Metformin is the biguanide of hepatic insulin sensitizer for patients with non-alcohol fatty liver disease (NAFLD). Findings regarding its efficacy in restoring blood lipids and liver histology have been contradictory. In this study, we explore metformin's preventive effects on NAFLD in leptin-insensitive individuals. We used liver tissue, serum exosomes and isolated hepatocytes from high-fat diet (HFD)-induced Zucker diabetic fatty (ZDF) rats and leptin receptor (Lepr) knockout rats to investigate the correlation between hepatic Lepr defective and liver damage caused by metformin. Through immunostaining, RT-PCR and glucose uptake monitoring, we showed that metformin treatment activates adenosine monophosphate (AMP)-activated protein kinase (AMPK) and its downstream cytochrome C oxidase (CCO). This leads to overactivation of glucose catabolism-related genes, excessive energy repertoire consumption, and subsequent hepatocyte pyroptosis. Single-cell RNA sequencing further confirmed the hyper-activation of glucose catabolism after metformin treatment. Altogether, we showed that functional Lepr is necessary for metformin treatment to be effective, and that long-term metformin treatment might promote NAFLD progression in leptin-insensitive individuals. This provides important insight into the clinical application of metformin.

Diabetic encephalopathy (DE) is one of the major chronic complications of diabetes mellitus. This study aims to investigate the inhibitory effect of berberine (BBR) on the damage of PC12 cells induced by high glucose (HG). Differentiated PC12 cells were treated with different concentrations of glucose/BBR. The cell morphology, cell viability, lactate dehydrogenase (LDH) activity, apoptosis, oxidative stress (OS), mitochondrial structure, mitochondrial membrane potential (MMP), mitochondrial complex I-V activity, and adenosine triphosphate (ATP) levels were evaluated. The mRNA and protein levels of the Keap1/Nrf2/ARE pathway-related genes were assessed by RT-qPCR and Western blot. High-dose BBR and HG jointly treated-PC12 cells were treated with Nrf2-specific inhibitor ML385 to further verify whether Nrf2 was the target of BBR. The results showed that BBR inhibited cell damage, OS, and mitochondrial dysfunction induced by HG. The inhibitory effect of high BBR was more significant. The Keap1/Nrf2/ARE pathway was inhibited in PC12 cells induced by HG. BBR could activate the Keap1/Nrf2/ARE pathway, thus up-regulating the expression levels of antioxidant enzymes. ML385 antagonized the ameliorating effect of BBR on OS and mitochondrial dysfunction. The conclusion is that BBR can activate the Keap1/Nrf2/ARE pathway, upregulate the expression patterns of antioxidant enzymes, and reduce cell damage, OS, and mitochondrial dysfunction of PC12 cells induced by HG.

Inherited defects in the Adenosine deaminase (ADA) gene typically cause severe combined immunodeficiency (SCID). In addition to infections, ADA-deficient patients can present with neurodevelopmental, behavioral, hearing, skeletal, lung, heart, skin, kidney, urogenital, and liver abnormalities. Some patients also suffer from autoimmunity and malignancies. In recent years, there have been remarkable advances in the management of ADA deficiency. Most ADA-deficient patients can be identified by newborn screening for SCID, which facilitates early diagnosis and treatment of asymptomatic infants. Most patients benefit from enzyme replacement therapy (ERT). Allogenic hematopoietic cell transplantation (HCT) from an HLA-matched sibling donor (MSD) or HLA-matched family member donor (MFD) with no conditioning is currently the preferable treatment. When MSD or MFD are not available, autologous ADA gene therapy (GT) with nonmyeloablative conditioning and ERT withdrawal, which is in reported in recent studies to result in 100% overall survival and 90-95% engraftment, should be pursued. If GT is not immediately available, ERT can be continued for a few years, although its excessive cost might be prohibitive. The recent improved outcome of HCT using HLA-mismatched family related donors or HLA-matched unrelated donors, after reduced intensity conditioning, suggest that such procedures might also be considered rather than continuing ERT for prolonged periods. Long-term follow-up will further assist in determining the optimal treatment approach for ADA-deficient patients.

