Endogenous Activity Research Articles

Conserved Cis-Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals.

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used in vivo enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range. We uncover a novel conserved cis-acting element, Range EXtender (REX), that confers extreme-distance regulatory activity and is located next to a long-range enhancer of Sall1. The REX element itself has no endogenous enhancer activity. However, addition of the REX to other short- and mid-range enhancers substantially increases their genomic interaction range. In the most extreme example observed, addition of the REX increased the range of an enhancer by an order of magnitude, from its native 71kb to 840kb. The REX element contains highly conserved [C/T]AATTA homeodomain motifs. These motifs are enriched around long-range limb enhancers genome-wide, including the ZRS, a benchmark long-range limb enhancer of Shh. Mutating the [C/T]AATTA motifs within the ZRS does not affect its limb-specific enhancer activity at short range, but selectively abolishes its long-range activity, resulting in severe limb reduction in knock-in mice. In summary, we identify a sequence signature globally associated with long-range enhancer-promoter interactions and describe a prototypical REX element that is necessary and sufficient to confer extreme-distance gene activation by remote enhancers.

LPA3 Receptor Phosphorylation Sites: Roles in Signaling and Internalization.

Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, confirming that the main phosphorylation sites are present in both domains and their role in receptor phosphorylation eliminated by substitution and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. The agonist, Ki16425, diminished baseline intracellular calcium, which suggests some receptor endogenous activity. Similarly, baseline ERK1/2 phosphorylation was diminished by Ki16425. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with β-arrestin 2 and LPA3 internalization were severely diminished in cells expressing the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated. Our data experimentally show that receptor phosphorylation in the distinct domains is relevant for LPA3 receptor function.

Epigenome editing revealed the role of DNA methylation of T-DMR/CpG island shore on Runx2 transcription

RUNX2 is a transcription factor crucial for bone formation. Mutant mice with varying levels of Runx2 expression display dosage-dependent skeletal abnormalities, underscoring the importance of Runx2 dosage control in skeletal formation. RUNX2 activity is regulated by several molecular mechanisms, including epigenetic modification such as DNA methylation. In this study, we investigated whether targeted repressive epigenome editing including hypermethylation to the Runx2-DMR/CpG island shore could influence Runx2 expression using Cas9-based epigenome-editing tools. Through the transient introduction of CRISPRoff-v2.1 and gRNAs targeting Runx2-DMR into MC3T3-E1 cells, we successfully induced hypermethylation of the region and concurrently reduced Runx2 expression during osteoblast differentiation. Although the epigenome editing of Runx2-DMR did not impact the expression of RUNX2 downstream target genes, these results indicate a causal relationship between the epigenetic status of the Runx2-DMR and Runx2 transcription. Additionally, we observed that hypermethylation of the Runx2-DMR persisted for at least 24 days under growth conditions but decreased during osteogenic differentiation, highlighting an endogenous DNA demethylation activity targeting the Runx2-DMR during the differentiation process. In summary, our study underscore the usefulness of the epigenome editing technology to evaluate the function of endogenous genetic elements and revealed that the Runx2-DMR methylation is actively regulated during osteoblast differentiation, subsequently could influence Runx2 expression.

Diverse avenues of research support the transmethylation theory of psychosis: implications for neuroprotection

Transmethylation in the context of psychiatry has historically referred to the enzymatic transfer of a methyl group from one biochemical to another, whose resulting function can change so dramatically that a biochemical like tryptamine, for example, is converted into the hallucinogen dimethyltryptamine. Central to endogenous methylation activity is the folate cycle, which generates the primary transferable methyl groups in mammalian biochemistry. The relevance of this cycle to mental health becomes clear when the cycle is dysregulated, often leading to a buildup of both homocysteine and S-adenosylhomocysteine (SAH), while accompanied by a transient reduction in the intended physiologic target, S-adenosylmethionine (SAM). This paper includes an in-depth review of the causes of folate cycle perturbations associated with psychotic symptoms, expounding on alternative downstream pathways which are activated and pointing toward potential etiologic agents of the associated psychosis, the methylated tertiary amines N-methyl-salsolinol, N-methyl-norsalsolinol, and adrenochrome, which appear in scientific reports concerning their association with hallucinogenic and/or neurotoxic outcomes. Electrotopological state (E-state) data has been generated for these compounds, illustrating a strong similarity with hallucinogens, particularly in terms of the E-state of the nitrogen in their tertiary amine moieties. In light of the role the folate cycle plays in transmethylation, neuroprotective strategies to prevent the transition to psychosis are suggested, including the advisory that folate supplementation can be harmful depending on the status of other relevant biochemicals.

