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This letter to the editor discusses a rare case of daratumumab-induced rhabdomyolysis in a 63-year-old male with multiple myeloma. Daratumumab is a monoclonal antibody used to treat myeloma, and this is the first reported case of rhabdomyolysis associated with its use. The patient experienced muscle enzyme elevation after daratumumab treatment, which resolved upon discontinuation of the drug and hydration. The mechanism of rhabdomyolysis caused by daratumumab is not fully understood and requires further study. [Extracted from the article]

[Display omitted] Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca2+-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins. [ABSTRACT FROM AUTHOR]

We sought to quantify exposure to and financial impacts of poly (adenosine diphosphate ribose) polymerase inhibitor (PARPi) treatments for eventually withdrawn ovarian cancer indications. We identified in Optum's deidentified Clinformatics® Data Mart database 1695 patients with ovarian cancer diagnoses who received olaparib, rucaparib, or niraparib between January 2015 and September 2021. We describe PARPi use and out-of-pocket, total healthcare, and PARPi spending among patients with ovarian cancer with 3 or more previous lines of therapy. Of the 1695 patients who received PARPi, 254 were estimated to have been heavily pretreated and exposed to eventually withdrawn indications. Cumulative total medical and pharmacy costs for these patients were $53 392 184; PARPi costs accounted for 34%. Median PARPi cost per patient was $43 347. Cumulative out-of-pocket costs totaled $533 281. Potential patient harm, including financial toxicity, might have been mitigated through more stringent drug approval requirements. • Starting in 2014, 3 poly (adenosine diphosphate ribose) polymerase inhibitors were approved without evidence of overall survival benefit for ovarian cancer treatment of heavily pretreated patients with recurrent disease. Ovarian cancer treatment indications were withdrawn in 2022, after trials demonstrating shorter survival among heavily pretreated patients with recurrent disease. • In addition to potential patient harm from drug exposure, conservative estimates of the number of patients treated with poly (adenosine diphosphate ribose) polymerase inhibitor for the withdrawn indications demonstrated significant financial impact for patients and the healthcare system. • Financial toxicity of Food and Drug Administration cancer drug approvals without evidence of clinical benefit must be considered. [ABSTRACT FROM AUTHOR]

Metastatic castrate-resistant prostate cancer (mCRPC) is associated with poor prognosis. DNA damage response (DDR) genes are commonly altered in mCRPC rendering them as promising therapeutic targets. Poly (ADP ribose) polymerase inhibitors (PARPi) demonstrated antitumor activity in mCRPC patients with DDR gene mutations through synthetic lethality. Multiple clinical trials with PARPi monotherapy exhibited encouraging clinical outcomes in selected patients with mCRPC. More recently, three Phase III randomized clinical trials (RCTs) combining PARPi with androgen receptor signaling inhibitors (ARSIs) demonstrated improved antitumor activity compared to ARSI monotherapy in mCRPC patients as the first-line therapy. Clinical benefit was more pronounced in patients harboring DDR alterations, specifically BRCA1/2. Interestingly, antitumor activity was also observed irrespective of DDR gene mutations, highlighting BRCAness phenotype with androgen receptor blockade resulting in synergistic activity between ARSIs and PARPi. In this review, we discuss the clinical efficacy and safety data of the combination of PARPi plus ARSI in all Phase 3 randomized controlled trials (RCTs), emphasizing strategies for patient selection and highlighting emerging trends based on clinical trial data. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) encompasses many different histological and molecular subtypes. It is a major driver of cancer mortality because of poor survival and limited treatment options. Personalised medicine in the form of patient-derived organoids (PDOs) represents a promising approach for improving therapeutic outcomes. The goal of this study was to overcome the limitations of current models by ameliorating organoid cultivation. Methods: Organoids derived from cancer tissue were evaluated by haematoxylin and eosin staining, immunohistochemistry, mRNA, and whole-exome sequencing. Three representative chemotherapy drugs, 5-fluorouracil, docetaxel, and oxaliplatin, were compared for their efficacy against different subtypes of gastric organoids by ATP assay and apoptosis staining. In addition, drug sensitivity screening results from two publicly available databases, the Genomics of Drug Sensitivity in Cancer and Cancer Cell Line Encyclopaedia, were pooled and applied to organoid lines. Once key targeting genes were confirmed, chemotherapy was used in combination with poly (ADP ribose) polymerase (PARP)-targeted therapy. Results: We successfully constructed GC PDOs surgically resected from GC patient tissue. PDOs closely reflected the histopathological and genomic features of the corresponding primary tumours. Whole-exosome sequencing and mRNA analysis revealed that changes to the original tumour genome were maintained during long-term culture. The drugs caused divergent responses in intestinal, poorly differentiated intestinal, and diffuse gastric cancer organoids, which were confirmed in organoid lines. Poorly differentiated intestinal GC patients benefited from a combination of 5-fluorouracil and veliparib. Conclusion: The present study demonstrates that combining chemotherapy with PARP targeting may improve the treatment of chemotherapy-resistant tumours. [ABSTRACT FROM AUTHOR]

Objective: To determine the useful biomarker for predicting the effects of poly-(ADP ribose)- polymerase (PARP) inhibitors in Japanese patients with ovarian cancer. Methods: We collected clinical information and performed molecular biological analysis on 42 patients with ovarian, fallopian tube, and primary peritoneal carcinomas who received PARP inhibitors. Results: Among the analyzed patients with ovarian cancer, 23.8% had germline BRCA mutation ( gBRCAm), 42.9% had homologous recombination repair-related gene mutation (HRRm), and 61.1% had a genomic instability score (GIS) of ≥42. Patients with HRRm had a significantly longer progression-free survival (PFS) than those without HRRm (median PFS 35.6 vs. 7.9 months; p=0.009), with a particularly marked increase in PFS in patients with gBRCAm (median PFS 42.3 months). Similarly, among patients with recurrent ovarian cancer, those with HRRm had a longer PFS than those without HRRm (median PFS 42.3 vs. 7.7 months; p=0.040). Multivariate Cox proportional hazards regression analysis found that performance status and gBRCAm status were independent factors associated with prolonged PFS with PARP inhibitors. In recurrent ovarian cancer, multivariate regression analysis identified platinum-free interval (PFI) in addition to performance status as a significant predictor of PFS. On the contrary, no significant association was observed between PFS and a GIS of ≥42 used in clinical practice. Conclusion: We found that HRRm can be a useful biomarker for predicting the effects of PARP inhibitors in treating ovarian cancer and that the PFI can also be useful in recurrent ovarian cancer. [ABSTRACT FROM AUTHOR]

