. Thus, cells resort to several mechanisms to preserve the structure and composition of membrane domains: From directing vesicle trafficking to the domain, to heterogeneously distributing specific lipids and proteins/enzymes, or reducing the mobility of molecules.The IS is a clear example of spatial regulation. The structure of the IS varies depending on the type of T cells and APCs involved , their stage of maturation, and the outcome of their interaction ,3,89. FoThroughout the maturation of the IS, signaling molecules are sorted into different SMACs. After initial triggering, TCRs at the plasma membrane diffuse laterally across the membrane and aggregate, forming multiple microclusters . InitialTargeted vesicle trafficking to specific areas of the plasma membrane generates membrane domains with a unique protein composition. This is a common strategy in cells with very active and sustained signaling, mediated by ligand–receptor interactions that rely on a high recycling rate. T cells polarize their exocytic machinery towards the IS by reorganizing the actin and tubulin cytoskeletons. The centrosome, that is associated to the Golgi Apparatus and centralizes the vesicle traffic of the cell, relocates underneath the IS ,100, accMolecules recruited to the IS are commonly targeted to specific membrane domains. It is unclear whether TCR nanoclusters localize to highly ordered lipid domains prior to activation ,103 but 2 produced by PIP5K. Redistribution of these kinases during T cell activation leads to drastic changes in localization of PIP2 and, subsequently, in the activity of PIP2 effectors. After TCR engagement, PLC-γ moves to activated TCR areas, reducing the levels of PIP2, which is, in turn, replaced by DAG [2 directly interacts with several actin-related proteins  proteins) or the nucleating-promotor factor, Wiskott–Aldrich Syndrome Protein (WASP)), its redistribution has a striking effect on actin cytoskeleton dynamics. Thus, loss of PIP2 leads to clearance of filamentous actin from the center of the IS [3 also drives the formation of the F-actin ring (3 precedes and leads the formation of the contractile actin ring in cytotoxic T cells [2 into PI(4)P, whereas INPP5F/OCRL1 metabolizes PIP3 down to PIP2 [2, while the plasma membrane surrounding the cilium is characterized by higher levels of PIP3 and PIP2 [2 mediates actin recruitment that could interfere with vesicle traffic to the cilia [Specialized membrane domains can be originated by allocating enzymes with antagonistic activities in strategic sites, which leads to compartments composed by gradients of different enzymatic products a,b. The d by DAG ,115. Sinf the IS  P2 ,118. TheIP2 ), leadinIP2 ,121,122.he cilia . Phosphahe cilia .In highly ordered lipid domains or lipid rafts, where content of cholesterol and sphingomyelins is higher than in the surrounding membrane and contains highly packed lipids, proteins diffuse slower than outside of the domain, increasing the potential interaction between proteins localized to the same domain b. SimilaC. elegans’ primary neurons [All the energy cells employ to generate specialized domains would be inefficient if molecules diffused freely after being targeted to a specific domain. Thus, diffusion barriers contribute to maintain a stable molecular composition over time. Proteins of the cortical actin cytoskeleton such as spectrins, ankyrin, and occasionally adhesion proteins, can interfere with the lateral diffusion of membrane components (reviewed in ) Figureb. Simila neurons , linkingAlthough there is still little evidence that links the cSMAC, pSMAC, and dSMAC compartments and the contractile actin ring, we believe that the actin ring potentially acts as a picket fence to impair free diffusion between the SMACs. Translocation of ICAM1 relies on actin retrograde flow from the edge of the synapse to inner areas, but ICAM1 is restricted from the mature cSMAC, supporting the existence of a barrier that limits access of ICAM1 to the center of the synapse . This baWe have examined the relevance of signaling dynamics, focusing on T cells since their fast and organized activation utilize a wide range of mechanisms for signal regulation. T cells have developed a multitude of tools to speed up signaling without compromising specificity; and have efficiently exploited signaling compartments and functional membrane domains to combine stability with fast recycling and interchange of molecules to keep signaling active over time.A clear conclusion is that all the mechanisms of spatiotemporal regulation described here work together in tight regulation. Ciliary proteins, like UNC119 and its regulators, have been overlooked in the context of T cell activation until very recently. Although we still do not fully understand the role that these proteins might play in the temporal and spatial regulation of T cell signaling, we believe they might be adding a layer of regulation to LCK activity and localization aside from their recruitment by lateral diffusion and vesicular trafficking. To address this, future studies should focus on obtaining reliable quantitative information of the molecular dynamics of lipid-modified proteins like LCK and their solubilizing factors during the very early stages of T cell activation. It is worth investigating further the contribution of temporal and spatial regulation of signaling to the immunity of individuals. Are these pathways redundant and so interconnected that the cell bypasses any potential defect or deficiency? Most studies have focused on the importance of the targeting of signaling proteins at the synapse in T cell response, but little is known regarding the importance of a timely response. How do changes in the speed of the early T cell response affect immunity? There is need for further studies that focus on the dynamics of signaling in immunity.


We expressed A1R and CB1 fusions with G‐peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G‐peptides. We used agonists N6‐Cyclopentyladenosine (CPA) and 5’‐N‐Ethylcarboxamidoadenosine (NECA) for A1R and 2‐Arachidonoylglycerol (2‐AG) and WIN 55,212‐2 mesylate (WN) for CB1. G‐peptides derived from Gαi and Gαq enhance agonist‐dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi‐coupled signaling. In contrast, both G‐peptides suppress agonist‐dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq‐coupled signaling. Taken together with our previous studies on Gs‐coupled receptors, this study provides an extended model for the allosteric effects of G‐peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand‐binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C‐terminus of the Gα subunit (G‐peptides) on signaling from two Gi‐coupled receptors, adenosine A1 receptor (A 1) receptors.Gα peptides differentially impact Gs and Gi signaling in Cannabinoid (CB Likewise, the Gq‐coupled V1 vasopressin receptor(V1R) shows enhanced IP1 levels in the presence of the noncognate Gs protein.adenosine type 1(A1R) and cannabinoid type 1(CB1) using G‐peptides derived from Gs, Gi, and Gq subtypes.In this study, we examine the potential for the G protein α subunit C‐terminus (G‐peptide) to serve as an allosteric modulator of GPCR signaling. The G‐peptide is a well‐established determinant of GPCR‐G protein coupling selectivity.1R principally signal through Gs, and A1R and CB1 primarily signal through Gi. However, A1R and CB1 display signaling through multiple G proteins with A1R signaling through Gi and Gq, and CB1 signaling through Gi, Gq, and Gs.1, the most widely expressed GPCR in the central nervous system, primarily signals through Gi producing euphoria and analgesia upon binding tetrahydrocannabinol (THC) in the brain.1 has also been shown to signal through Gq in human embryonic kidney (HEK) 293 cells after treatment with WIN55,212‐2(WN)1 signaling through Gs, Gq, and non‐G protein‐mediated pathways is less clear since there have not been biased ligands identified that specifically target these pathways. A1R is another example of a promiscuous receptor that can activate different signal transduction pathways in an agonist‐dependent manner. A1R is ubiquitously expressed and most well known for being antagonized