Antioxidant and Hepatoprotective Effect of Rosa davurica Pall Seed Oil on CCl4-Induced Acute Liver Injury in Mice.

The liver, being the most metabolically active organ, is highly vulnerable to damage caused by oxidative stress. Rosa davurica Pall. seed oil (RDPO), a novel vegetable oil, and its bioactive components have been extensively researched in the field of antioxidants. In this research, the antioxidant properties and hepatoprotection by RDPO were evaluated. A series of antioxidant evaluation systems and a CCl4-induced acute liver injury model in mice were used to investigate the antioxidant activity and hepatoprotective efficacy of RDPO. The results showed that the extraction rate of RDPO was 11.12% using the optimal extraction process. Three major unsaturated fatty acids of the oil were α-linolenic acid (11.89 ± 0.017%), linoleic acid (18.52 ± 0.072%), and oleic acid (11.54 ± 0.425%). Furthermore, its antioxidant small-molecule compounds were β-sitosterol (1.429 ± 0.002 μg/g), α-tocopherol (1.273 ± 0.079 μg/g), β-carotene (0.012 ± 0.001 μg/g), lycopene (0.108 ± 0.002 μg/g), squalene (178.950 ± 0.794 μg/g), total polyphenols (1.114 ± 0.032 μg GAE/mg), and total flavonoids (0.504 ± 0.009 mg RU/g), respectively. In vitro, RDPO significantly inhibited the production of ABTS+•, DPPH•, O2•-, and hydroxyl radicals, as well as Fe3+. In vivo, RDPO significantly reversed the activity of total superoxide-dismutase, catalase, L-glutathione, and the level of malondialdehyde (MDA) in liver tissue. It also obviously inhibited the activity of aspartate transaminase (AST) and the level of MDA in the serum. Therefore, RDPO has demonstrated excellent antioxidant activity and a potential liver protective effect. This effect may be ascribed to its capacity for decreasing AST activity, inhibiting lipid peroxidation, and boosting endogenous antioxidant enzyme activity. Therefore, RDPO has significant application value in the biopharmaceutical industry and as a dietary supplement.

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis. The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors- Gata4, Foxa3 , and Hnf1a -or alternatively, the expression of two transcription factors, Gata4 and Foxa3 . In vivo , we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER ; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Activation of only two factors, Gata4 and Foxa3 , via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Prediction of endogenous mineralocorticoid receptor activity by depressor effects of mineralocorticoid receptor antagonists in patients with primary aldosteronism

Prediction of endogenous mineralocorticoid receptor activity by depressor effects of mineralocorticoid receptor antagonists in patients with primary aldosteronism

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation

BackgroundElevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates.MethodsThe senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation.ResultsThe high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG.ConclusionsFSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis (“FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients.Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

An intranasal nanoparticle vaccine elicits protective immunity against Mycobacterium tuberculosis

Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1− lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille–Calmette–Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.

Lysine Acetylation of Aquaporin-3 in Renal Principal Cell Mediates Water Homeostasis