In order to analyze the effect and mechanism of Moscatilin on sevoflurane-induced HT22 hippocampal neuron cell (HT22) injury, sevoflurane was applied to HT22 cells to create an in vitro model of nerve damage, and Moscatilin was given to observe its effect on indicators of nerve injury. The effect of Moscatilin on sevoflurane-induced cell injury was measured based on cell viability (Cell Counting Kit-8), the expression of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the cells was detected by Enzyme-Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR). Reactive oxygen species (ROS) production in the neuronal cells was detected by oxidative stress measurements based on the 2′,7′ -Dichlorofluorescein (DCF) assay. Using flow cytometry, the rate of apoptosis was determined, and the expression of apoptosis-related markers cleaved-caspase3 and cleaved-Poly Adp Ribose Polymerase (cleaved-PARP), and nuclear factor-k-gene binding (NF-κB)-related proteins were detected by western-blot analysis. The results showed that sevoflurane caused a significant decrease in HT22 cell viability, an increase in inflammatory factors TNF-α, IL-6 and IL-1β, ROS and apoptosis, an increase in apoptosis-related markers, cleaved-caspase3 and cleaved-PARP, as well as in the pp65/p65 ratio of NF-κB-related proteins, and a decrease in the expression of inhibitor of NF-κBα (IκBα) protein. Moscatilin treatment counteracted sevoflurane-induced reduction in cell viability, decreased inflammatory factors and ROS production, inhibited sevoflurane-induced apoptosis, suppressed p-p65/p65 ratio, and increased IκBα protein expression. In conclusion, Moscatilin inhibits the NF-κB pathway and ameliorates sevoflurane-induced inflammatory responses, ROS production and apoptosis in HT22 hippocampal neuronal cells. [ABSTRACT FROM AUTHOR]

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Introduction: During thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called "browning" and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced fromNAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis. Methods: Wild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the b-adrenergic receptor agonist CL316,243. Results: Trpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation. Discussion: Our data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in genes encoding the Krebs cycle enzymes Fumarate Hydratase (FH) and Succinate Dehydrogenase (SDH) induce accumulation of fumarate and succinate, respectively and predispose patients to hereditary cancer syndromes including the development of aggressive renal cell carcinoma (RCC). Fumarate and succinate competitively inhibit αKG-dependent dioxygenases, including Lysine-specific demethylase 4A/B (KDM4A/B), leading to suppression of the homologous recombination (HR) DNA repair pathway. In this study, we have developed new syngeneic Fh1- and Sdhb-deficient murine models of RCC, which demonstrate the expected accumulation of fumarate and succinate, alterations in the transcriptomic and methylation profile, and an increase in unresolved DNA double-strand breaks (DSBs). The efficacy of poly ADP-ribose polymerase inhibitors (PARPis) and temozolomide (TMZ), alone and in combination, was evaluated both in vitro and in vivo. Combination treatment with PARPi and TMZ results in marked in vitro cytotoxicity in Fh1- and Sdhb-deficient cells. In vivo, treatment with standard dosing of the PARP inhibitor BGB-290 and low-dose TMZ significantly inhibits tumor growth without a significant increase in toxicity. These findings provide the basis for a novel therapeutic strategy exploiting HR deficiency in FH and SDH-deficient RCC with combined PARP inhibition and low-dose alkylating chemotherapy.

A recently characterized RNA modification is NAD+-modified RNAs (NAD-RNAs). Various enzymes decap NAD-RNAs, and Wang and Yu et al. now describe another, namely Toll/interleukin-1 receptor (TIR) domain-containing proteins of bacteria and Archaea. TIR decapping products are a specific variant of cyclic ADP ribose (ADPR)-RNAs (v-cADPR-RNAs), opening a new window to the NAD-RNA world. [ABSTRACT FROM AUTHOR]

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease. Dormant hematopoietic stem cells (dHSC) represent a stem cell reserve that is important for hematopoiesis throughout life. In this study, CD38 is identified as a surface marker and novel regulator of mouse HSCs that promotes their dormancy. Interestingly, while human HSCs lack CD38 expression, CD38 expression in neighboring cells similarly promotes human HSC quiescence. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy, a lysosome-dependent self-degradative process, is a critical mechanism for the clearance of misfolded proteins and dysfunctional organelles in neurons. In the peripheral nervous system, autophagic stress is associated with the development of peripheral neuropathy. However, the molecular mechanism of axonal neuropathy induced by autophagic stress due to dysfunction of autophagy in peripheral neurons in vivo is still unclear. We found that dorsal root ganglion (DRG) neuron-specific atg7 (autophagy related 7) knockout (atg7-cKO) mice employing two different Cre recombinase systems exhibited sensory neuropathy approximately 2 months after birth. In electron microscopy analysis, axon degeneration was clearly observed in the myelinated fibers of the sciatic nerve before the appearance of neuronal cell death. Dystrophic axons filled with abnormal vesicular accumulations and amorphous inclusions were specifically localized in the myelinated axons within the DRG in atg7-cKO mice, indicating the presence of autophagic stress in proximal axons. In line with the EM findings, the mutant mice showed preferential induction of axonal injury-associated genes, including ATF3 (activating transcription factor 3), in large-size DRG neurons that constitute myelinated fibers without axotomy. SARM1 (sterile alpha and HEAT/Armadillo motif containing 1), the central executioner of Wallerian degeneration, was activated in the sciatic nerves of atg7-cKO mice, and axonal degeneration and sensory neuropathy in atg7-cKO mice were prevented via expression of a dominant-negative Sarm1 transgene. Our findings demonstrate the importance of SARM1-dependent axon degeneration in the development of peripheral sensory neuropathy induced by impairment of autophagy. AAV: adeno-associated virus; ATF3: activating transcription factor 3; ATG7: autophagy related 7; AVIL: advillin; cADPR: cyclic ADP ribose; CALC: calcitonin/calcitonin-related polypeptide; CMT: Charcot-Marie-Tooth disease; cKO: conditional knockout; DEG: differentially expressed gene; DRG: dorsal root ganglion; FE-SEM: field emission scanning electron microscopy; IF: immunofluorescence; NCV: nerve conduction velocity; PVALB: parvalbumin; RAG: regeneration-associated gene; ROS: reactive oxygen species; SARM1: sterile alpha and HEAT/Armadillo motif containing 1; SYN1: synapsin I [ABSTRACT FROM AUTHOR]

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergenchallenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca21 flux to trigger airway smooth muscle contraction. Intracellular Ca21 flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca21 concentration ([Ca21]i) (143nM) and a sustained plateau that included slow oscillations in [Ca21]i. Sustained [Ca21]i elevation was ablated in Ca21-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca21]i was unaffected in Ca21-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca21]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca21 release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca21 flux. OxPAPC induced -15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca21 in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness. [ABSTRACT FROM AUTHOR]

The effective treatment of breast cancer remains a profound clinical challenge, especially due to drug resistance and metastasis which unfortunately arise in many patients. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), as a selective inhibitor of cyclin-dependent kinase 9, was shown to be effective in inducing apoptosis in various hematopoietic malignancies. However, the anticancer efficacy of DRB against breast cancer is still unclear. Herein, we demonstrated that administration of DRB to the breast cancer cell line led to the inhibition of cellular proliferation and induction of the typical signs of apoptotic cells, including the increases in Annexin V-positive cells, DNA fragmentation, and activation of caspase-7, caspase-9, and poly (ADP ribose) polymerase (PARP). Treatment of DRB resulted in a rapid decline in the myeloid cell leukemia 1 (Mcl-1) protein, whereas levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 decreased the DRB-induced PARP cleavage, whereas knockdown of Mcl-1 enhanced the effects of DRB on PARP activation, indicating that loss of Mcl-1 accounts for the DRB-mediated apoptosis in MCF-7 cells, but not in T-47D. Furthermore, we found that co-treatment of MCF-7 cells with an inhibitor of AKT (LY294002) or an inhibitor of the proteasome (MG-132) significantly augmented the DRB-induced apoptosis. These data suggested that DRB in combination with LY294002 or MG-132 may have a greater therapeutic potency against breast cancer cells. [ABSTRACT FROM AUTHOR]