by caffeine, producing stimulant effects.1R canonically signals through Gi, there is evidence that A1R has a diverse G protein‐activating profile where A1R can adopt agonist‐specific conformations, arising from small changes in ligand structure, which lead to the differential activation of G proteins including Gi and Gq.While β2‐AR and D1R and CB1). The C‐termini of three G proteins, Gαs, Gαi, and Gαq, will be referred to as s‐pep, i‐pep, and q‐pep (or collectively as G‐peptides) throughout this manuscript. We expressed A1R and CB1 fusions with the s‐, i‐, or q‐pep in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking this G‐peptide, referred to henceforth as no‐pep. We have extensively reported on this SPASM technique, which allows systematic control of the intramolecular interaction between a GPCR and a G‐peptide.The goal of this focused study is to examine the allosteric effects of G‐peptides derived from three distinct Gα C‐termini peptides  on signaling from two promiscuous Gi‐coupled receptors (AN6‐Cyclopentyladenosine(CPA) and 5’‐N‐Ethylcarboxamidoadenosine (NECA) for A1R and 2‐Arachidonoylglycerol (2‐AG) and WIN 55,212‐2 mesylate (WN) for CB1. Our current study confirms what we previously found in Gs‐coupled receptors β2‐AR and D1‐R, where s‐pep and q‐pep positively modulate canonical Gs signaling.1 is enhanced by s‐pep (~40% increase). In contrast, i‐pep diminishes cAMP response from CB1 for both 2‐AG and WN . At low concentrations of 2‐AG, WN, and CPA (nmol/L) we observed inhibition of cAMP, associated with signaling through Gi. We found that the presence of q‐pep or i‐pep enhanced canonical Gi signaling in A1R after activation by CPA (~35% increase), and in CB1 after activation by WN (~700% increase) and 2‐AG (~125% increase), respectively. These findings extend our previously reported allosteric effects of G‐peptides to Gi‐coupled signaling.1) is observed, associated with signaling through Gq. We found that the presence of different G‐peptides universally inhibits IP1 signaling through Gq (decreases ranging from 30% to 65%), with the exception of s‐pep (~50% increase) on CB1 following activation by WN. Taken together, our data provide an extended model for the allosteric effects of distinct G‐peptides on signaling through Gs, Gi, and Gq pathways and highlight the ability of G‐peptides to differentially impact signaling in a receptor and ligand‐dependent manner.To investigate the allosteric effects of G‐peptides on Gi‐coupled receptors, we used 22.1N‐Ethylcarboxamidoadenosine (NECA), pertussis toxin (PTX), and forskolin were purchased from Sigma‐Aldrich. 2‐Arachidonoylglycerol (2‐AG), N6‐Cyclopentyladenosine (CPA), WIN 55,212‐2 mesylate (WN), SCH 442416(SCH), and PSB 1115(PSB) were purchased from Tocris. cDNA encoding Gαi2 isoform 1, Gαq, and the long splice variant of Gαs were acquired from GE (Open Biosystems). Human A1R was acquired from DNASU Plasmid Repository. Mus musculus CB1 was acquired from transOMIC technologies. DNA transfection reagents X‐tremeGENE HP and Mirus‐LT DNA were purchased from Roche and Mirus, respectively. Buffer A is phosphate‐buffered saline , 800 μmol/L ascorbic acid, and 0.2% dextrose (w/v). Buffer B  is 10 mmol/L HEPES, 1 mmol/L CaCl2, 0.5 mmol/L MgCl2, 4.2 mmol/L KCl, 146 mmol/L NaCl, 5.5 mmol/L glucose, 50 mmol/L LiCl2, pH 7.4.5’‐2.21R or CB1), mCitrine, 10 nm ER/K linker, mCerulean, and a Gα subunit C‐terminal peptide corresponding to Gαs, Gαi, Gαq,  or a control peptide (no‐pep), consisting of repeating (Gly‐Ser‐Gly)4 residues. A (Gly‐Ser‐Gly)4 linker was inserted between all protein domains as part of the primer sequence to allow for free rotation between domains. All sensors also contained either an N‐terminal HA‐tag or a His‐tag. All constructs were confirmed by sequencing.For mammalian HEK 293 expression, all GPCR and Gα constructs were cloned into a PCDNA5/FRT vector (ThermoFisher). GPCR sensors were cloned with a modular scheme. Each GPCR sensor contained (from N‐ to C‐terminus): a full length GPCR (A2.32. HEK293T cells were plated onto six‐well tissue culture treated plates at ~30% confluence. Cells were transfected 16‐20 hours later with X‐tremeGENE HP DNA transfection reagent. Transfection conditions including the amount of DNA (1.4‐4 μg DNA + 4.2‐6 μL reagent) and the length of transfection  were optimized to consistently yield equivalent levels of sensor expression across different conditions. Where indicated, 12 hours after transfection, cells were incubated with 100 ng/mL PTX for 16 hours. Experiments were conducted at 60%‐80% transfection efficiency . At the time of the experiment, 60%‐90% of transfected cells expressed predominantly plasma membrane localized sensor with minimal localization to the intracellular compartments. Sensor integrity, localization, and sensor expression were tracked for all experiments to ensure consistency. Each experiment was performed at equivalent sensor expression and matched OD of the cell suspension using the following steps. Cells were first resuspended by gentle pipetting into their original media, spun down , and washed once with Buffer A or B for cAMP or IP1 assays, respectively. Subsequently, cells were resuspended in an appropriate volume of the same buffer to reach a 0.3 OD measured at A600 nm. Sensor expression was measured by mCitrine fluorescence. mCitrine fluorescence was held within 1.6‐2.4 × 106 counts‐per‐second (cps) for a cell OD of 0.3. Sensor integrity was confirmed by measuring the mCitrine  to mCerulean fluorescence ratio . Experiments were conducted at mCitrine to mCerulean fluorescence ratio of 1.7‐2.1.HEK293T‐Flp‐In  cells were cultured in DMEM media (ThermoFisher) supplemented with 10% FBS (v/v) (Millipore Sigma), 4.5/gL D‐glucose, 1% Glutamax (ThermoFisher), 20 mmol/L HEPES, pH 7.5 at 37°C in a humidified atmosphere at 5% CO2.46 cells/mL density. Cell suspensions were aliquoted into 384‐well opaque plates (5 μL per well). Where indicated, cells were preincubated with 100 nmol/L of the adenosine type 2A receptor(A2AR) selective antagonist, SCH 442416 (SCH), and 1 μmol/L of the adenosine type 2B receptor(A2BR) selective antagonist, PSB 1115 (PSB) in 10 μmol/L forskolin for 15 minutes at 37°C. Cells were incubated with CPA or NECA (for A1R) or 2‐AG or WN (for CB1) for 15 minutes with 10 μmol/L forskolin at 37°C. Subsequently, cells were lysed and the protocol was followed according to the manufacturer's recommendation (Promega). Luminescence was measured using a microplate reader . cAMP levels were evaluated by subtracting relative luminescence units (RLUs) in the absence and presence of agonists. Each experiment was performed in quadruplicate and independently repeated at least three times (N > 3). For experiments involving comparisons between multiple sensors, equivalent sensor expression was first verified using fluorescence measurements (see previous section) and data for all four sensors were collected together  to assess cAMP levels using the bioluminescent cAMP Glo assay (Promega). Cells were gently suspended in their original media, counted using a hemocytometer, and spun down . Cells were resuspended in an appropriate volume of Buffer A to reach 4 × 10r Figure .2.51 levels using the IP‐One HTRF assay kit (Cisbio). Cells were gently suspended in their original media, counted using a hemocytometer, and spun down . An appropriate volume of Buffer B (StimB buffer) was added to reach 3 × 106 cells/mL density. Where indicated, cells were preincubated with 1 μmol/L of the A2BR selective antagonist, PSB 1115 (PSB) for 30 minutes at 37°C. Cells were incubated with 100 μmol/L of CPA or NECA (for A1R) or 100 μmol/L of 2‐AG or WN (for CB1) at 37°C for a total incubation time of 30 or 120 minutes. The manufacturer's protocol was modified to achieve a high signal to noise ratio as follows: 70 μL of suspension was incubated for 1 hour with 2 μL IP1 conjugated to d2 dye diluted in 13 μL of lysis buffer (Cisbio) and 2 μL terbium cryptate‐labeled anti‐IP1 monoclonal antibody also diluted in 13 μL of lysis buffer. 