Since the early days of foundational studies of nucleic acids, many chemical moieties have been discovered to decorate RNA and DNA in diverse organisms. In mammalian cells, one of these chemical modifications, N6-methyl adenosine (m6A), is unique in a way that it is highly abundant not only on RNA polymerase II (RNAPII) transcribed, protein-coding transcripts but also on non-coding RNAs, such as ribosomal RNAs and snRNAs, mediated by distinct, evolutionarily conserved enzymes. Here, we review RNA m6A modification in the light of the recent appreciation of nuclear roles for m6A in regulating chromatin states and gene expression, as well as the recent discoveries of the evolutionarily conserved methyltransferases, which catalyze methylation of adenosine on diverse sets of RNAs. Considering that the substrates of these enzymes are involved in many important biological processes, this modification warrants further research to understand the molecular mechanisms and functions of m6A in health and disease.

The striatal dopamine D2 receptor (D2R) is generally accepted to be involved in positive symptoms of schizophrenia and is a main target for clinically used antipsychotics. D2R are highly expressed in the striatum, where they form heteromers with the adenosine A2A receptor (A2AR). Changes in the density of A2AR-D2R heteromers have been reported in postmortem tissue from patients with schizophrenia, but the degree to which A2R are involved in schizophrenia and the effect of antipsychotic drugs is unknown. Here, we examine the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay. After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization. The bulky tricyclic moiety of clozapine displaces TM 5 of D2R, inducing a clash with A2AR, while the extended binding mode of haloperidol and aripiprazole stabilizes a specific conformation of the second extracellular loop of D2R that enhances the interaction with A2AR. It is proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects (EPS) of antipsychotics and that the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low EPS liability.

Herein, we develop a strategy of matched spectral and temporal light management to improve photosynthetic efficiency by co-assembling natural thylakoid membrane (TM) with artificial long afterglow particle (LAP). To be specific, LAP with excellent stability and biocompatibility possesses the capabilities of light conversion and storage, optically-matched with the absorption of TM. These favorable features permit LAP as an additional well-functioned light source of photosynthesis performed by TM. As a consequence, enhanced photosynthesis is achieved after co-assembly, compared with pure TM. Under light, the rates of electron transfer, oxygen yield and adenosine triphosphate (ATP) production in this biohybrid architecture are boosted owing to down-conversion fluorescence emission from LAP. Under dark, persistent phosphorescence emission in charged LAP facilitates continual photosynthesis of TM, while that of pure TM almost stops immediately. This proof-of-concept work opens a new route to augment the photosynthetic efficiency of green plants by utilizing precise light-managed materials.

Background - The inspIRE study evaluated safety and effectiveness of a fully integrated biphasic pulsed field ablation (PFA) system with a variable loop circular catheter for the treatment of drug-refractory paroxysmal atrial fibrillation (AF). Methods - Subjects underwent pulmonary vein isolation (PVI) with the PFA system, using at least 12 applications per vein; adenosine/isoproterenol was administered to confirm entrance block. Wave I assessed initial safety, including for esophageal lesions, silent cerebral lesions (SCLs), and PV stenosis. Wave II (pivotal phase) tested i) primary safety - incidence of early onset primary adverse events (PAEs), and primary effectiveness - confirmed PVI with freedom from documented atrial arrhythmia at 12-months (12M). The study design specified an interim analysis to determine early success once 30 subjects reached 12M follow-up (FU) and all subjects reached 3M FU. Results - Across 13 centers in Europe/Canada, 226 subjects were enrolled, met criteria for safety and effectiveness evaluations and received PFA (Wave I: 40; Wave II: 186). Wave I demonstrated no esophageal thermal lesions or PV stenosis. Among 39 subjects with cerebral MRI, SCLs were detected in 4 of the first 6 subjects, after which workflow enhancements, including a 10s pause between PFA applications was implemented; subsequently, only 4 of 33 subjects had SCLs. In the Wave II phase, no PAE was reported. Upon declaring early success, 83 subjects reached 12M FU. With 100% entrance block, PVI without acute reconnection was achieved in 97.1% of targeted veins. For Wave II, the primary effectiveness endpoint per Kaplan Meier at the time of interim analysis was 70.9%; 12M freedom from symptomatic AF/atrial flutter/atrial tachycardia recurrence and repeat ablation was 78.9% and 92.3%, respectively. Total procedure and transpired PFA times were 70.1 ± 27.7 min and 26.7 ± 14.0 min, respectively. Conclusions - The inspIRE trial confirmed the safety and effectiveness of the novel mapping-integrated PFA system.