Alphaviruses are emerging and re-emerging viruses that cause severe disease and threaten public health worldwide. To advance the understanding of the underlying mechanisms of alphavirus replication and identify new host restriction factors, we performed RNA-seq and identified antiviral host factors on the Getah virus (GETV), a re-emerging alphavirus. We identified tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase (TIPARP) as a host antiviral factor. TIPARP is downregulated in GETV-infected Vero cells, and its overexpression significantly inhibits GETV replication, while TIPARP deficiency results in significantly increased viral titers. We demonstrated that TIPARP interacts with the viral E2 glycoprotein, inducing k48-linked ubiquitination and subsequent proteasomal degradation. Additionally, we found that TIPARP recruits the E3 ubiquitin ligase membrane-associated RING-CH 8 (MARCH8) to modify the ubiquitination, leading to the degradation of E2. Lys253 in E2 was identified as the TIPARP-facilitated ubiquitination site. A mutation in Lys253 resulted in the loss of TIPARP’s anti-GETV effect. Our study demonstrated for the first time that host TIPARP is a restricting factor against GETV replication. Investigating the underlying mechanisms and understanding TIPARP for GETV will be essential to fully understanding viral pathogenesis and developing novel broad-spectrum therapeutic strategies against alphavirus infection. IMPORTANCE Alphaviruses threaten public health continuously, and Getah virus (GETV) is a re-emerging alphavirus that can potentially infect humans. Approved antiviral drugs and vaccines against alphaviruses are few available, but several host antiviral factors have been reported. Here, we used GETV as a model of alphaviruses to screen for additional host factors. Tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase was identified to inhibit GETV replication by inducing ubiquitination of the glycoprotein E2, causing its degradation by recruiting the E3 ubiquitin ligase membrane-associated RING-CH8 (MARCH8). Using GETV as a model virus, focusing on the relationship between viral structural proteins and host factors to screen antiviral host factors provides new insights for antiviral studies on alphaviruses. [ABSTRACT FROM AUTHOR]

Brain concentrations of nicotinamide adenine dinucleotide (NAD+), an important cellular co‐factor, tend to decrease with age and in neurodegeneration. As the NADase cluster of differentiation 38 (CD38) significantly contributes to NAD+ consumption, we reasoned that CD38 inhibition may be of therapeutic value for CNS disorders. The new pyrazole compound was designed based on a known CD38 inhibitor and showed good inhibitory potency. Several attempts to co‐crystallise this pyrazole with CD38 and cyclic adenosine diphosphate ribose (cADPR) culminated in a high‐resolution X‐ray structure, in which the pyrazolyl group in the new compound formed a covalent bond with one of the ribosyl units of cADPR. This reaction proceeded under retention of configuration and resulted in a neutral ribosyl‐pyrazole conjugate that is embedded within the active site of the enzyme. An analysis of this structural complex gave rise to design principles that enabled the preparation of more potent CD38 inhibitors with drug‐like properties. [ABSTRACT FROM AUTHOR]

AIM: To investigate the effects of abdominal Tuina on functional dyspepsia (FD) in rats, and to explore its mechanisms focusing on metabolism using liquid chromatographymass spectrometry (LCMS). METHODS: Thirty rats were randomly divided into 3 groups: normal control (NC) group, FD model group and Tuina group, with 10 rats in each group. The rats in FD model group and Tuina group were administered 0. 1% sucrose iodoacetamide solution via gavage and subjected to the small platform standing method to induce the FD model. After modeling, gastric antrum tissue samples were obtained, and hematoxylineosin (HE) staining was used to observe histopathologic changes. Following 7 d of intervention, samples were collected from each group to measure gastric emptying rate, detect metabolite content in gastric antrum tissues, and screen for differential metabolites through correlation analysis. These differential metabolites were then subjected to further examination using MetaboAnalyst 5. 0 and KEGG online database for metabolic pathway analysis. RESULTS: The HE staining results revealed that the gastric mucosa glands of the rats in all groups exhibited a well-organized arrangement with intact structures, no obvious inflammatory cell infiltration was observed, and no significant difference was observed in the histopathology of the gastric antrum. Compared with NC group, the gastric emptying rate of the rats in FD model group was significantly decreased (P<0.05). Conversely, the gastric emptying rate of the rats in Tuina group displayed a significant increase compared with FD model group (P<0.05). Metabolomics analysis revealed notable differences in the levels of various metabolites in gastric antrum tissues among different groups. In comparison with NC group, the rats in FD model group exhibited significant increases in the levels of retinal, argininosuccinic acid, N-methyl-α-aminoisobutyric acid, acetyl phosphate, phosphonoacetate, 2-phenylbutyric acid, creatine, 4-coumarate, and phenylpyruvate. Conversely, the levels of adenine, glutathione, adenosine, β-nicotinamide adenine dinucleotide, adenosine diphosphate ribose and stachyose in gastric antrum tissues of the rats in FD model group were significantly decreased. Compared with FD model group, the rats in Tuina group exhibited decreased levels of adenosine monophosphate, argininosuccinic acid, glutamate 5′-methylcytosine, methylthioadenosine, L-proline, guanine, creatine, oxoproline, tyrosine, glutamine, N-methylnicotinamide and indoleacrylic acid in gastric antrum tissues. CONCLUSION: Abdominal Tuina can improve the gastric emptying rate and regulate gastric tissue metabolism in FD rats. Its mechanism may be associated with the arginine biosynthesis, the metabolism of alanine, aspartic acid and glutamic acid, and the metabolism of D-glutamine and D-glutamic acid. [ABSTRACT FROM AUTHOR]

BST-1 (bone marrow stromal cell antigen-1) is thought to be a key molecule involved in regulating the functional activity of cells in various tissues and organs. BST-1 can catalyze the hydrolysis of nicotinamide adenine dinucleotide (NAD+) to produce cyclic ADP ribose (cADPR), which activates the activity of intracellular Ca2+ signaling. Currently, the role of BST-1 regulation of Ca2+ signaling pathway in pathological myocardial hypertrophy is unclear. We found elevated expression of BST-1 in cardiac hypertrophy tissues of spontaneously hypertensive rats in our vivo study, subsequently; the mechanism of BST-1 action on myocardial hypertrophy was explored in vitro experiment. We used aldosterone (ALD) to induce H9C2 cellular hypertrophy. cADPR levels and intracellular Ca2+ concentrations declined and calcium-regulated neurophosphatase (CaN) activity and protein expression were decreased after BST-1 knockdown. And then activated T-cell nuclear factor (NFATc3) entry nucleus was inhibited. All of the above resulted in that H9C2 cells size was reduced by rhodamine-phalloidin staining. Thus, BST-1 may exacerbate cardiac hypertrophy by activating the Ca2+/CaN/NFATc3 pathway. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) is a recent pandemic that caused serious global emergency. To identify new and effective therapeutics, we employed a drug repurposing approach. The poly (ADP ribose) polymerase inhibitors were used for this purpose and were repurposed against the main protease (Mpro) target of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). The results from these studies were used to design compounds using the 'Grow Scaffold' modules available on Discovery Studio v2018. The three designed compounds, olaparib 1826 and olaparib 1885, and rucaparib 184 demonstrated better CDOCKER docking scores for Mpro than their parent compounds. Moreover, the compounds adhered to Lipinski's rule of five and demonstrated a synthetic accessibility score of 3.55, 3.63, and 4.30 for olaparib 1826, olaparib 1885, and rucaparib 184, respectively. The short-range Coulombic and Lennard-Jones potentials also support the potential binding of the modified compounds to Mpro. Therefore, we propose these three compounds as novel SARS-CoV-2 inhibitors. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. his study indicates that a cationic environment may drive recognition specificity of PAR. [ABSTRACT FROM AUTHOR]