The kidneys function to maintain water homeostasis. High plasma osmolality activates the release of the antidiuretic hormone vasopressin (AVP), which acts on the kidneys through its receptor AVPR2 on the principal cell (PC) to increase apical aquaporin-2 (AQP2) and basolateral AQP3 abundance for enhanced water reabsorption. However, unlike AQP2, the regulation mechanism(s) of AQP3 is unclear. We previously identified a novel AQP3 post-translational modification: acetylation of lysine 282 (K282), which we hypothesize to positively regulate PC water permeability. We developed mouse models of acetylated AQP3 by mutating K282 to glutamine (K282Q; Q mutant) and deacetylated AQP3 by mutating K282 to arginine (K282R; R mutant), on C57BL/6J background to determine the functional consequences of lysine acetylated AQP3. Male and female mice (10-15 weeks old) received a standard chow diet and free access to water. We first investigated the responses to endogenous AVPR2 activation by subjecting the mice to 18-hr overnight water deprivation and compared to mice with ad libitum water access (ad lib). All dehydrated mice (males WT 3087 ± 291 mOsm/kg, n = 8; Q 3688 ± 113 mOsm/kg, n = 6; R 3447 ± 171 mOsm/kg, n = 11; females WT 3393 ± 266 mOsm/kg, n = 8; Q 3586 ± 206 mOsm/kg, n = 9; R 3749 ± 126 mOsm/kg, n = 10) had higher urine osmolality than ad lib mice (males WT 2409 ± 165 mOsm/kg, n = 8; Q 2428 ± 316 mOsm/kg, n = 7; R 1960 ± 196 mOsm/kg, n = 7; females WT 2281 ± 96 mOsm/kg, n = 8; Q 1935 ± 190 mOsm/kg, n = 11; R 2279 ± 249, n = 7, two-way ANOVA p[treatment] <0.0001 for both sexes). However, there were no significant differences among the genotypes in both sexes [two-way ANOVA p[sex] > 0.05). For plasma osmolality, all dehydrated mice in both sexes (males WT 332 ± 2 mOsm/kg, n = 10; Q 324 ± 2 mOsm/kg, n = 8; R 323 ± 2 mOsm/kg n = 11; females WT 316 ± 3 mOsm/kg, n =10; Q 320 ± 2 mOsm/kg, n = 10; R 308 ± 3 mOsm/kg, n = 11) had higher values than ad lib mice (males WT 314 ± 3 mOsm/kg, n = 10; Q 309 ± 1 mOsm/kg, n = 9; R 313 ± 3 mOsm/kg n = 10; females WT 312 ± 2 mOsm/kg, n =11; Q 305 ± 2 mOsm/kg, n = 11; R 301 ± 4 mOsm/kg, n = 10, 2-way ANOVA p[treatment] < 0.0001 for males and = 0.0006 for females). Female R mutants had lower plasma osmolality than WTs and Q in both ad lib and dehydrated groups (2-way ANOVA p[genotype] = 0.0045). In males, compared to WTs, Q and R mutants had similar plasma osmolality in the ad lib but lower in dehydrated group (2-way ANOVA p[genotype] = 0.0359). This suggests that deacetylated AQP3 may result in a reset osmostat, although this remains to be explored further. We next compared the responses of WT and Q mutants to 4-hour exogenous AVPR2 activation by dDAVP (10 ng, i.p). Q females exhibited lower plasma osmolality than WT females (286 ± 4 vs. 305 ± 2 mOsm/kg, n = 11 for both, Student’s t-test p = 0.0005) suggesting lysine acetylation of AQP3 leads to acute enhancement of water permeability. However, there were no significant differences between Q and WT males (300 ± 5 (n = 7) vs. 303 ± 2 mOsm/kg (n = 8), Student’s t-test p > 0.05). Therefore, we conclude that lysine acetylation of AQP3 contributes to renal PC water permeability and this may be sex-dependent. Thus, acetylated AQP3 is a novel mechanism to maintain water homeostasis. R01DK128001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Polycyclic aromatic hydrocarbon (PAH) activation of AhR inhibits glutathione S-transferase (GST) activity in primary human small airway epithelial cells (hSAECs)