Complement activation is thought to underline the pathologic progression of obesity-related metabolic disorders; however, its role in adaptive thermogenesis has scarcely been explored. Here, we identify complement C3a receptor (C3aR) and C5a receptor (C5aR) as critical switches to control adipocyte browning and energy balance in male mice. Loss of C3aR and C5aR in combination, more than individually, increases cold-induced adipocyte browning and attenuates diet-induced obesity in male mice. Mechanistically, loss of C3aR and C5aR increases regulatory T cell (Treg) accumulation in the subcutaneous white adipose tissue during cold exposure or high-fat diet. Activated Tregs produce adenosine, which is converted to inosine by adipocyte-derived adenosine deaminases. Inosine promotes adipocyte browning in a manner dependent on activating adenosine A2a receptor. These data reveal a regulatory mechanism of complement in controlling adaptive thermogenesis and suggest that targeting the C3aR/C5aR pathways may represent a therapeutic strategy in treating obesity-related metabolic diseases.

Food-derived bioactive peptides able to regulate neuronal function have been intensively searched and studied for their potential therapeutic application. Our previous study showed that a polypeptide complex yolkin, isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), improved memory and cognitive functions in rats. However, the mechanism activated by the yolkin is not explained. The goal of the present study was to examine what molecular mechanism regulating brain-derived neurotrophic factor (BDNF) expression is activated by the yolkin complex, using in vitro models of PC12 cell line and fetal rat hippocampal cell line H19-7. It was shown that yolkin increased the proliferative activity of rat hippocampal precursor cells H19-7 cells and upregulated the expression/production of BDNF in a cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-dependent manner. Additionally the upregulation of carboxypeptidase E/neurotrophic factor-α1 (CPE/(NF-α1) expression was shown. It was also determined that upregulation of CREB phosphorylation by yolkin is dependent on cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) signaling pathway activation. Moreover, the impact of yolkin on the level of intracellular Ca2+, nitric oxide, and activation of extracellular signal-regulated kinases 1/2 (ERK 1/2 kinase) was excluded. These results emphasize that yolkin can act comprehensively and in many directions and may participate in the regulation of neurons' survival and activity. Therefore, it seems that the yolkin specimen can be used in the future as a safe, bioavailable, natural nutraceutical helping to improve the cognition of older people.

One-pot co-precipitation of target molecules e.g. organic dyes and the synthesis of a crystal containing microporous-mesoporous regimes of zeolitic imidazolate frameworks-8 (ZIF-8) are reported. The synthesis method can be used for cationic (rhodamine B (RhB), methylene blue (MB)), and anionic (methyl blue (MeB)) dyes. The crystal growth of the ZIF-8 crystals takes place via an intermediate phase of zinc hydroxyl nitrate (Zn5(OH)8(NO3)2) nanosheets that enabled the adsorption of the target molecules i.e., RhB, MB, and MeB into their layers. The dye molecules play a role during crystal formation. The successful encapsulation of the dye molecules was proved via diffuse reflectance spectroscopy (DRS) and electrochemical measurements e.g., cyclic voltammetry (CV), linear sweep voltammetry (LSV), and electrochemical impedance spectroscopy (EIS). The materials were investigated for carbon dioxide (CO2) adsorption and adenosine triphosphate (ATP) biosensing. ZIF-8, RhB@ZIF-8, MB@ZIF-8, and MeB@ZIF-8 offered CO2 adsorption capacities of 0.80, 0.84, 0.85, and 0.53 mmol g-1, respectively. The encapsulated cationic molecules improved the adsorption performance compared to anionic molecules inside the crystal. The materials were also tested as a fluorescent probe for ATP biosensing. The simple synthesis procedure offered new materials with tunable surface properties and the potential for multi-functional applications.