Ischemia and perfusion (I/R) induce inflammation and oxidative stress, which play a notable role in tissue damage. The aim of this study was to investigate the role of an NADPH oxidase inhibitor (apocynin) in the protection of the heart from I/R injury. Hearts isolated from Wistar rats (n = 8 per group) were perfused with a modified Langendorff preparation. Left ventricular (LV) contractility and cardiovascular hemodynamics were evaluated by a data acquisition program, and infarct size was evaluated by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining. Furthermore, the effect of apocynin on the pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and anti-inflammatory cytokine (IL-10) was evaluated using an enzyme linked immunosorbent assay (ELISA). Hearts were subjected to 30 min of regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery, followed by 30 min of reperfusion. Hearts were infused with apocynin before ischemia, during ischemia or at reperfusion. To understand the potential pathways of apocynin protection of the heart, a nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP), nitric oxide blocker (N (gamma)-nitro-L-arginine methyl ester, L-Name), nicotinic acid adenine dinucleotide phosphate (NAADP) inhibiter (Ned-K), cyclic adenosine diphosphate ribose (cADPR) agonist, or CD38 blocker (Thiazoloquin (az)olin (on)e compound, 78c) was infused with apocynin. Antioxidants were evaluated by measuring superoxide dismutase (SOD) and catalase (CAT) activity. Apocynin infusion before ischemia or at reperfusion protected the heart by normalizing cardiac hemodynamics and decreasing the infarct size. Apocynin treatment resulted in a significant (p < 0.05) decrease in pro-inflammatory cytokine levels and a significant increase (p < 0.05) in anti-inflammatory and antioxidant levels. Apocynin infusion protected the heart by improving LV hemodynamics and coronary vascular dynamics. This treatment decreased the infarct size and inflammatory cytokine levels and increased anti-inflammatory cytokine and antioxidant levels. This protection follows a pathway involving CD38, nitric oxide and acidic stores. [ABSTRACT FROM AUTHOR]

Disuse atrophy of skeletal muscle is associated with a severe imbalance in cellular Ca2+ homeostasis and marked increase in nuclear apoptosis. Nuclear Ca2+ is involved in the regulation of cellular Ca2+ homeostasis. However, it remains unclear whether nuclear Ca2+ levels change under skeletal muscle disuse conditions, and whether changes in nuclear Ca2+ levels are associated with nuclear apoptosis. In this study, changes in Ca2+ levels, Ca2+ transporters, and regulatory factors in the nucleus of hindlimb unloaded rat soleus muscle were examined to investigate the effects of disuse on nuclear Ca2+ homeostasis and apoptosis. Results showed that, after hindlimb unloading, the nuclear envelope Ca2+ levels ([Ca2+]NE) and nucleocytoplasmic Ca2+ levels ([Ca2+]NC) increased by 78% (p < 0.01) and 106% (p < 0.01), respectively. The levels of Ca2+-ATPase type 2 (Ca2+-ATPase2), Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP3R1), Cyclic ADP ribose hydrolase (CD38) and Inositol 1,4,5-tetrakisphosphate (IP3) increased by 470% (p < 0.001), 94% (p < 0.05), 170% (p < 0.001), 640% (p < 0.001) and 12% (p < 0.05), respectively, and the levels of Na+/Ca2+ exchanger 3 (NCX3), Ca2+/calmodulin dependent protein kinase II (CaMK II) and Protein kinase A (PKA) decreased by 54% (p < 0.001), 33% (p < 0.05) and 5% (p > 0.05), respectively. In addition, DNase X is mainly localized in the myonucleus and its activity is elevated after hindlimb unloading. Overall, our results suggest that enhanced Ca2+ uptake from cytoplasm is involved in the increase in [Ca2+]NE after hindlimb unloading. Moreover, the increase in [Ca2+]NC is attributed to increased Ca2+ release into nucleocytoplasm and weakened Ca2+ uptake from nucleocytoplasm. DNase X is activated due to elevated [Ca2+]NC, leading to DNA fragmentation in myonucleus, ultimately initiating myonuclear apoptosis. Nucleocytoplasmic Ca2+ overload may contribute to the increased incidence of myonuclear apoptosis in disused skeletal muscle. [ABSTRACT FROM AUTHOR]

Peritoneal metastasis (PM) is often regarded as a less frequent pattern of spread; however, collectively across all spectra of primary tumors, the consequences of PM impact a large population of patients annually. Unlike other modes of metastasis, symptoms at presentation or during the treatment course are common, representing an additional challenge in the management of PM. Early efforts with chemotherapy and incomplete surgical interventions transiently improved symptoms, but durable symptom control and survival extension were rare, which established a perspective of treatment futility for PM through most of the 20th century. Notably, the continued development of better systemic therapy combinations, optimization of cytoreductive surgery (CRS), and rigorous investigation of combining regional therapy—specifically hyperthermic intraperitoneal chemotherapy—with CRS, have resulted in more effective multimodal treatment options for patients with PM. In this article, the authors provide a comprehensive review of the data establishing the contemporary approach for tumors with a high frequency of PM, including appendix, colorectal, mesothelioma, and gastric cancers. The authors also explore the emerging role of adding hyperthermic intraperitoneal chemotherapy to the well established paradigm of CRS and systemic therapy for advanced ovarian cancer, as well as the recent clinical trials identifying the efficacy of poly(adenosine diphosphate ribose) polymerase maintenance therapy. Finally, recent data are included that explore the role of precision medicine technology in PM management that, in the future, may help further improve patient selection, identify the best systemic therapy regimens, detect actionable mutations, and identify new targets for drug development. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer is more aggressive than other types of breast cancer. Protein kinase R (PKR), which is activated by dsRNA, is known to play a role in doxorubicin-mediated apoptosis; however, its role in DNA damage-mediated apoptosis is not well understood. In this study, we investigated the roles of PKR and its downstream players in doxorubicin-treated HCC1143 triple-negative breast cancer cells. Doxorubicin treatment induces DNA damage and apoptosis. Interestingly, doxorubicin treatment induced the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PKR, whereas the inhibition of PKR with inhibitor C16 reduced eIF2α phosphorylation. Under these conditions, doxorubicin-mediated DNA fragmentation, cell death, and poly(ADP ribose) polymerase and caspase 7 levels were recovered. In addition, phosphorylation of checkpoint kinase 1 (CHK1), which is known to be involved in doxorubicin-mediated DNA damage, was increased by doxorubicin treatment, but blocked by PKR inhibition. Protein translation was downregulated by doxorubicin treatment and upregulated by blocking PKR phosphorylation. These results suggest that PKR activation induces apoptosis by increasing the phosphorylation of eIF2α and CHK1 and decreasing the global protein translation in doxorubicin-treated HCC1143 triple-negative breast cancer cells. [ABSTRACT FROM AUTHOR]