Background: PAH represents the organic phase of particulate matter (PM) in air pollution emitted via the incomplete combustion of various compounds (i.e. diesel fuel, wood, and plastics). It is widely regarded that PAH activation of aryl hydrocarbon receptor (AhR) plays an important role in pro-inflammatory signaling, however the mechanisms remain unknown. In contrast- endogenous AhR agonists, such as 6-Formylindolo[3,2-b]carbazole (FICZ), exert pleiotropic protective effects against inflammation, fibrosis, and oxidative stress. The objective of our research program is to delineate the differences between activation of AhR by PAH (with adverse effects) compared to its beneficial homeostatic activation by endogenous FICZ. PM may induce oxidative stress in part through inhibition of antioxidant defense mechanisms. GSTs are a superfamily of enzymes that plays an important role in protecting cells from oxidative damage (by conjugating reduced glutathione to substrates under pro-oxidizing conditions). We hypothesize that PAH activation of AhR contributes to oxidative stress via inhibition of GST activity in primary hSAECs. Methods: Primary hSAECs were subjected to varying dosages of PAH [0, 250, 500, 1000, 5000 ng/mL] to model airborne PM exposure levels, and to increasing doses of FICZ [0, 0.50, 10, 100, 1000 nM] to model normal and excessively high endogenous ligand activation of AhR. We determined the relative potencies of PAH and FICZ by measuring CYP1A1 and CYP1B1 gene expression. Oxidative stress was measured using DHE-labeling and GST activity was measured with enzymatic kits (Sigma Aldrich). AhR activity was pharmacologically inhibited using 10μM CH223191. Statistical analysis was conducted using SigmaPlot 14.0 (Systat Software). Results: PAH and FICZ maximally stimulate hSAEC CYP1A1 and CYP1B1 expression at 1000ng/mL and 100nM, respectively, and are therefore defined as equipotent concentrations used in our studies. In a representative experiment in hSAECs, PAH (1000ng/mL) significantly increased hSAEC oxidative stress vs FICZ (100nM) n=6, p<0.01), while significantly decreasing GST specific activity vs equipotent FICZ activation of AhR in primary hSAECs from a 66-year-old female without lung disease (26.6±0.874 vs 28.3±.611 nmol/mL/min); n=6 from 2 independent assays; p=0.032. Similar differential effects of PAH vs FICZ activation of AhR on GST were seen in additional experiments with male hSAECs and Calu-3 cells (male). Importantly, CH223191 inhibition of GST enzymatic activity suggests that AhR plays an important role in protecting cells from oxidative stress. Conclusions: Our studies indicate that airborne PAH exposure can have detrimental health effects on the lung via aberrant AhR signaling. Significance: Understanding activity of AhR signaling can leads to novel therapeutic options for redox disorders in the lung. Funding: VA 5I01BX001777-05 (RP) and R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of differentially expressed Gpr116 (Adgrf5) transcript variants in mouse kidney

Adhesion G protein-coupled receptors (aGPCRs) are important and understudied modulators of physiological processes. Unlike class A GPCRs that are activated by soluble ligands, aGPCRs are self-activated by a cryptic tethered agonist after holoreceptor dissociation. We recently demonstrated that kidney-specific ADGRF5/Gpr116 knockout causes luminal membrane accumulation of V-ATPase in acid-secreting A-type intercalated cells (AICs) in the collecting ducts and a significant reduction in urine pH (Zaidman et al. 2020). Previous work suggests that aGPCRs, and Gpr116 in particular, undergo significant tissue-specific mRNA processing that results in holoreceptors with unique and variable N-terminal structures (Knierim et al. 2019). These aGPCR splice variants possess singular structure-function relationships that are integral to endogenous self-activation. Therefore, identifying tissue-specific transcript variants is critical to understanding the physiological roles of aGPCRs. Besides robust expression in AICs, Gpr116 is also transcribed in endothelial cells (ECs) in the kidney. We hypothesized that unique Gpr116 transcript variants are expressed in renal AICs and ECs. To test this hypothesis, we enzymatically digested kidneys from Gpr116flag V1B1eGFP mice and enriched for ICs (CD31−, GFP+) and ECs (CD31+, GFP−) by FACS. Gpr116 was not detected in CD31− GFP− sorted cells. IC enrichment was confirmed by qPCR by detection of Atp6v0d2, c-kit (CD117), Slc4a1, and Slc4a9 transcripts. EC enrichment was confirmed by detection of Kdr and Nrp1 transcripts. Next, we assayed cDNA from these cell populations for exon variants by PCR, and subsequent nanopore sequencing. We detected and aligned three exons that undergo differential expression in the kidney: exons 2, 12, and 22. Both IC and EC populations were positive for all three differentially expressed exons, but also expressed Gpr116 transcripts without the variable regions, suggesting multiple Gpr116 isoforms in each cell type. However, EC markers were detected in GFP+ ICs, demonstrating some EC contamination in the sorted ICs. To test the tissue specificity of these differentially expressed exons, we assayed 7 other tissues from Gpr116flag V1B1eGFP mice. We detected exon 2 and 12 in lung, heart and fat; we did not detect it in pancreas, spleen, thymus or liver. We detected exon 22 in lung and fat; we did not detect it in pancreas, heart, spleen, thymus or liver. We detected Gpr116flag in the lung, heart, spleen, fat, thymus, kidney and liver, but not pancreas. In summary, we have identified three variable exons within the open reading frame of Gpr116. Future studies will characterize the roles of differentially expressed exons on the endogenous activation of Gpr116 and determine if transcript variants are cell-type specific. This study is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (4R00DK127215). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nature of Perovskite Mineralization of Silicate-Carbonate Veins in the Margins of Kusinsko-Kopanskaya Layered Intrusion (South Urals, Russia)