Purinergic signalling is involved in the control of several processes related to brain development, such as neurogenesis and gliogenesis, migration and differentiation of neuronal precursors, synaptogenesis and synaptic elimination to achieve a fully wired and efficient mature brain. Therefore, any deregulation of purine-dependent signalling mediated by stimulation of specific adenosine and purinergic receptor subtypes: P1, P2X, or P2Y, can lead to functional deficits and the development of neuropsychiatric disorders, including autism spectrum disorders (ASD). In this study, we investigated the changes in expression and activity of selected purinergic receptors during rat brain development in an animal model of ASD. Pregnant dams received an intraperitoneal injection of valproic acid (VPA; 450 mg/kg body weight) at embryonic day (ED) 12.5, around the time of neural tube closure. Subsequently, changes in the expression and activity of specific purinergic receptor subtypes were analysed at ED19, an important prenatal stage of brain development. Our results suggest that prenatal VPA exposure leads to a significant increase in the level and activity of adenosinergic receptors A1, A2b and A3, which are involved in the regulation of progenitor cell proliferation and nerve growth, and upregulation of purinergic P2X2/P2X3 receptors, which in turn may contribute to the postnatal neuroanatomical abnormalities and synaptic dysfunction. Conversely, the significant downregulation of P2Y1 and P2X7 receptors, together with their reduced activity in the embryonic VPA brain, may indicate disturbances in the processes of neuronal precursor migration and differentiation, dendritic and axonal formation, and glutamate/GABA imbalance, thereby altering neuronal excitability. In conclusion, defects in purinergic signalling induced by prenatal VPA exposure could have a profound impact on brain development during embryogenesis and on intellectual and behavioural functions after birth. These observations could provide clues for future implementation of potential therapeutic strategies for ASD.

Adenosine-to-inosine (A-to-I) RNA editing is a major source of nucleotide diversification that has significant mechanistic implications in cancer progression and treatment response. However, its activity and prevalence have not yet been systematically determined at a single-cell resolution. Chan and colleagues revealed widespread A-to-I RNA editing events in single cancer cells through an in-depth analysis of a public lung adenocarcinoma single-cell transcriptome dataset. Edits significantly enriched in cancer cells compared to other cell types have the potential to inhibit innate immune response and to predict poor therapeutic response and prognosis in patients treated with targeted therapies. See related article by Chan et al., p. 374.

The conversion of adenosine to inosine at the wobble position of select tRNAs is essential for decoding specific codons in bacteria and eukarya. In eukarya, wobble inosine modification is catalyzed by the heterodimeric ADAT complex containing ADAT2 and ADAT3. Human individuals homozygous for loss of function variants in ADAT3 exhibit intellectual disability disorders. We created a flexible computational tool to scan the human, mouse, nematode, fruit fly, and yeast exomes for genes either enriched or depleted in ADAT-dependent codons as compared to background models of codon bias derived from the exomes themselves. We find that many genes are enriched or depleted for ADAT-dependent codons as compared to the genomic background in all five species. Among those genes enriched for ADAT-dependent codons in humans, we find there is significant Gene Ontology (GO) enrichment for genes involved in diverse neurological processes. This pattern persists in the mouse exome but not the fruit fly or nematode exome. In the nematode exome, genes enriched in ADAT-dependent codons are GO enriched for translation associated genes, and in yeast there is GO enrichment for genes involved in metabolic functions. There is also GO-term overlap between yeast and fruit flies. Importantly, in its generalized form, ADATscan can also be used to scan any exome for genes enriched in any subset of codons specified by the user.

Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory T cells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

Activation of the renin-angiotensin system is associated with podocyte injury and has been well demonstrated as a pivotal factor in the progression of chronic kidney disease. Podocyte energy metabolism is crucial for maintaining their physiological functions. However, whether renin-angiotensin system activation promotes chronic kidney disease progression by disturbing the energy metabolism of podocytes has not been elucidated. Angiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Here, we demonstrate a rapid decrease in the expression of pyruvate kinase M2, a key glycolytic enzyme, and reduced glycolytic flux in podocytes exposed to angiotensin II in vivo and in vitro. Podocyte-specific deletion of pyruvate kinase M2 in mice aggravated angiotensin II-induced glomerular and podocyte injury with foot process effacement and proteinuria. The inhibition of glycolysis was accompanied by adenosine triphosphate deficiency, cytoskeletal remodeling and podocyte apoptosis. Mechanistically, we found that angiotensin II-induced glycolysis impairment contributed to an insufficient energy supply to the foot process, leading to podocyte injury. Additionally, pyruvate kinase M2 expression was found to be reduced in podocytes from kidney biopsies of patients with hypertensive nephropathy and diabetic kidney disease. Thus, our findings suggest that glycolysis activation is a potential therapeutic strategy for podocyte injury.

Epilepsy is characterized by the manifestation of spontaneous and recurrent seizures. The high prevalence of comorbidities associated with epilepsy, such as cognitive dysfunction, affects the patients quality of life. Adenosine signaling modulation might be an effective alternative to control seizures and epilepsy-associated comorbidities. This study aimed to verify the role of adenosine modulation on the seizure development and cognitive impairment induced by pentylenetetrazole (PTZ) in zebrafish. At first, animals were submitted to a training session in the inhibitory avoidance test and, after 10 min, they received an intraperitoneal injection of valproate, adenosine A1 receptor agonist cyclopentyladenosine (CPA), adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A2A receptor antagonist ZM 241385, adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nony1)-adenine hydrochloride (EHNA) or the nucleoside transporter inhibitor dipyridamole. Thirty min after the intraperitoneal injection, the animals were exposed to 7.5 mM PTZ for 10 min, where they were evaluated for latency to reach the seizure stages (I, II, and III). Finally, 24 h after the training session, the animals were submitted to the inhibitory avoidance test to verify their cognitive performance during the test session. Valproate, CPA, and EHNA showed antiseizure effects and prevented the memory impairment induced by PTZ exposure. DPCPX, ZM 241385, and dipyridamole pretreatments caused no changes in seizure development; however, these drugs prevented memory impairment without altering locomotion. Our results reinforce the antiseizure effects of adenosine signaling and support the idea that the involvement of adenosine in memory processes may be a target for preventive strategies against cognitive impairment associated with epilepsy.

Dysfunctional autophagy induced by excessive reactive oxygen species (ROS) load and inflammation accelerates the development of Alzheimer's disease (AD). Recently, there has been an increasing interest in selenium-enriched ingredients (SEIs), such as selenoproteins, selenoamino acids and selenosugars, which could improve AD through antioxidant and anti-inflammation, as well as autophagy modulating effects. This review indicates that SEIs eliminate excessive ROS by activating the nuclear translocation of nuclear factor erythroid2-related factor 2 (Nrf2) and alleviate inflammation by inhibiting the mitogen-activated protein kinases (MAPKs)/nuclear factor kappa-B (NF-κB) pathway. Furthermore, they can activate the adenosine 5'-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, and subsequently promote amyloid beta (Aβ) clearance and reduce memory impairments. SEIs are ubiquitous in many plants and microorganisms, such as Brassicaceae vegetables, yeast, and mushroom. Enzymatic hydrolysis, as well as physical processing, such as thermal, high pressure and microwave treatment, are the main techniques to modify the properties of dietary selenium. This work highlights the fact that SEIs can inhibit inflammation and oxidative stress and provides evidence that supports the potential use of these dietary materials to be a novel strategy for improving AD.