Patients with epithelial ovarian cancer (EOC) can benefit from poly- (ADP ribose) polymerase inhibitors (PARPi) therapy. However, PARPi resistance has become a challenge in clinical practice, and its mechanism requires further exploration. We established three PARPi-resistant cell strains following olaparib exposure. CCK-8, clonogenic survival, transwell, wound healing, cell cycle, RT-qPCR and western blot assays were performed to explore the functional phenotype of the resistant cells. Whole-exome sequencing and RNA-sequencing were performed to identify the altered genes. Stable knockdown and overexpression were used to investigate the role of EP300, an upstream regulator of E-cadherin and epithelial-mesenchymal transition (EMT), in cell lines. We further validated the finding in clinical ovarian cancer samples by immunohistochemistry. We combined public datasets to obtain an integrated PARPi sensitivity profile in EOC cells, which indicated that primary PARPi resistance could not be fully explained by mutations in BRCA1/2 or homologous recombination deficiency related genes. Genomic and transcriptome analyses revealed distinct mechanisms between primary and acquired resistance. Long-term PARPi treatment induced accumulation of de novo single nucleotide variants (SNV), and the complete frame-shift deletion of PARP1 was detected in the A2780 resistant strain. Additionally, the depressed histone acetyltransferase of EP300 could cause resistant phenotype through activated EMT process in vitro, and associated with PARPi-resistance in EOC patients. Long-term PARPi treatment led to evolutionary genomic and transcriptional alterations that were associated with acquired resistance, among which depressed EP300 partly contributed to the resistant phenotype. • Primary PARPi resistance could not be explained completely by mutations in BRCA1/2 or HRD related genes. • Multiple and heterogeneous processes could be involved in the development of PARPi resistance • The acquired resistance by long-term treatment selection had evolutional dynamics to tolerate the stress of PARPi. • Long-term PARPi treatment could induce accumulating de nove SNVs. • Depressed EP300 could cause resistant phenotype through activated epithelial-mesenchymal transition process. [ABSTRACT FROM AUTHOR]

Coronavirus Disease-2019 (COVID-19) is a highly contagious disease caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). The World Health Organization (WHO) classified the disease a as global public health hazard on 11 March 2020. Currently, there are no adequate measures to combat viral infections, including COVID-19, and the medication guidelines for the management of COVID-19 are dependent on previous findings from SARS-CoV and MERS-CoV research. Natural products have achieved widespread acceptance around the world as a means of enhancing healthcare and disease prevention. Plants are a potential source of antiviral factors such as flavonoids, phenolic acids, terpenoids, and others. Some of these agents exhibit a broad spectrum of antiviral activity. This study aimed to screen herbal leads for possible inhibitors of the SARS-CoV-2 ADP Ribose Phosphatase enzyme (ARP). Guggulsterone was found to be highly stabilized within the active site of the viral ARP enzyme by molecular dynamic simulation with very little fluctuation throughout the simulation timeframe of 100 ns. Thus, guggulsterone can be further used to develop a safe and competent medication for evolving therapy against SARS-CoV-2 in post-preclinical and clinical trials. [ABSTRACT FROM AUTHOR]

The transient receptor potential melastatin type 2 (TRPM2) channel is a non‐selective cation channel that has high Ca2+ permeability. TRPM2 is sensitive to warm temperatures and is expressed in cells and tissues that are maintained at core body temperature. TRPM2 activity is also regulated by endogenous factors including redox signalling, cytosolic Ca2+ and adenosine diphosphate ribose. As a result of its wide expression and function at core body temperature, these endogenous factors could regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. We previously reported that cellular redox signalling can lower TRPM2 temperature thresholds, although the mechanism that regulates these thresholds is unclear. Here, we used biochemical and electrophysiological techniques to explore another regulatory mechanism for TRPM2 temperature thresholds that is mediated by TRPM2 phosphorylation. Our results show that: (1) the temperature threshold for TRPM2 activation is lowered by cytosolic Ca2+; (2) protein kinase C‐mediated phosphorylation of TRPM2 counteracts the effect of cytosolic Ca2+; and (3) Thr738 in mouse TRPM2 that lies near the Ca2+ binding site in the cytosolic cleft of the transmembrane domain is a potential phosphorylation site that may be involved in phosphorylation‐mediated elevation of TRPM2 thresholds. These findings provide structure‐based evidence to understand how temperature thresholds of thermo‐sensitive TRP channels (thermo‐TRPs) are determined and regulated. Key points: The transient receptor potential melastatin type 2 (TRPM2) ion channel is temperature‐sensitive and Ca2+‐permeable.Endogenous factors and pathways such as redox signalling can regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions.In the present study, we report the novel finding that cytosolic Ca2+ lowers the temperature threshold for TRPM2 activation in a concentration‐dependent manner.Protein kinase C‐mediated phosphorylation of TRPM2 at amino acid Thr782 elevates the temperature threshold for activation by counteracting the effects of cytosolic Ca2+.These findings provide structure‐based evidence to understand how temperature thresholds of thermo‐sensitive TRP channels are determined and regulated. [ABSTRACT FROM AUTHOR]

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ~800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes. [ABSTRACT FROM AUTHOR]

Current anti–hepatitis B virus (HBV) therapies have little effect on covalently closed circular DNA (cccDNA) and fail to eliminate HBV. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been reported to directly target cccDNA and exert antiviral effects. In this study, we hypothesized that the inhibition of the DNA repair machinery, which is important for the repair of CRISPR‐induced double‐strand breaks, may enhance the effect of CRISPR targeting cccDNA, and we investigated the antiviral effect of potential combination therapy. The antiviral effect of CRISPR targeting cccDNA (HBV‐CRISPR) was evaluated in HBV‐susceptible HepG2‐hNTCP‐C4 cells expressing Cas9 (HepG2‐hNTCP‐C4‐iCas9) or primary human hepatocytes (PHHs) expressing Cas9. Following HBV infection, HBV‐CRISPR reduced cccDNA levels, accompanied by decreases in pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen and hepatitis B e antigen levels in HepG2‐hNTCP‐C4‐iCas9 cells, and PHHs. HBV‐CRISPR induced indel formation in cccDNA and up‐regulated poly(adenosine diphosphate ribose) polymerase (PARP) activity in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells. The suppression of PARP2‐Histone PARylation factor 1 (HPF1) (involved in the initial step of DNA repair) with small interfering RNA (siRNA) targeting either PARP2 or HPF1 increased the reduction in pgRNA and cccDNA by HBV‐CRISPR in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells. The suppression of DNA Ligase 4 (LIG4) (essential for nonhomologous end joining [NHEJ]) but not breast cancer susceptibility gene (BRCA) (essential for homologous recombination) enhanced the antiviral effect of HBV‐CRISPR in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells. Finally, the clinically available PARP inhibitor olaparib increased the reductions in pgRNA and cccDNA levels induced by HBV‐CRISPR in HBV‐infected HepG2‐hNTCP‐C4‐iCas9 cells and PHHs. Conclusion: The suppression of the NHEJ‐mediated DNA repair machinery enhances the effect of CRISPR targeting cccDNA. The combination of CRISPR and olaparib may represent a therapy for HBV elimination. [ABSTRACT FROM AUTHOR]