80 μL of each reaction suspension was then transferred and split between 4 wells (20 μL/well) on a 384‐well opaque plate. IP1 spectra were collected by exciting samples at 340 nm (bandpass 15 nm). Emission counts were recorded from 600 to 700 nm using a long pass 475 nm filter . Raw IP1 signal was calculated as the ratio of fluorescence emissions at 665nm and 620nm. Data were corrected by subtracting the untransfected IP1 ratio from cells expressing transfected sensor. Data are presented as a change in IP1 ratio following drug treatment. Each experiment included four repeats per condition and was independently repeated at least three times (N > 3).HEK293T cells expressing the indicated sensor were harvested 28‐32 h posttransfection (X‐tremeGENE HP) to assess IP2.6P‐values *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001; *****P ≤ .00001.Data are represented as mean values ± SEM. All experiments were repeated for at least three independent trials, with three to six technical repeats per condition (N > 3). Statistical analysis was performed using GraphPad Prism 7.0c . To assess significance across experimental repeats, pooled or un‐pooled data underwent subsequent pairwise ANOVA analysis. Tukey's post hoc test was performed to assess significance when evaluating comparisons between multiple conditions with 2.7http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY,Key protein targets and ligands in this article are hyperlinked to corresponding entries in 33.11 receptor (A1R) and cannabinoid type 1 (CB1)  and endogenous G proteins in cells, allowing one to study the impact of the tethered Gα peptides on canonical GPCR signaling.1R and CB1 constructs lacking a C‐terminal peptide (no‐pep) were used to measure background cAMP and IP1 levels and for characterization of ligand dose‐response.SPASM sensors were developed for two cognate Gi‐coupled receptors, adenosine A) Figure . From N‐) Figure , left.2) Figure . We ther3.21 sensor display potentiation of forskolin‐stimulated cAMP accumulation with signaling dominated by Gs in response to 30 μmol/L of the CB1 agonists 2‐Arachidonoylglycerol (2‐AG)  or WIN 55,212‐2 mesylate (WN) .green) or WN (purple). We observed no potentiation of forskolin‐stimulated cAMP accumulation in untransfected HEK 293 cells in response to a range of 2‐AG and WN concentrations  or 300 nmol/L WN . To characterize Gi signaling in CB1, dose‐response curves were performed for both 2‐AG and WN  or absence (gray lines) of pertussis toxin (PTX). cAMP levels increased in response to PTX treatment in 2‐AG‐stimulated CB1  to characterize the impact of peptides on cAMP stimulation and Gs signaling  to characterize cAMP inhibition and signaling through Gi in CB1 . SPASM sensors with s‐, i‐, or q‐pep fusions, in addition to a no‐pep control (−), were expressed in HEK 293 cells as shown previously.1 sensors were treated with high concentrations (30 μmol/L) of 2‐AG  or WN (300 nmol/L), conditions resulting in cAMP inhibition  sensor display Gi‐mediated inhibition of forskolin‐stimulated cAMP accumulation after stimulation by 50 nmol/L of the A1R agonist, N6‐Cyclopentyladenosine (CPA) . To address potential variability in individual sensor response, for each experiment equivalent sensor expression was verified using fluorescence measurements (see methods) and data for all four peptide sensors were collected together , and 1 μmol/L of the A2BR selective antagonist, PSB 1115 (PSB), resulted in complete inhibition of cAMP production. The slight decrease in forskolin‐stimulated cAMP accumulation in cells transfected with A1R without antagonist pretreatment can likely be attributed to increased Gi signaling by transfected A1R receptors. We performed the same control experiments with untransfected HEK 293 cells treated with 30 uM NECA and found equivalent potentiation of forskolin‐stimulated cAMP accumulation as compared to A1R‐transfected cells  but not the α1 adrenergic receptor(α1‐AR).1 production in A1R and CB1 receptors. A dose‐response study of NECA  and CPA (gray lines) with A1R no‐pep (−) sensors revealed maximum IP1 signal at 100 μmol/L ligand (blue dotted line). To rule out Gβγ‐dependent PLC‐β activation, we performed IP1 dose‐response assays in the absence  and presence (light gray line) of pertussis toxin (PTX) treatment. Regardless of CPA concentration, no reduction in IP1 production was observed in PTX‐treated cells compared to untreated cells, suggesting the observed IP1 production is due to A1R signaling through the PTX‐insensitive Gq pathway. Additionally, to rule out Gq signaling through endogenous HEK 293 A2BR receptors, IP1 levels were assessed in untransfected HEK 293 cells after stimulation by 100 μmol/L CPA or NECA , significant IP1 production occured in A1R transfected cells but not in untransfected HEK 293 cells, suggesting IP1 production resulted from Gq signaling through A1R and not endogenous A2BR  sensor lacking a peptide, were expressed in HEK 293 cells to equivalent levels. IP1 assays were performed with each of the A1R sensor constructs after stimulation by 100 μmol/L CPA  and 2‐AG (gray lines) on CB1 no‐pep (−) sensors revealed maximum IP1 signal at 100 μmol/L ligand (blue dotted line). To rule out Gβγ‐dependent PLC‐β activation, we performed IP1 dose‐response assays in the absence  and presence (light gray line) of pertussis toxin (PTX) treatment. Regardless of 2‐AG concentration, no reduction in IP1 production was observed in PTX‐treated cells compared to untreated cells, suggesting the observed IP1 production is due to CB1 signaling through the PTX‐insensitive Gq pathway. An IP1 assay was performed on HEK 293 cells expressing SPASM sensors with s‐, i‐, or q‐pep fusions, in addition to a sensor lacking a peptide no‐pep (‐), after stimulation by 100 μmol/L 2‐AG  or WN (right). Consistent with A1R, the i‐pep and q‐pep inhibited signaling through Gq in CB1, as evidenced by reduction of IP1 signal . G‐peptides derived from Gαi and Gαq (i‐pep and q‐pep) enhance agonist‐dependent cAMP inhibition, demonstrating their function as positive allosteric modulators of Gi‐coupled signaling. In contrast, i‐pep and q‐pep suppress agonist‐dependent IP1 levels suggesting that they function as negative allosteric modulators of Gq‐coupled signaling. Taken together with our previous studies focused on Gs‐coupled receptors, our findings reinforce the potential of G‐peptides to allosterically modulate signaling from class A GPCRs.In this study we demonstrate the allosteric modulation of two Gi‐coupled receptors, A1 and A1R) examined in this study have also been reported to signal to varying degrees through other G proteins.1R can couple to Gs and Gq in response to CPA or NECA, suggesting A1R can adopt agonist‐specific conformations arising from small differences in ligand structure leading to differential G protein activation.1R signaling through Gi and contradict signaling through Gs.1R, measured by potentiation of forskolin‐induced cAMP production, cannot be distinguished from activation of endogenous A2AR or A2BR receptors by A1R agonists. We saw significantly higher potentiation of forskolin‐induced cAMP production in untransfected HEK 293 cells compared to A1R‐transfected HEK 293 cells in response to CPA, suggesting CPA is likely stimulating endogenous Gs‐coupled receptors  are presented at equal effective concentrations, it is unlikely that inhibition of Gq signaling stems from a simple competitive inhibition mechanism. Instead, the differential effects of G‐peptides likely stem from the dynamic conformational landscape of GPCRs.Our data contrast with previous studies that report inhibition of GPCR signaling by native cognate G‐peptides.1R‐Gi fusions.We propose a model wherein transient interactions with G‐peptides alter receptor conformation. The receptor does not form a stable ternary complex with the G‐peptide and therefore at low concentrations (10 μmol/L) does not interfere with the kinetics of the receptor‐cognate G protein interaction.5No animals, human tissue, human volunteers, or patients were used in this study.Touma, Malik, and Sivaramakrishnan participated in research design. Touma, Malik, and Gupte conducted experiments.Touma, Malik, Gupte, and Sivaramakrishnan performed data analysis.Touma and Sivaramakrishnan wrote manuscript.Figure S1‐S6Click here for additional data file.