Immunotherapy is an attractive treatment strategy for cancer, while its efficiency and safety need to be improved. A dual-cascade activatable nanopotentiator for sonodynamic therapy (SDT) and chemodynamic therapy (CDT)-cooperated immunotherapy of deep tumors via reshaping adenosine metabolism is herein reported. This nanopotentiator (NPMCA) is constructed through crosslinking adenosine deaminase (ADA) with chlorin e6 (Ce6)-conjugated manganese dioxide (MnO2) nanoparticles via a reactive oxygen species (ROS)-cleavable linker. In the tumor microenvironment with ultrasound (US) irradiation, NPMCA mediates CDT and SDT concurrently in deep tumors covered with 2-cm tissues to produce abundant ROS, which results in dual-cascade scissoring of ROS-cleavable linkers to activate ADA within NCMCA to block adenosine metabolism. Moreover, immunogenic cell death (ICD) of dying tumor cells and upregulation of the stimulator of interferon genes (STING) is triggered by the generated ROS and Mn2+ from NPMCA , respectively, leading to activation of antitumor immune response. The potency of immune response is further reinforced by reducing the accumulation of adenosine in tumor microenvironment by the activated ADA. As a result, NPMCA enables CDT and SDT-cooperated immunotherapy, showing an obviously improved therapeutic efficacy to inhibit the growths of bilateral tumors, in which the primary tumors are covered with 2-cm tissues.

The physiopathology and neurotransmission of pain are of an owe inspiring complexity. Our ability to satisfactorily suppress neuropathic or other forms of chronic pain is limited. The number of pharmacodynamically distinct and clinically available medications is low and the successes achieved modest. Pain Medicine practitioners are confronted with the ethical dichotomy imposed by Hippocrates: On one hand the mandate of primum non nocere, on the other hand, the promise of heavenly joys if successful divinum est opus sedare dolorem. We briefly summarize the concepts associated with nociceptive pain from nociceptive input (afferents from periphery), modulatory output [descending noradrenergic (NE) and serotoninergic (5-HT) fibers] to local control. The local control is comprised of the "inflammatory soup" at the site of pain origin and synaptic relay stations, with an ATP-rich environment promoting inflammation and nociception while an adenosine-rich environment having the opposite effect. Subsequently, we address the transition from nociceptor pain to neuropathic pain (independent of nociceptor activation) and the process of sensitization and pain chronification (transient pain progressing into persistent pain). Having sketched a model of pain perception and processing we attempt to identify the sites and modes of action of clinically available drugs used in chronic pain treatment, focusing on adjuvant (co-analgesic) medication.

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.