Background: Vascular leakage is a deadly complication of severe infections, ranging from bacterial sepsis to malaria. Worldwide, septicemia is among the top 10 causes of lethality because of the shock and multiorgan dysfunction that arise from the host vascular response. In the monoclonal gammopathy-associated capillary leak syndrome (MG-CLS), even otherwise mundane infections induce recurrent septic-like episodes of profound microvascular hyperpermeability and shock. There are no defined genetic risk factors for MG-CLS or effective treatments for acute crises., Methods: We characterized predicted loss-of-function mutations in PARP15 (poly[ADP-ribose] polymerase 15), a protein of unknown function that is absent in mice, in patients with MG-CLS. We analyzed barrier function in PARP15-deficient vascular endothelial cells and vascular leakage in mice engineered to express WT or loss-of-function variant human PARP15., Results: We discovered several loss-of-function PARP15 variants associated with MG-CLS. These mutations severely reduced PARP15 enzymatic function. The presence of the most frequently detected variant (G628R) correlated with clinical markers of severe vascular leakage. In human microvascular endothelial cells, PARP15 suppressed cytokine-induced barrier disruption by ADP-ribosylating the scaffold protein JIP3 (c-Jun N-terminal kinase-interacting protein 3) and inhibiting p38 MAP kinase activation. Mice expressing enzymatically inactive human PARP15(G628R) were significantly more prone to inflammation-associated vascular leakage than mice expressing WT PARP15 in a p38-dependent fashion., Conclusions: PARP15 represents a previously unrecognized genetic susceptibility factor for MG-CLS. PARP15-mediated ADP ribosylation is an essential and genetically determined mechanism of the human vascular response to inflammation.

Ulcerative colitis (UC) is a complex, multifactorial disease driven by a dysregulated immune response against host commensal microbes. Despite rapid advances in our understanding of host genomics and transcriptomics, the metabolic changes in UC remain poorly understood. We thus sought to investigate distinguishing metabolic features of the UC colon (14 controls and 19 patients). Metabolomics analyses revealed inflammation state as the primary driver of metabolic variation rather than diagnosis, with multiple metabolites differentially regulated between inflamed and uninflamed tissues. Specifically, inflamed tissues were characterized by reduced levels of nicotinamide adenine dinucleotide (NAD+) and enhanced levels of nicotinamide (NAM) and adenosine diphosphate ribose (ADPr). The NAD+/NAM ratio, which was reduced in inflamed patients, served as an effective classifier for inflammation in UC. Mitochondria were also structurally altered in UC, with UC patient colonocytes displaying reduced mitochondrial density and number. Together, these findings suggest a link between mitochondrial dysfunction, inflammation, and NAD+ metabolism in UC. [ABSTRACT FROM AUTHOR]

Auranofin, as a thioredoxin reductase (TrxR) inhibitor, has promising anti-cancer activity in several cancer types. However, little is known about the inhibitory effect of auranofin on lung cancer cell growth. We, therefore, investigated the antigrowth effects of auranofin in various lung cancer cells with respect to cell death, reactive oxygen species (ROS), and glutathione (GSH) levels. Treatment with 0~5 µM auranofin decreased cell proliferation and induced cell death in Calu-6, A549, SK-LU-1, NCI-H460, and NCI-H1299 lung cancer cells at 24 h. In addition, 0~5 µM auranofin increased ROS levels, including O2•−, and depleted GSH levels in these cells. N-acetyl cysteine (NAC) prevented growth inhibition and mitochondrial membrane potential (MMP, ∆Ψm) loss in 3 and 5 µM auranofin-treated Calu-6 and A549 cells at 24 h, respectively, and decreased ROS levels and GSH depletion in these cells. In contrast, L-buthionine sulfoximine (BSO) enhanced cell death, MMP (∆Ψm) loss, ROS levels, and GSH depletion in auranofin-treated Calu-6 and A549 cells. Treatment with 3 and 5 µM auranofin induced caspase-3 activation and poly (ADP ribose) polymerase (PARP) cleavage in Calu-6 and A549 cells, respectively. Both were prevented by NAC, but enhanced by BSO. Moreover, TrxR activity was reduced in auranofin-treated Calu-6 and A549 cells. That activity was decreased by BSO, but increased by NAC. In conclusion, these findings demonstrate that auranofin-induced cell death is closely related to oxidative stress resulted from increased ROS levels and GSH depletion in lung cancer cells. [ABSTRACT FROM AUTHOR]

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxiainducible factor-1a (HIF-1a) in the Notch3 response are still unclear. In this study, we found that HIF-1a and poly [ADPribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1a knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1a and also another molecule that binds the Notch3 promoter's guaninerich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1a associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1a, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Lymphoid neoplasms, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and NK/T cell neoplasms, are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies, like daratumumab, have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials. Methods: We generated chimeric antigen receptor (CAR) T cells targeting CD38 and tested its cytotoxicity against multiple CD38high and CD38low lymphoid cancer cells. We evaluated the synergistic effects of all-trans retinoic acid (ATRA) and CAR T cells or daratumumab against cancer cells and xenograft tumors. Results: CD38-CAR T cells dramatically inhibited the growth of CD38high MM, mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia (WM), T-cell acute lymphoblastic leukemia (T-ALL), and NK/T-cell lymphoma (NKTCL) in vitro and in mouse xenografts. ATRA elevated CD38 expression in multiple CD38low cancer cells and enhanced the anti-tumor activity of daratumumab and CD38-CAR T cells in xenograft tumors. Conclusions: These findings may expand anti-CD38 immunotherapy to a broad spectrum of lymphoid malignancies and call for the incorporation of ATRA into daratumumab or other anti-CD38 immunological agents for cancer therapy. [ABSTRACT FROM AUTHOR]

Infection with schistosomes (blood flukes) can result in the debilitating disease schistosomiasis. These parasites survive in their host for many years, and we hypothesize that proteins on their tegumental surface, interacting with the host microenvironment, facilitate longevity. One such ectoenzyme -- the nucleotide pyrophosphatase/phosphodiesterase SmNPP5 can cleave ADP (to prevent platelet aggregation) and NAD (likely preventing Treg apoptosis). A second tegumental ectoenzyme, the glycohydrolase SmNACE, also catabolizes NAD. Here, we undertake a comparative biochemical characterization of these parasite ectoenzymes. Both are GPI-linked and exhibit different optimal pH ranges. While SmNPP5 requires divalent cations, SmNACE does not. The KM values of the two enzymes for NAD at physiological pH differ: SmNPP5, KM = 340 mM± 44; SmNACE, KM = 49 mM± 4. NAD cleavage by each enzyme yields different products. SmNPP5 cleaves NAD to form nicotinamide mononucleotide (NMN) and AMP, whereas SmNACE cleaves NAD to generate nicotinamide (NAM) and adenosine diphosphate ribose (ADPR). Each enzyme can process the other's reaction product. Thus, SmNACE cleaves NMN (to yield NAM and ribose phosphate) and SmNPP5 cleaves ADPR (yielding AMP and ribose phosphate). Metabolomic analysis of plasma containing adult worms supports the idea that these cleavage pathways are active in vivo. We hypothesize that a primary function of SmNPP5 is to cleave NAD to control host immune cell function and a primary function of SmNACE is to cleave NMN to generate the vital nutrient nicotinamide (vitamin B3) for convenient uptake by the worms. Chemical inhibition of one or both ectoenzymes could upset worm metabolism and control schistosome infection. [ABSTRACT FROM AUTHOR]

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with kcat /Km values >10,000 M-1  s-1 : Q92EH0_LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1_BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported kcat /Km values exhibited against these canonical substrates are well under 105 M-1  s-1 . By contrast, several Nudix enzymes show much larger kcat /Km values (in the range of 105 to >107 M-1  s-1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc.