Inner afferent arteriolar diameter was measured locally and upstream to evaluate conducted vasoconstriction. Electrical stimulation induced local vasoconstriction in all groups. The local vasoconstriction was significantly smaller when Cx40 was absent. The vasoconstriction decreased in magnitude with increasing distance from the stimulation site. In both Cx40 and Cx45 deficient mice, the vasoconstriction conducted a shorter distance along the vessel compared to wild-type mice. In Cx40 deficient arterioles, this may be caused by a smaller local vasoconstriction. Collectively, these findings imply that Cx40 and Cx45 are central for normal vascular reactivity and, therefore, likely play a key role in TGF-induced regulation of afferent arteriolar resistance.The juxtaglomerular apparatus (JGA) is an essential structure in the regulation of renal function. The JGA embodies two major functions: tubuloglomerular feedback (TGF) and renin secretion. TGF is one of the mechanisms mediating renal autoregulation. It is initiated by an increase in tubular NaCl concentration at the macula densa cells. This induces a local afferent arteriolar vasoconstriction and a conducted response that can be measured several 100 μm upstream from the juxtaglomerular segment. This spread of the vasomotor response into the surrounding vasculature likely plays a key role in renal autoregulation, and it requires the presence of gap junctions, intercellular pores based on connexin (Cx) proteins. Several Cx isoforms are expressed in the JGA and in the arteriolar wall. Disruption of this communication pathway is associated with reduced TGF, dysregulation of renin secretion, and hypertension. We examine if the absence of Cx40 or Cx45, expressed in the endothelial and vascular smooth muscle cells respectively, attenuates afferent arteriolar local and conducted vasoconstriction. Afferent arterioles from wildtype and Cx-deficient mice (Cx40 and Cx45) were studied using the isolated perfused juxtamedullary nephron preparation. Vasoconstriction was induced  The juxtaglomerular apparatus (JGA) is a specialized structure, formed by the afferent and efferent arteriole, thick limb of the ascending loop of Henle, and mesangial cells. The JGA embodies two major renal functions: tubuloglomerular feedback (TGF), which is part of the mechanism underlying renal autoregulation, and renin secretion. The JGA consists of different cell types with specialized functions: the macula densa (MD) cells, juxtaglomerular cells, mesangial cells, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). The MD cell of the thick limb of the ascending loop of Henle functions as a NaCl sensor. An increase in NaCl concentration in the tubular fluid eventually causes the VSMC in the afferent arteriole to contract to reduce glomerular filtration rate (GFR) and the juxtaglomerular cells of the afferent arteriole to reduce renin secretion. Mesangial cells are located between the MD cells (the sensor) and the afferent arteriole (the effector). They are believed to play an important role in the signal transduction from the MD to the afferent arteriole .The TGF signal originates in the MD cells and elicits local vasoconstriction in the juxtaglomerular part of the afferent arteriole. The response, however, encompasses the entire afferent arteriole, the distal part of the interlobular artery, and travels into the afferent arterioles of neighboring nephrons . This inGJs are fluid-filled pores that allow the movement of small molecules and current between neighboring cells. GJs are made up of two hemichannels, connexons, built from connexins (Cxs) . Six CxsIn the vasculature, Cx40 is the most widely expressed isoform and is predominantly found in the ECs , as wellvia GJs .Procedures were approved by the Danish National Animal Experiments Inspectorate. All animals were kept in the animal facility at University of Copenhagen and received tap water and standard chow infrafrontier.eu, Munich, Germany). Kidneys from a total of 15 adult mice were used for the studies . The age of the mice ranged from 4 to 14 months.Cx40 wildtype (Cx40 WT) and Cx40 knockout (Cx40 KO) mice were on a mixed C57Bl/6 × 129S4/SvJae genetic background. Mice were purchased for breeding from the European Mouse Mutant Archive  were mated with homozygous Cx45fl/fl:Nestin-Cre males, >87% C57Bl/6 background. Breeding pairs were a generous gift from Dr. Klaus Willecke, University of Bonn, Germany; the mice have been described previously . The Cx45fl/fl:Nestin-Cre mice are used as our knockout (KO). Kidneys from 20 adult mice were used for the studies , ranging from 2 to 6 months of age.Homozygous female mice with floxed Cx45 gene . The abdominal aorta was catheterized and immediately perfused with Tyrode’s solution  containing 5% BSA  and an amino acid mixture  at pH 7.4. Kidneys were excised, and the cannula was advanced into the renal artery of the left kidney. The kidney was decapsulated and sectioned longitudinally exposing the intact papilla, which was reflected revealing the inner cortical surface. Venous tissue on the cortical surface was cut, allowing access to the arterial vasculature. Perfusion was isolated to the inner cortical vessels by ligating larger arterial vessels .Experiments were performed using the 2-5% CO2 gas mixture to the perfusion solution reservoir.The cannula system comprised of a 27-gauge blunt needle and a polyethylene (PE-10) line, connected to a servo-nulling pressure system , allowing for continuous RPP monitoring. RPP was maintained at 95 mmHg throughout the experiment by adjusting the regulator, controlling the flow of 95% OBorosilicate glass micropipettes  were pulled on a horizontal Flaming/Brown micropipette puller  and back-filled with a 2 mol/L NaCl solution containing Lissamine green . The microelectrode was connected to a stimulus isolator  connected to a stimulator  and an oscilloscope . The microelectrode (0.5–0.8 MΩ resistance) was placed at the glomerular entrance of the afferent arteriole, where local afferent vasoconstriction was induced by electrical pulse stimulation  as well as every 50 μm upstream from the glomerulus  electrical stimulation induced a minimum of 5% constriction in the vessel diameter , (2) post-stimulation, the vessel recovered to at least 50% of its baseline diameter, and (3) diameters were measurable at a minimum of three positions along the vessel (including stimulation site).t-test testing against 0 was used to evaluate significant constriction at each measurement point.Graphical presentation and statistical analyses were performed using SigmaPlot , MatLab , and Rstudio . Vessel diameter during stimulation was normalized to the respective mean resting diameter at the given distance from the stimulation site. Normalized and pooled data for contraction at the stimulation site (0 μm) from each experimental series were evaluated using a one-way ANOVA to test for differences between WT and KO. A one-sample y = bxae, where y is the change in diameter at upstream distance x from the stimulation site, a is the y-intercept, and b is the decay rate of the response along the vessel. When conducted vasoconstriction reached 0 or a negative value, constriction was set to 0 for the remaining measurements. The maximum distance from the stimulation point at which the individual vessel could be visualized varied from 100 to 500 μm. Consequently, there were more measurements at the shorter distances. All measurements at a given distance were pooled for each group. Therefore, in each fit, the shorter distances with more measurements weighs in more heavily than longer distances with fewer measurements  for each type of animal by a non-linear least squares fit of the data to the equation urements . Each grurements . In briep ≤ 0.05 was considered statistically significant. All values are reported as means ± SEM unless otherwise stated.Previous studies have shown no difference in the measured renal vascular responses between male and female mice, and data were therefore pooled for statistical comparison . p ≤ 0.0p = 0.04) and in Cx45 WT mice (27.4 ± 1.3 g) compared to Cx45 KO mice . Mice from the Cx40 strain were significantly heavier than the Cx45 strain. Age was not different within the groups; however, mice from the Cx40 strain were older than the mice from the Cx45 strain. Resting vessel diameter measured at the glomerular entrance was significantly different between Cx40 WT and Cx40 KO (p = 0.04), but not between Cx45 WT and Cx45 KO, or between groups. Maximal traceable vessel length (afferent arteriole and in some cases interlobular arteries) was