This study presents the first comprehensive investigation of perovskite from its type locality (Mineral Mines of Southern Urals, Russia), where this mineral was first described by Gustav Rose in 1839. The new data includes results from precise chemical analyses (electron-probe microanalyzer, LA-ICP-MS) and U-Pb ages (SHRIMP-II) of perovskite. Perovskite occurs in silicate-carbonate veins that transect the marginal parts of the Middle Riphaean Kusinsko-Kopanskaya layered intrusion, previously thought to be skarns. The perovskite crystals range from micrometer-scale grains to up to 11 cm in size. Chemical investigations revealed a low content of trace elements (rare earth elements, Y, Nd, U, Th) compared to perovskites from alkaline ultramafic rocks, silica-undersaturated basic rocks, carbonatites, and kimberlites. The determined age of the perovskite, 535 ± 43 Ma, significantly differs from the 1379 ± 8 Ma age of the Kusinsko-Kopanskaya intrusion, challenging the skarn-origin hypothesis for perovskite. Instead, the findings suggest a carbonatite origin for the perovskite mineralization. This timing indicates a previously unknown stage of endogenic activity on the Western Slope of the Southern Urals.

Phytochemical analysis and evaluation of antioxidant properties of extracts from leaves, stem-barks and roots of <i>Pterocarpus Erinaceus</i>

Oxidative stress is found to be associated with the etiology of several human diseases. Free radicals are the causative agent of oxidative stress, and can be scavenged by either natural (plants) or synthetic antioxidants. Synthetic antioxidants have some side effects which push a lot of research focuses on natural antioxidants. Pterocarpus erinaceus is a medicinal plant employed as traditional remedy for the treatment of several diseases associated with oxidative stress. This basis, the present study aimed at screening phytochemical and evaluating antioxidant properties of extracts from leaves, stem-barks and roots of Pterocarpus erinaceus. Leaves, stem-barks and roots of Pterocarpus erinaceous after collection were air-dried and pulverized. Each was extracted with methanol and the methanolic extracts were used. Phytochemicals were screened, in vitro, and in vivo antioxidant studies conducted. Rats were grouped into; Group 1: Normal control (liquid paraffin, vehicle 1 ml.kg), Group 2: Negative control (received 1 ml/kg CCl4), Group 3: Positive control (received 1ml/kg CCl4 +1 ml/kg Silymarin), Group 4-6: Extract treated rats (received 1 ml/kg CCl4 + varied doses of Extracts at 100, 200, and 400mg/kg body weight of rats). The treatment was done daily via oral means for 14 days. The results of the study showed Pterocarpus erinaceus rich in different phytochemicals which possess free radicals scavenging properties. In vivo study revealed that Pterocarpus erinaceus could ameliorate CCl4 toxicity in a way that seem to be via elevation of endogenous antioxidant activities. In conclusion, the leaves of Pterocarpus Erinaceus possessed more of the components rich with antioxidant properties hence its recommendation for further study to identify molecules enshrined therein.

Targeted Thrombolysis with Magnetic Nanotherapeutics: A Translational Assessment.