Skin metastasis from ovarian cancer is rare, and its prognosis is poor. Effective therapeutic strategies are currently lacking, but the combination of various treatment methods shrink the tumor and relieve symptoms. The present study reports a rare case of advanced ovarian cancer with skin metastases and intestinal wall thickening, along with a BRCA1 DNA repair associated (BRCA1) mutation. After standard first-line treatment and non-standard second-line treatment, the patient developed skin metastases. The patient's skin itching, pain and lesions were completely relieved after administering bevacizumab in combination with paclitaxel and carboplatin. After 4 months, skin metastases recurred along with anal distension during maintenance treatment with oral poly(ADP ribose) polymerase (PARP) inhibitors. The patient was treated again with bevacizumab combined with docetaxel, and the anal distension was significantly relieved. Angiogenesis therapy combined with chemotherapy is effective, but that the disease-free survival time is short, and PARP inhibitor maintenance effect is limited even in cases with a BRCA1 gene mutation. [ABSTRACT FROM AUTHOR]

Thrombospondin-1 (Tsp-1), a matricellular protein, could protect retinal neurons from endogenous or exogenous insults; however, its underlying mechanism remains unclear. Thus, this study aimed to investigate Tsp-1-mediated neuron-protection effect in retinal cells. Our data showed that Tsp-1 downregulation would aggravate UV irradiation-induced DNA damage in 661 W cells and cone photoreceptor cells. The increasing levels of poly (ADP ribose) polymer (PAR) and γ-H2AX in Tsp-1-silenced 661 W cells indicate severe DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). By utilizing an error-prone substrate, Tsp-1 silencing significantly increased deleted DNA end joining in 661 W cells with spontaneous DNA damage (SDD). Moreover, Tsp-1 is indirectly involved in DNA stability in 661 W cells as UV treatment caused a significant Tsp-1 decreasing in cytoplasm, but no obvious Tsp-1 alteration in cell nuclear of 661 W cells. Furthermore, our data indicate that Tgf-β1 activation domain in Tsp-1 plays a critical role in DNA stability in 661 W cells through expressing mutated exogenous Tsp-1 and Tgf-β inhibitor, LSKL. Therefore, this study provides new insights into the mechanism of the neuroprotective action positively mediated by Tsp-1, which might be a therapeutic target for the treatment of retinal pathology. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) removes damaged bases by generating single‐strand breaks (SSBs), gap‐filling by DNA polymerase β (POLβ), and resealing SSBs. A base‐damaging agent, methyl methanesulfonate (MMS) is widely used to study BER. BER increases cellular tolerance to MMS, anti‐cancer base‐damaging drugs, temozolomide, carmustine, and lomustine, and to clinical poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib. The poisons stabilize PARP1/SSB complexes, inhibiting access of BER factors to SSBs. PARP1 and XRCC1 collaboratively promote SSB resealing by recruiting POLβ to SSBs, but XRCC1−/− cells are much more sensitive to MMS than PARP1−/− cells. We recently report that the PARP1 loss in XRCC1−/− cells restores their MMS tolerance and conclude that XPCC1 facilitates the release of PARP1 from SSBs by maintaining its autoPARylation. We here show that the PARP1 loss in XRCC1−/− cells also restores their tolerance to the three anti‐cancer base‐damaging drugs, although they and MMS induce different sets of base damage. We reveal the synthetic lethality of the XRCC1−/− mutation, but not POLβ−/−, with olaparib and talazoparib, indicating that XRCC1 is a unique BER factor in suppressing toxic PARP1/SSB complex and can suppress even when PARP1 catalysis is inhibited. In conclusion, XRCC1 suppresses the PARP1/SSB complex via PARP1 catalysis‐dependent and independent mechanisms. [ABSTRACT FROM AUTHOR]

Adenosine diphosphate ribose cyclase (ADPRC) exists widely in eukaryotes and lower metazoans cells. It can degrade nicotinamide adenine dinucleotide (NAD) into cyclic ADP ribose (cADPR) and nicotinamide, and subsequently hydrolyses cADPR to ADP ribose (ADPR). In this paper, we have summarized the relative subcellular localization of ADPRC and enzymes with ADPRC activity in organisms, related enzyme family members of ADPRC are also described. In addition, we discussed the main biological functions of ADPRC, the regulation of Ca2+ signal, the regulation of insulin and glucagon secretion, oxytocin secretion, and the effects of renal and pulmonary vasomotor tension. Finally, we expounded the relationship between ADPRC and human health and disease occurrence. It provides a theoretical basis for the targeted treatment of ADPRC as a pharmacological tool for related diseases, and has important significance in clinical diagnosis and disease intervention. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of microRNA-182 (miR-182) on hepatocellular carcinoma (HCC) and its mechanisms of action. Methods: Sixty-five HCC tissues and matched adjacent tissues were obtained, and miR-182 and sexdetermining region on the Y Chromosome (SRY)-related HMG box 11 (SOX11) expression were quantified using reverse transcription-polymerase chain reaction (RT-PCR). SOX11-positive cells were identified by immunohistochemistry and the correlation between SOX11 and miR-182 expression was analyzed by Spearman correlation analysis. Huh-7 cells were transfected with the miR-182 mimic, the miR-182 inhibitor, and/or si-SOX11, and cell proliferation was measured by cell counting kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry. Luciferase reporter assay was used to investigate the putative target gene of miR-182. A xenograft nude mouse model was established by transfection with miR-182 antagomir, while tumor volume and weight were calculated. SOX11, cleaved caspase-3, cleaved poly (ADP ribose) polymerase (PARP), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) protein levels were analyzed by western blot. Results: MiR-182 expression increased and SOX11 expression was decreased in HCC tissues (p < 0.05). Luciferase reporter assay data confirmed that miR-182 directly targets SOX11. Inhibition of miR-182 repressed proliferation and Bcl-2 expression, but increased protein expression of cleaved caspase-3, cleaved RAPP, and Bax in Huh-7 cells (p < 0.05). In addition, suppression of SOX11 reversed the effects of miR-182 on cell proliferation and apoptosis. The miR-182 antagomir decreased tumor growth and miR-182 expression but increased SOX11 expression in vivo (p < 0.05). Conclusion: MiR-182 and SOX11 may be novel therapeutic targets for HCC patients. [ABSTRACT FROM AUTHOR]