not significantly different between or within groups.Mean values of body weight, age, pre-stimulated vessel diameter (measured at 95 mmHg), and traceable vessel length are summarized in A typical recording of the afferent arteriolar diameter from a Cx40 WT mouse is shown in p < 0.05) was significantly different. Albeit numerically different, no significant difference was found between Cx45 WT and KO . This is in line with the fitted data (y-intersection (a) is equivalent to the local vasoconstriction.Comparing measurements at the stimulation site directly between groups (one-way ANOVA) showed that relative vasoconstriction between Cx40 WT and KO  of the conducted vasoconstriction evaluated using bootstrapping was significantly smaller in Cx45KO compared to Cx45 WT (p < 0.05, The decay rate (In all groups, vasoconstriction decreases with increasing distance. Comparing the relative afferent arteriolar constriction in each group against 0, the data showed that in Cx40 WT a significant constriction was measurable up to 200 μm , in Cx40via GJs, through Cx40 and Cx45, in the conduction of afferent arteriolar vasoconstriction in response to local electrical stimulation. We have previously shown that lack of Cx40 or Cx45 significantly reduces TGF (The present study investigated the importance of intercellular communication uces TGF . Here, wVSMCs and ECs work together to coordinate and propagate constriction and dilation along the vessel wall . The conBoth mouse strains used in the present study have been characterized previously. The Cx40 KO was hypertensive , but RBFElectrical pulse stimulation induced a local and a conducted vasoconstriction response in afferent arterioles from Cx40 and Cx45 WT and KO mice. The local response measured at the stimulation site was significantly smaller in Cx40 KO compared to Cx40 WT. However, in cremaster arterioles from Cx40 KO mice, local vasoconstriction elicited with electrical stimulation or KCl was similar to that observed in WT mice . As show2+ measured 500 μm from the stimulation site (2+ or vasoconstriction as parameters. Comparing the constriction at a given distance against 0 revealed that in Cx40 KO the constriction was only significant at 50 μm. This could be due to the smaller local constriction.Although the decay rate of vasoconstriction seemed slightly increased in Cx40 KO, no significant difference was found between Cx40 WT and KO. This is in accordance with results obtained in skeletal muscle arterioles, where KCl-induced conducted vasoconstriction was unaffected in Cx40 KO mice . It is gion site . However2+ signaling as Cx45 is known to be involved in the propagation of Ca2+ waves in VSMC isolated from afferent arterioles (In contract, in Cx45 KO, the decay rate was significantly increased compared to WT. Possibly, Cx45 is part of the MEGJ in the afferent arteriolar wall, and its removal by conditional knockout could reduce communication between the VSMC layer and the endothelium, hence reducing the mechanical response from the VSMC layer. Myoendothelial junctions are unevenly distributed along the wall. The spread of depolarization from VSMCs that are coupled to the endothelium, to VSMCs that are not, could be central for a distant mechanical response to electrical stimulation. In addition, although the endothelium is normally considered the main path for a longitudinal spread of current in the microcirculation, the presence of VSMC-VSMC coupling by Cx45-containing GJs may also contribute. A possible proposed mechanism is through Caterioles . CompariThe structure of the vascular wall may affect both the local vasoconstriction and conduction properties. The vascular wall in renal vessels with a diameter <40 μm is thicker compared to vessels from skeletal muscles , which w2+ channels, significantly increased afferent arteriolar diameter (Differences in myogenic activity at the chosen RPP of 95 mmHg may also influence the obtained results. Resting membrane potential of VSMCs in the afferent arteriole in isolated perfused rat kidneys was approximately −40 mV at 80 mmHg RPP . We assudiameter . In Cx40diameter  and in Cdiameter , suggestdiameter  it does A number of limitations are present in our experiments. Due to the experimental setup, it is not possible to position the stimulation pipette next to the vascular wall. A varying layer of tubular tissue will separate the pipette tip and the vascular wall. This limitation has been mitigated by only including arterioles with a minimum local constriction of 5%. In addition, the preparation is not fixed and the arterioles have some vasomotion that could affect the measurements. Consequently, we used an average of the diameter measured over 30 s. Vasoconstriction is not the most sensitive measure to quantify vascular conduction. Changes in membrane potential can be measured using sharp electrodes or patch-clamp . This meIn conclusion, we have shown that knockout of Cx40 reduces local vasoconstriction but does not significantly change the conduction of vasoconstriction. Vascular deletion of Cx45 significantly reduces the vascular conduction induced by electrical stimulation in afferent arterioles. Consequently, vasoconstriction in response to a comparable stimulus is conveyed shorter when intercellular communication between cells in the vascular wall is reduced in the renal microcirculation. This may in turn affect the ability to regulate and autoregulate renal perfusion.All datasets generated and source code for this study are included in the article/The animal study was reviewed and approved by the Danish National Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark, and Danish Veterinary and Food Administration.SM and CS designed the animal experiments. SM performed animal experiments and drafted manuscript. SM, CS, and JJ analyzed data and prepared figures and tables. SM, JJ, N-HH-R, and CS interpreted results, edited and revised manuscript and approved final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adenosarcoma is an extremely rare malignancy of the female genital tract composed of stromal sarcoma with a benign epithelial component. Current treatment recommendations include total hysterectomy with bilateral salpingo-oophorectomy, precluding future fertility. Although most frequently diagnosed in postmenopausal women, it is occasionally present in younger women of reproductive age with desire for future fertility. In 2015, we reported the case of a 23-year-old patient diagnosed with uterine adenosarcoma, who having strong desire of future fertility, opted for fertility sparing surgery. At a follow-up five years later, we can now report her case of spontaneous pregnancy and livebirth. A review of the literature concerning fertility outcomes in patients with uterine adenosarcoma undergoing fertility sparing therapeutic options is presented. Uterine adenosarcoma (UA) is a rare gynaecological malignancy composed of sarcomatous stroma and a benign epithelial component . While tLee et al.  describeA proposed fertility sparing treatment protocol includes fertility sparing surgery (FSS) consisting of a complete resection of the lesion followed by medical treatment with megestrol acetate 160 mg/day for at least 6 months. Once the desire for fertility is fulfilled, hysterectomy with bilateral salpingo-oophorectomy is recommended. A fertility sparing treatment protocol must be performed only in low-risk cases  and close longterm follow-up is needed . PatientIn 2015, a nulliparous 23-year-old woman referred to our institution was diagnosed with UA stage IA according to FIGO 2009 criteria. Due to her strong desire of future fertility, after extensive counselling she opted for FSS. Hysteroscopic complete resection of the tumour was performed, followed by oral megestrol acetate 160 mg/d as adjuvant therapy . This waMullerian adenosarcoma is a rare gynaecological cancer composed of a benign epithelial component with sarcomatous stroma . Total hAdenosarcoma is an extremely rare gynaecological malignancy with only a few cases reported in patients of reproductive age. The current recommended treatment is hysterectomy with bilateral salpingo- oophorectomy, however, fertility sparing treatment options are feasible in selected patients.In cases of focal tumours without sarcomatous overgrowth and without sign of infiltration or metastasis, a fertility sparing approach may be proposed to young women diagnosed with UA and who strongly wish to preserve their fertility. There is currently insufficient evidence regarding the ideal follow up protocol for patients diagnosed with UA who are managed with FSS.