Plasminogen activators, such as recombinant tissue-type plasminogen activators (rtPAs), while effective in treating thromboembolic diseases, often induce hemorrhagic complications due to non-specific enzyme activities in the systemic circulation. This study evaluated the targeting efficiency, efficacy, biodistribution, and potential toxicity of a rtPA covalently attached to chitosan-coated magnetic nanoparticles (chitosan-MNP-rtPA). The thrombolytic activity of a chitosan-MNP-rtPA was preserved by protection from an endogenous plasminogen activator inhibitor-1 (PAI-1) in whole blood and after circulation in vivo, as examined by thromboelastometry. Single-photon emission computed tomography (SPECT) demonstrated real-time retention of a 99mTc-MNP-rtPA induced by magnet application in a rat embolic model; an 80% reduction in rtPA dosage for a chitosan-MNP-rtPA with magnetic guidance was shown to restore blood flow. After treatment, iron deposition was observed in the reticuloendothelial systems, with portal edema and neutrophil infiltration in the liver at a ten-fold higher dose but not the regular dose. Nevertheless, no liver or renal toxicity was observed at this higher dose. In conclusion, the liver may still be the major deposit site of rtPA nanocomposites after targeted delivery; chitosan-coated MNPs are potentially amenable to target therapeutics with parenteral administration.

Therapeutic developments for neurodegenerative GM1 gangliosidosis.

GM1 gangliosidosis (GM1) is a rare but fatal neurodegenerative disease caused by dysfunction or lack of production of lysosomal enzyme, β-galactosidase, leading to accumulation of substrates. The most promising treatments for GM1, include enzyme replacement therapy (ERT), substrate reduction therapy (SRT), stem cell therapy and gene editing. However, effectiveness is limited for neuropathic GM1 due to the restrictive nature of the blood-brain barrier (BBB). ERT and SRT alleviate substrate accumulation through exogenous supplementation over the patient's lifetime, while gene editing could be curative, fixing the causative gene, GLB1, to enable endogenous enzyme activity. Stem cell therapy can be a combination of both, with ex vivo gene editing of cells to cause the production of enzymes. These approaches require special considerations for brain delivery, which has led to novel formulations. A few therapeutic interventions have progressed to early-phase clinical trials, presenting a bright outlook for improved clinical management for GM1.

Effects of Etching Time and Ethanol Wet Bonding on Bond Strength and Metalloproteinase Activity in Radicular Dentin.

(1) Background: The objective of this in vitro study was to evaluate the impact of different etching times and ethanol pre-treatments on the immediate bond strength of a hydrophilic multi-mode universal adhesive (Clearfil Universal Bond Quick, Kuraray, UBQ) and on the consequent gelatinolytic activity of metalloproteinases (MMPs) on radicular dentin. (2) Methods: Sixty single-root teeth were selected and divided into four groups according to the adhesive protocol applied for fiber post cementation: (G1) 15 s H3PO4 application + UBQ; (G2) 30 s H3PO4 application + UBQ; (G3) 15 s H3PO4 application + ethanol pre-treatment + UBQ; (G4) 30 s H3PO4 + ethanol pre-treatment + UBQ. After adhesive procedures, fiber posts were luted into the post space with a dual-curing cement (DC Core, Kuraray) and light-cured for 40 s. To perform the push-out test and nanoleakage analyses for both coronal end apical areas, 1 mm slices were prepared, following a 24 h storage period in artificial saliva. Additionally, an in situ zymographic assay was conducted to explore endogenous MMP activity within the radicular layer. Results were statistically analyzed with ANOVA and Tukey post hoc tests. Statistical significance was set at p < 0.05. (3) Result: ANOVA revealed a statistically significant difference in push-out bond strength related to the pre-treatment variable but did not highlight any significance of etching time. Specimens pre-treated with ethanol wet bond application showed higher bond strength (p < 0.01). In situ zymography quantification analyses revealed that all tested groups, independently of etching time end ethanol pre-treatment, activated MMP gelatinolytic activity. A significant increase in MMP activity was detected for the 30 s etching time. However, ETOH pre-treatment significantly reduced MMP activity within the adhesive interface (p < 0.01). (4) Conclusions: The tested adhesive showed similar results regardless of the etching time protocol. The gelatinolytic activity of MMPs was observed in all the groups. Further investigations and extended follow-ups are required to validate the results of the present study in vivo.