Cyclic adenosine diphosphate ribose (cADPR) is a cyclic nucleotide involved in the Ca2+ homeostasis. In its structure, the northern ribose, bonded to adenosine through an N1 glycosidic bond, is connected to the southern ribose through a pyrophosphate bridge. Due to the chemical instability at the N1 glycosidic bond, new bioactive cADPR derivatives have been synthesized. One of the most interesting analogues is the cyclic inosine diphosphate ribose (cIDPR), in which the hypoxanthine replaced adenosine. The efforts for synthesizing new linear and cyclic northern ribose modified cIDPR analogues led us to study in detail the inosine N1 alkylation reaction. In the last few years, we have produced new flexible cIDPR analogues, where the northern ribose has been replaced by alkyl chains. With the aim to obtain the closest flexible cIDPR analogue, we have attached to the inosine N1 position a 2″,3″-dihydroxypentyl chain, possessing the two OH groups in a ribose-like fashion. The inosine alkylation reaction afforded also the O6-alkylated regioisomer, which could be a useful intermediate for the construction of new kinds of cADPR mimics. [ABSTRACT FROM AUTHOR]

Poly (ADP-ribose) polymerase (PARP) inhibition by pharmacological agents has been a useful cancer treatment in recent times. PARP inhibitors represent a category of anticancer drugs that function by blocking PARP catalytic activity. The design of highly selective and effective PARP inhibitors has significant therapeutic advantages and remains an integral part of several PARP-based drug discovery research. We employed conformational landscape and binding energy analyses to decipher the mechanisms underlying the inhibitory potencies and selectivity of Cpd81 towards PARP enzymes. Relatively, the thermodynamics analysis revealed that pp2-cpd81 (−55.38 kcal/mol) had a higher ΔGbind than pp1-cpd81 (−27.97 kcal/mol), indicating high selective binding activity of Cpd81 towards PARP-2. The binding activity of Cpd81 to PARP-2 was potentiated by the high effects of van der Waal. Moreso, the NAM-AD positioning of Cpd81 facilitated its optimal orientation for multiple high-affinity complementary interactions with key site residues. Resultantly, Cpd81 could span the two subsites of the PARP binding pocket effectively, as seen in the multiple interactions exhibited by the compound. These interactions serve as the basis for the selective inhibitory potential of Cpd81. We believe the findings here would pave the way for developing PARP inhibitors that are selective in breast cancer treatment. [ABSTRACT FROM AUTHOR]

This work explores the ADP-ribosyltransferase activity of Pseudomonas (P.) aeruginosa exotoxin A using the guanyl hydrazone derivative, nitrobenzylidine aminoguanidine (NBAG) and the impact of gamma radiation on its efficacy. Unlike the conventional detection methods, NBAG was used as the acceptor of ADP ribose moiety instead of wheat germ extract elongation factor 2. Exotoxin A was extracted from P. aeruginosa clinical isolates and screened for toxA gene using standard PCR. NBAG was synthesized using aminoguanidine bicarbonate and 4-nitrobenzaldehyde and its identity has been confirmed by UV, FTIR, Mass and 13C-NMR spectroscopy. The ADP-ribosyl transferase activity of exotoxin A on NBAG in the presence of Nicotinamide adenine dinucleotide (NAD+) was recorded using UV spectroscopy and HPLC. In vitro ADP-ribosyl transferase activity of exotoxin A protein extract was also explored by monitoring its cytotoxicity on Hep-2 cells using sulforhodamine B cytotoxicity assay. Bacterial broths were irradiated at 5, 10, 15, 24 Gy and exotoxin A protein extract activity were assessed post exposure. Exotoxin A extract exerted an ADP-ribosyltransferase ability which was depicted by the appearance of a new ʎmax after the addition of exotoxin A to NBAG/NAD+ mixture, fragmentation of NAD+ and development of new peaks in HPLC chromatograms. Intracellular enzyme activity was confirmed by the prominent cytotoxic effects of exotoxin A extract on cultured cells. In conclusion, the activity of Exotoxin A can be monitored via its ADP-ribosyltransferase activity and low doses of gamma radiation reduced its activity. Therefore, coupling radiotherapy with exotoxin A in cancer therapy should be carefully monitored. [ABSTRACT FROM AUTHOR]

Background: Patients with germline/somatic BRCA1/BRCA2 mutations (g/sBRCA1/2) comprise a distinct biologic subgroup of pancreas ductal adenocarcinoma (PDAC). Methods: Institutional databases were queried to identify patients who had PDAC with g/sBRCA1/2. Demographics, clinicopathologic details, genomic data (annotation sBRCA1/2 according to a precision oncology knowledge base for somatic mutations), zygosity, and outcomes were abstracted. Overall survival (OS) was estimated using the Kaplan‐Meier method. Results: In total, 136 patients with g/sBRCA1/2 were identified between January 2011 and June 2020. Germline BRCA1/2 (gBRCA1/2) mutation was identified in 116 patients (85%). Oncogenic somatic BRCA1/2 (sBRCA1/2) mutation was present in 20 patients (15%). Seventy‐seven patients had biallelic BRCA1/2 mutations (83%), and 16 (17%) had heterozygous mutations. Sixty‐five patients with stage IV disease received frontline platinum therapy, and 52 (80%) had a partial response. The median OS for entire cohort was 27.6 months (95% CI, 24.9‐34.5 months), and the median OS for patients who had stage IV disease was 23 months (95% CI, 19‐26 months). Seventy‐one patients received a poly(adenosine diphosphate ribose) polymerase (PARP) inhibitor (PARPi), and 52 received PARPi monotherapy. For maintenance PARPi, 10 patients (36%) had a partial response, 12 (43%) had stable disease, and 6 (21%) had progression of disease as their best response. Six patients (21%) received maintenance PARPi for >2 years. For those with stage IV disease who received frontline platinum, the median OS was 26 months (95% CI, 20‐52 months) for biallelic patients (n = 39) and 8.66 months (95% CI, 6.2 months to not reached) for heterozygous patients (n = 4). The median OS for those who received PARPi therapy was 26.5 months (95% CI, 24‐53 months) for biallelic patients (n = 25) and 8.66 months (95% CI, 7.23 months to not reached) for heterozygous patients (n = 2). Conclusions: g/sBRCA1/2 mutations did not appear to have different actionable utility. Platinum and PARPi therapies offer therapeutic benefit, and very durable outcomes are observed in a subset of patients who have g/sBRCA1/2 mutations with biallelic status. In a cohort of 136 patients with pancreas cancer and a germline or somatic BRCA mutation, the median overall survival is 27.6 months (95% CI, 24.9‐34.5 months) for the entire cohort and 23 months for the stage IV cohort (n = 81). Biallelic zygosity enriches the response to platinum and poly(adenosine diphosphate ribose) polymerase inhibitor therapies. [ABSTRACT FROM AUTHOR]

Abstract Background Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca2+). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca2+ signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N 6-Benzoyladenosine-3′,5′-cyclic monophosphate (N 6-Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants. Methods Ten ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N 6-Bnz-cAMP drug treatments by flow cytometry. Results Baseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56DimCD16+ subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56BrightCD16Dim/− subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group. Conclusion Overexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca2+ homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca2+]i mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site.