Mesenchymal stem cells (MSCs) give rise to adipocytes, osteocytes, and chondrocytes and reside in various tissues, including bone marrow and adipose tissue. The differentiation choices of MSCs are controlled by several signaling pathways, including the Wnt/β-catenin signaling. When MSCs undergo adipogenesis, they first differentiate into preadipocytes, a proliferative adipocyte precursor cell, after which they undergo terminal differentiation into mature adipocytes. These two steps are controlled by the Wnt/β-catenin pathway, in such a way that when signaling is abrogated, the next step in adipocyte differentiation can start. This sequence suggests that the main role of Wnt/β-catenin signaling is to suppress differentiation while increasing MSC and preadipocytes cell mass. During later steps of MSC differentiation, however, active Wnt signaling can promote osteogenesis instead of keeping the MSCs undifferentiated and proliferative. The exact mechanisms behind the various functions of Wnt signaling remain elusive, although recent research has revealed that during lineage commitment of MSCs into preadipocytes, Wnt signaling is inactivated by endogenous Wnt inhibitors. In part, this process is regulated by histone-modifying enzymes, which can lead to increased or decreased Wnt gene expression. The role of Wnt in adipogenesis, as well as in osteogenesis, has implications for metabolic diseases since Wnt signaling may serve as a therapeutic target. Decades of research has established a main role of Wnt signaling in stem cell fate and control Nusse, . For exaAdipose tissue is a potential source of easily accessible MSCs  and Wnt/Calcium pathways. Wnt signaling plays important roles during embryonic development as well as in adults Nusse, . When WnSecreted Wnt inhibitors factors include Wnt inhibitor protein (WIF) and secreted Frizzled-related proteins (sFRPs). Dickkopf (DKK) is a secreted factor that binds and inhibits the LRP5/6 co-receptors  and brown adipose tissue (BAT). These are both derived from adipose-residing MSCs and are generated in a stepwise differentiation program. MSCs first differentiate into an intermediate cell type called preadipocytes, during which the cells undergo growth arrest and mitotic clonal expansion , resulting in the upregulation of the adipogenic transcription factors PPARγ and C/EBPα  marks the promoters of Additionally, the activity of epigenetic enzymes also affects how MSCs and preadipocytes respond to Wnt signaling. Histone Acetyltransferase GCN5 (GCN5), an enzyme that acetylates histone 3 on lysine 9 (H3K9ac), is known to prime the activation of Wnt genes, resulting in reduced adipogenesis , happens at the expense of adipocyte differentiation, and this bifurcation is regulated by Wnt signaling. Two distinct mechanisms are involved in skeletal bone development, intramembranous and endochondral ossification. The first is characterized by direct formation of osteoblasts while the latter proceeds through an intermediate step of chondrocytes transplantation. Although isolated human MSCs are self-renewing stem cells, they have limited doubling potential when cultured TCF7L2) (Chen et al., In vivo experiments with inactive mutations of TCF7L2 results in impaired adipogenesis characterized by increased subcutaneous adipose tissue mass, adipocyte hypertrophy, and inflammation. All mechanisms that are manifested when there is an excess of lipids. Moreover, this was accompanied by whole-body glucose intolerance and hepatic insulin resistance, a T2D hallmark (Kahn et al., Impaired Wnt signaling can lead to disease, and this is also true for mesenchymal tissues. Since mesenchymal tissue types are constantly renewed (Alfaro et al., Adipogenesis, the process of the formation of adipocytes from MSCs, is tightly regulated by signaling pathways. The Wnt signaling pathways are among these and have been proven to be crucial for MSC lineage specification. It is widely documented that canonical Wnt signaling regulates several steps in this differentiation process, but the precise underlying mechanisms remain poorly understood. In recent years, MSCs have been shown to be promising for use in regenerative medicine (Hoeppner et al., In adipose tissue, Wnt signaling promotes stem cell maintenance of preadipocytes. The stem cell niche of preadipocytes, however, does not solely consist of preadipocytes and adipocytes. Other cell types that reside in the cell type niche are fibroblasts, immune cells, endothelial cells, and a small fraction of MSCs. Preadipocytes can be found in the stromal vascular fraction of adipose tissue and this is believed to be in close proximity of the blood vessels (Tsuji et al., A limitation of many studies related to adipogenesis and MSCs biology is the use of non-human permanent cell lines 3T3-L1 and C3H10T1/2 (Ross et al., In conclusion, Wnt signaling mainly plays a role in suppressing adipogenesis during several stages of differentiation. It seems that the main role of suppression is to increase the MSC and preadipocytes cell mass. Epigenetic and transcriptional regulators have a significant impact on this process, but it is still poorly understood how transcriptional regulation and Wnt signaling interact during adipogenesis.TW performed the literature review, analyzed and interpreted the findings, and wrote the manuscript. RN supervised the writing process and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Osteoarthritis (OA) is a heterogeneous disease that is extremely hard to cure owing to its complex regulation network of pathogenesis, especially cartilage degeneration. FBXO21 is a subunit of ubiquitin E3 ligases that degrades P‐glycoprotein and EID1 by ubiquitination and activates the JNK and p38 pathways; however, its role in OA remains unknown. Here, the main objective of this study was to evaluate the potential effects and mechanism of FBXO21 in OA degeneration, we revealed that FBXO21 is upregulated in the cartilage of patients with OA, aging, and monosodium iodoacetate‐induced OA rats, and chondrocytes treated with interleukin‐1β, tumor necrosis factor‐α, and lipopolysaccharide. Moreover, the in vivo and in vitro knockdown of FBXO21 suppressed OA‐related cartilage degeneration, as evidenced by activated autophagy, upregulated anabolism, alleviated apoptosis, and downregulated catabolism. In contrast, its overexpression promoted OA‐related cartilage degeneration. In addition, using mass spectrometry and co‐immunoprecipitation assay, we demonstrated that the downstream mechanism of FBXO21 inhibits autophagy by interacting with and phosphorylating ERK. Furthermore, FBXO21 alleviated anabolism and enhanced apoptosis and catabolism by inhibiting autophagy in rat chondrocytes. Interestingly, for its