Head and neck cancers represent a significant portion of cancer diagnoses, with head and neck cancer incidence increasing in some parts of the world. Typical treatment of early-stage head and neck cancers includes either surgery or radiotherapy; however, advanced cases often require surgery followed by radiation and chemotherapy. Salivary gland damage following radiotherapy leads to severe and chronic hypofunction with decreased salivary output, xerostomia, impaired ability to chew and swallow, increased risk of developing oral mucositis, and malnutrition. There is currently no standard of care for radiation-induced salivary gland dysfunction, and treatment is often limited to palliative treatment that provides only temporary relief. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that activates catabolic processes and has been shown to influence the cell cycle, proliferation, and autophagy. In the present study, we found that radiation (IR) treatment decreases tissue levels of phosphorylated AMPK following radiation and decreases intracellular NAD+ and AMP while increasing intracellular adenosine triphosphate. Furthermore, expression of sirtuin 1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) was lower 5 d following IR. Treatment with AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, attenuated compensatory proliferation (days 6, 7, and 30) following IR and reversed chronic (day 30) salivary gland dysfunction post-IR. In addition, treatment with metformin or AICAR increased markers of apical/basolateral polarity (phosphorylated aPKCζT560-positive area) and differentiation (amylase-positive area) within irradiated parotid glands to levels similar to untreated controls. Taken together, these data suggest that AMPK may be a novel therapeutic target for treatment of radiation-induced salivary damage.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Mammalian cells generate ATP through mitochondrial respiration and glycolysis. Mitochondria not only play a key role in cell energy metabolism but also in cell cycle regulation. As a neurotoxic pollutant, benzo(a)pyrene (BaP) can trigger neuronal oxidative damage and apoptosis. However, the features of BaP-induced energy metabolism disturbance in SH-SY5Y cells has rarely been addressed. This study aimed to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as indications of respiratory activities and glycolytic. SH-SY5Y cells were treated with BaP to establish a cytotoxicity model, and butylated hydroxy anisole (BHA) was used to alleviate the damages induced by BaP. Using the Seahorse Extracellular Flux analyzer (XFp), we found that BaP significantly reduced basal respiration, ATP-linked OCR in SH-SY5Y cells with dose- and time-dependent. BHA supplementation recovered the mitochondrial respiration, synchronously attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers, then rescued BaP-induced cell apoptosis. But long-term exposure to BaP or exposure to a high dosage of BaP could decrease OCR associated with maximal respiratory, spare capacity, and glycolysis metabolism. At the same time, the damage to cells is also more severe with the rate of apoptosis and mitochondrial membrane potential (ΔΨm) loss rising sharply, which were not entirely reversed by BHA. This study provides energy metabolism-related, indicative biomarkers of cytotoxicity induced by BaP, which might provide information for early prevention and intervention.

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N6-(Δ2-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N6-(Δ2-isopentenyl)adenosine-5'-monophosphate (iPRMP) → N6-(Δ2-isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that acidify specific intracellular compartments in almost all eukaryotic cells. These multi-subunit enzymes consist of a soluble catalytic V1 region and a membrane-embedded proton-translocating VO region. VO is assembled in the endoplasmic reticulum (ER) membrane, and V1 is assembled in the cytosol. However, V1 binds VO only after VO is transported to the Golgi membrane, thereby preventing acidification of the ER. We isolated VO complexes and subcomplexes from Saccharomyces cerevisiae bound to V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Electron cryomicroscopy shows how the Vma12-22p complex recruits subunits a, e, and f to the rotor ring of VO while blocking premature binding of V1. Vma21p, which contains an ER-retrieval motif, binds the VO:Vma12-22p complex, "mature" VO, and a complex that appears to contain a ring of loosely packed rotor subunits and the proteins YAR027W and YAR028W. The structures suggest that Vma21p binds assembly intermediates that contain a rotor ring and that activation of proton pumping following assembly of V1 with VO removes Vma21p, allowing V-ATPase to remain in the Golgi. Together, these structures show how Vma12-22p and Vma21p function in V-ATPase assembly and quality control, ensuring the enzyme acidifies only its intended cellular targets.

The kidney is a comparatively hostile microenvironment characterized by highsodium concentrations; however, lymphocytes infiltrate and survive therein in autoimmune diseases such as lupus. The effects of sodium-lymphocyte interactions on tissue injury in autoimmune diseases and the mechanisms used by infiltrating lymphocytes to survive the highsodium environment of the kidney are not known. Here, we show that kidney-infiltrating B cells in lupus adapt to elevated sodium concentrations and that expression of sodium potassium adenosine triphosphatase (Na+-K+-ATPase) correlates with the ability of infiltrating cells to survive. Pharmacological inhibition of Na+-K+-ATPase and genetic knockout of Na+-K+-ATPase γ subunit resulted in reduced B cell infiltration into kidneys and amelioration of proteinuria. B cells in human lupus nephritis biopsies also had high expression of Na+-K+-ATPase. Our study reveals that kidney-infiltrating B cells in lupus initiate a tissue adaption program in response to sodium stress and identifies Na+-K+-ATPase as an organ-specific therapeutic target.