upstream mechanism, JUNB promoted FBXO21 expression by directly targeting the FBXO21 promoter, thus further accelerating cartilage degeneration in SW1353 cells and rat chondrocytes. Overall, our findings reveal that the JUNB‐FBXO21‐ERK axis regulates OA apoptosis and cartilage matrix metabolism by inhibiting autophagy. Therefore, FBXO21 is an attractive target for regulating OA pathogenesis, and its knockdown may provide a novel targeted therapy for OA. FBXO21 is upregulated in osteoarthritis (OA). Knockdown and overexpression of FBXO21 suppresses and promotes OA‐related degeneration, respectively. Mechanistically, FBXO21 inhibits autophagy by interacting with and phosphorylating ERK, and enhances OA‐related degeneration by inhibiting autophagy. JUNB regulates FBXO21 expression via targeting FBXO21 promoter and accelerates OA‐related degeneration by promoting FBXO21. JUNB is elevated in human chondrocytes and cartilage with OA and mice cartilage with OA. In addition, JUNB can bind to the promoter of the matrix metalloproteinase13 (MMP13) that promotes the expression of MMP13 and reduces that of type II collagen (COL2A1) after the activation by cytokines such as interleukin (IL)‐1β  rats with OA of aging and monosodium iodoacetate (MIA) model, and chondrocytes with OA. Then, we investigated underlying mechanism that could regulate cartilage degeneration in OA. Subsequently, we identified the involvement of the JUNB‐FBXO21‐ERK axis in regulating apoptosis, anabolism, and catabolism of OA by inhibiting autophagy. To the best of our knowledge, this study is the first to explore the biological role of FBXO21 in OA, which sought to fill the gap in literature. Taken together, these results indicate that the novel functions of FBXO21 might provide a new therapeutic avenue for OA.22.1To evaluate the potential role of FBXO21 in OA, we first demonstrated the validity of the articular cartilage samples divided into undamaged (U) and damaged (D) area from 24 patients with knee OA by collecting clinical images Figure , performp = 0.045) (Table p = 0.004), obesity gradation (p = 0.012), and Kellgren–Lawrence gradation (p = 0.001)  Table . Hence, 2.2In the aging and MIA‐induced rat OA models, OA model was confirmed by gross imaging, plain radiograph, magnetic resonance imaging (MRI), and macroscopic score of gross imaging. We observed increased surface roughening, fibrillation, fissures, and erosions down to subchondral bone during OA aggravation. There were significant differences in the 12 month (M), 18 M, MIA 2 week (W), and MIA 3 W groups compared with control Figure . The relFurthermore, we found that FBXO21 level was enhanced in rat chondrocytes of IL‐1β OA model based on the results of immunofluorescence analysis Figure a. We als2.3We next explored the function of FBXO21 in OA pathogenesis. SD rats were preinjected with either knockdown‐sh‐FBXO21‐01/02 (KD‐01/02) or knockdown‐sh‐Negative control (KD‐NC) adenovirus three times a week into their knee joints one week before the MIA injection. Rats were then sacrificed two weeks after MIA injection as described in the methods section and schematic Figure . Using g2.4To further identify the role of FBXO21 in OA treatment, SD rats were preinjected with either overexpression‐FBXO21 (OE‐FBXO21) or overexpression‐NC (OE‐NC) adenovirus as described in Figure 2.5Then, we investigated the underlying mechanism of the FBXO21‐mediated enhanced cartilage degeneration of OA (stimulated with 20 ng/ml of IL‐1β). Initially, we performed immunoprecipitation (IP), Coomassie Brilliant Blue staining, and proteomic analysis (LC–MS/MS) to identify the FBXO21‐interacting proteins in chondrocytes of SD rats. Interestingly, the peptide sequence of the ERK protein was detected in the purified complex and there was no ERK protein in the 42‐kDa band of IgG binding protein Table , suggest2.6H. sapiens) , first, we used the JASPAR database to predict the potential TFs of FBXO21  Figure a and fou) Figure . To inve) Figure . To furt) Figure b and tha) Figure  and weak) Figure d. The re) Figure c. Next, ) Figure . Thus, tHence, based on our findings, we proposed a mechanism involving the JUNB/FBXO21/ERK axis that participates in the progression of OA cartilage degeneration by regulating autophagy Figure .3OA is extremely hard to cure owing to its complex pathogenesis involving the cross‐regulation of spatial networks having multiple pathways and phenotypes such as ERK pathway, autophagy, apoptosis, anabolism, and catabolism  and co‐immunoprecipitation (co‐IP) assays. ERK belongs to the same pathway as that of JNK and p38, which could be activated by FBXO21  using the JASPAR database . Informed consent was obtained from all patients. The cartilage tissues and clinicopathological features of 24 patients with knee OA who underwent total knee arthroplasty were collected. Articular cartilage samples were divided into two groups according to the International Cartilage Repair Society (ICRS) grade , and all animal procedures involving SD rats were performed in complete compliance with Health Guide of Laboratory Animals for the Care and Use from the National Institutes : 1 month old (1 M), 6 months old (6 M), 12 months old (12 M), and 18 months old (18 M). The MIA model   according to MIA treatment time: saline control (Ctrl), MIA treatment for 1 week (1 W), MIA treatment for 2 weeks (2 W), and MIA treatment for 3 weeks (3 W).We established two OA models consisting of Aging and MIA (Aike Reagent) induced to mimic OA in SD rats. The aging model was divided into four groups according to age  before the injection of MIA for 2 weeks (2 W) in 8 weeks old. Thus, rats with FBXO21 knockdown were divided into three groups (n = 6 per group): 2 W + sh‐NC (KD‐NC), 2 W + sh‐FBX021‐01 (KD‐01), and 2 W + sh‐FBX021‐02 (KD‐02). Rats with FBXO21 overexpression were divided into two groups (n = 6 per group): 2 W + Ad‐NC (OE‐NC) and 2 W + Ad‐FBXO21‐3xflag‐pAdTrack‐CMV (OE‐FBXO21).To identify the role of FBXO21, recombinant adenovirus with n = 6 per group). All SD rats were maintained in a specific pathogen‐free (SPF) environment kept at a constant temperature of 22°C and 70% humidity and fed with a standard diet.Overall, 78 male SD rats  provided by HFK Bioscience Cooperation were divided into thirteen groups . Plain radiographs of the knee joints were photographed using an X‐ray machine  operated by a technician, in which the bilateral ankles of SD rats were fixed on the pallet as previously described  operated by a technician, in which bilateral ankles of SD rats were fixed on the pallet. Briefly, knee joints were surveyed twice for accuracy of location after placing the special coil. Then, sagittal and coronal sections were scanned with the 3D_WATSc program. MRI scan evaluation was based on cartilage loss, osteophytes formation, and subchondral sclerosis . Macroscopic scoring was based on surface roughening, fibrillation, fissures, and erosions down to subchondral bone , and stored at −80°C for subsequent experiments.4.62) with 1% streptomycin, 1% penicillin (Hyclone), 10% fetal bovine serum (FBS) , and Dulbecco's modified Eagle's medium (DMEM) (Hyclone) in 5% CO2 atmosphere at 37 °C. Upon reaching 80% confluence, the primary chondrocytes were divided in a 1:3 ratio and stained with Alcian Blue (Solarbio), Safranin O (Solarbio), or Toluidine Blue (Solarbio) according to the manufacturer's instructions. Immunohistochemistry (IHC) staining for collagen II  was also performed.Primary articular chondrocytes were harvested from the hip and knee articular cartilage of 3‐week‐old male SD rats . After the cartilage was digested with pronase K  for 60 min at 37°C and collagenase D  for 60 min at 37°C in that order, the primary chondrocytes were seeded in culture flasks , recombinant rat TNFα for 12 h , and LPS for 6 h . Meanwhile, SW1353 cells were treated with recombinant human IL‐1β for 12 h , recombinant human TNFα for 12 h  and LPS for 6 h  , 10% FBS (Bioind), and DMEM (Hyclone) in 5% COFBXO21 knockdown or overexpression . Then, the primary chondrocytes were harvested after stimulation with IL‐1β (20 ng/ml) for 12 h. Thus, knockdown of FBXO21 was divided into three groups: IL‐1β + sh‐NC (KD‐NC), IL‐1β + sh‐FBX021‐01 (KD‐01), and IL‐1β + sh‐FBX021‐02 (KD‐02). Overexpression of FBXO21 was divided into two groups: IL‐1β + Ad‐NC (OE‐NC) and IL‐1β + Ad‐FBXO21‐3xflag‐pAdTrack‐CMV (OE‐FBXO21).To determine the role of FBXO21 in OA treatment, primary chondrocytes were transfected with recombinant adenovirus with To investigate whether FBXO21 regulates autophagy by phosphorylating ERK in vitro, chondrocytes were transfected with the KD‐02 adenovirus and harvested after simultaneous stimulation with IL‐1β (20 ng/ml) and the ERK activator Honokiol  for 12 h. To explore whether FBXO21 regulates OA‐related degeneration by autophagy, chondrocytes were transfected with the KD‐02 adenovirus and harvested after simultaneous stimulation with IL‐1β (20 ng/ml) and the autophagy inhibitor 3‐methyladenine  for 12 h.H. sapiens. Rat chondrocytes were transfected with JUNB knockdown or FBXO21 overexpression recombinant adenovirus  and harvested after stimulation with IL‐1β (20 ng/ml) for 12 h.To identify whether JUNB regulates cartilage degeneration via the transcriptional activation of FBXO21, the SW1353 cell line was transfected with the knockdown plasmid of NC (KD‐NC) and JUNB (KD‐JUNB) (GenePharma) and the overexpression plasmid of FBXO21‐pEX3 (OE‐FBXO21) (GenePharma) by lipo3000 (Invitrogen) according to the manufacturer's instructions. We used the human chondrocyte SW1353 cell line because the transcription factors of FBXO21 predicted by the JASPAR database is found in 4.7Primary chondrocytes were harvested after treating and fixing in 2.5% glutaraldehyde (pH 7.4) overnight and 1% osmium tetroxide for 2 h at 4°C. TEM (Hitachi) was performed after dehydration, infiltration, imbedding, and sectioning.4.8Primary chondrocytes were seeded on cell climbing slides and transfected with the mRFP‐GFP‐LC3 adenovirus  before or meanwhile. Chondrocytes were treated as described in 4.6 and fixed in 4% paraformaldehyde for 30 min at room temperature and imaged using a two‐photon fluorescence microscope (Zeiss).4.9Total protein was incubated with 1 μg of normal rabbit IgG  as control or 1 μg of anti‐FBXO21 antibody  overnight at 4°C after the extraction of primary chondrocytes using combination of RIPA buffer for immunoprecipitation , Phenylmethanesulfonyl fluoride , and phosphatase inhibitors (Beyotime) at a ratio of 100:1:1. The mixtures of protein and antibody were subsequently incubated with 40 μl of pre‐washed protein A/G PLUS‐Agarose beads  in gentle rotation for 4 h at 4°C. Next, the immunoprecipitates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on 10% agarose gel and stained by Coomassie Brilliant Blue . Mass spectrometry (Novogene) and immunoblotting were performed .4.10We used the NCBI gene database to search for the promoter region of FBXO21 and the JASPAR 2018 TFBS track of the University of California, Santa Cruz (UCSC) Genome Browser  according to the manufacturer's protocol. Briefly, SW1353 cells stimulated with IL‐1β were fixed in 1% formaldehyde at room temperature for 10 min to cross link DNA and protein. Then, they were treated with Buffer A, Buffer B, Micrococcal Nuclease , and ChIP buffer in this sequence to extract fragmented chromatins. They were incubated overnight with normal rabbit IgG  as control or anti‐JUNB antibody  at 4°C. The mixtures of protein–DNA and antibody were subsequently incubated with Protein G Agarose Beads of ChIP‐Grade  at 4°C for 3 h with shaking. After washing three times with low and once with high‐salt, the protein‐DNA crosslinks of the immunoprecipitates were uncoupled by reaction with proteinase K for 2 h, and DNA was purified with DNA purification columns. Purified DNA was subjected to qRT‐PCR  with the4.11t test (two groups), unpaired t test (two groups), Welch's t test , and one‐way analysis of variance (ANOVA) (multiple groups). For values with non‐normal distribution, non‐parametric analysis included Mann–Whitney test (two groups) and Kruskal–Wallis test (multiple groups). Simple linear regression was used to evaluate the relationship between JUNB and FBXO21. Chi‐square test and Spearman correlation analysis were used to evaluate the relationship between FBXO21 expression and the baseline characteristics of patients with OA. p < 0.05 was considered significant. R2 < 0.16 was considered low linear correlation; 0.16 ≤ R2 < 0.49 significant correlation; and 0.49 ≤ R2 < 1, high linear correlation.All experiments were conducted at least three times, and data were presented as the mean ± SD. Data analysis was conducted using SPSS v24.0 (SPSS Inc.) or GraphPad Prism v8 (GraphPad Inc.). Normality and homogeneity were evaluated using Shapiro–Wilk and Levene tests, respectively. For values with normal distribution, parametric analysis included paired None declared.ZML and LHB designed the study. MH and ZYW collected clinical data and samples. ZML and JNM performed the in vivo and in vitro experiments. ZML, TZ, and XYF analyzed the data and prepared the figures. ZML wrote the manuscript. LHB revised the manuscript. All authors have read and approved the final version of the manuscript for publication.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Fig S5Click here for additional data file.Fig S6Click here for additional data file.Supplementary MaterialClick here for additional data file.