Lithium salts have been in the therapeutic toolbox for better or worse since the 19th century, with purported benefit in gout, hangover, insomnia, and early suggestions that lithium improved psychiatric disorders. However, the remarkable effects of lithium reported by John Cade and subsequently by Mogens Schou revolutionized the treatment of bipolar disorder. The known molecular targets of lithium are surprisingly few and include the signaling kinase glycogen synthase kinase-3 (GSK-3), a group of structurally related phosphomonoesterases that includes inositol monophosphatases, and phosphoglucomutase. Here we present a brief history of the therapeutic uses of lithium and then focus on GSK-3 as a therapeutic target in diverse diseases, including bipolar disorder, cancer, and coronavirus infections. Lithium was discovered as a new element (atomic number 3) in 1817. Lithium is an alkali metal, the lightest solid element, and a monovalent cation in solution, used therapeutically as lithium carbonate (most common), acetate, citrate, chloride, or sulfate salts. Lithium salts first gained pharmacological popularity in the 19th century as treatment for gout and other “gouty” conditions thought to be caused by excessive uric acid, as lithium carbonate dissolved uric acid in vitro. By 1859, Sir Alfred Baring Garrod’s work on gout included his recommendation for lithium to treat “brain gout”, including mania. In 1871, lithium bromide was used to treat acute mania by William Hammond at Bellevue Hospital. Danish psychiatrists Carl and Frederik Lange recommended lithium carbonate as maintenance therapy to treat periodic depression, which they believed to be caused by uric acid. The “uric acid diathesis” hypothesis of disease was largely abandoned in the early 1900s ,3,4.Lithia water and lithium tablets were popular patent medicines and health supplements in the late 19th and early 20th centuries purported to prevent or treat uric acid-induced ailments. The carbonated beverage now known as 7Up was originally sold as “Bib-Label Lithiated Lemon-Lime Soda” and was marketed as a hangover cure. Lithium toxicity was described in several studies, but lithium salts remained popular in the United States until patent medicines were restricted by the FDA. In 1948, lithium chloride (LiCl) was marketed as a table salt substitute for low-sodium diets, resulting in multiple reports of lithium toxicity, including fatal cases by 1949 ,6.In 1949, Australian psychiatrist John Cade reported successfully using lithium citrate to treat patients institutionalized with mania after observing a sedative effect of lithium on guinea pigs in uric acid experiments and after assessing the safety of lithium by self-administration . Cade deThe direct targets of lithium in psychiatry are unknown, but basic science research gives rise to several viable candidate targets. While lithium induces wide-ranging physiological and developmental effects, lithium directly inhibits few known targets. Establishing which of these targets is responsible for a given effect of lithium can be challenging, especially for the therapeutic effects in humans. Criteria for validating direct targets of lithium have been previously discussed ,13. BrieDirect targets of lithium include inositol monophosphatase  and struWhile these three classes of lithium targets are structurally distinct, all are magnesium dependent, and in each case, lithium, which has an ionic radius similar to magnesium, competes for a magnesium binding site ,16,19,20Dictyostelium discoideum is sensitive to lithium: Upon starvation these free living amoeboid cells form multicellular aggregates comprising stalk and spore cells. Lithium exposure prior to aggregation inhibits spore cell fate and promotes stalk cell fate [Xenopus and zebrafish, expanding dorsal mesoderm and inducing a second dorsal axis [Lithium salts have been widely used experimentally to disrupt cell fate specification in diverse organisms from protozoans to vertebrates. Development of the slime mold ell fate ,24. Lithell fate ,26. Lithsal axis ,28,29. Lsal axis  and affeLithium also stimulates glycogen synthesis in muscle, hepatocytes, and adipocytes by increasing the activity of Glycogen Synthase (GS), enhancing glucose uptake and storage and mimicking insulin action, although the direct target for these effects was not identified in those early studies ,33,34,35gska in Dictyostelium caused expansion of stalk cell fate at the expense of spore cells [Gsk3b mimic the effect of lithium in Xenopus [Xenopus embryos, where Wnt activation also induces axis duplication [Each of these developmental and metabolic effects of lithium is mimicked by inhibition or genetic loss of glycogen synthase kinase-3 (GSK-3). GSK-3 was so named because it phosphorylates and inhibits GS; thus the effects of lithium on insulin signaling and glycogen synthesis can be explained by inhibition of GSK-3, as demonstrated later . Similarre cells  that wasre cells . Further Xenopus ,38,39. I Xenopus ,41,42, alication .Drosophila, and Dictyostelium [m magnesium binding site in GSK-3 with a Ki ~1–2 mM [Recognition that the metabolic and developmental effects of lithium parallel the effects of GSK-3 inhibition in these settings led to the hypothesis that lithium acts through direct inhibition of GSK-3, and this was demonstrated through in vitro assays using mammalian GSK-3β . Subsequostelium ,44. Lith ~1–2 mM ,20. Othe ~1–2 mM ,19,20,22 ~1–2 mM ,21.GSK3A and GSKB, which encode the proteins GSK-3α and GSK-3β [GSK-3 is a serine/threonine kinase expressed in most vertebrates as two highly similar and mostly redundant isoforms, d GSK-3β ,46 , periods of major depression, and a substantial risk of suicide . Type I In addition to directly inhibiting GSK-3 enzymatic action ,22, lithGsk3b haploinsufficiency mimics lithium treatment in mouse behavioral tests that are responsive to lithium [Gsk3b overexpression in the CNS blunts the response to lithium without affecting the animal’s health or activity levels [Gsk3 haploinsufficiency) in mouse behavior [The hypothesis that GSK-3 is the therapeutic target of lithium is supported by animal behavior studies using genetic manipulation of GSK-3.  lithium , while Gy levels , demonsty levels ,81,82,83behavior , nor didbehavior ,86,87. TWhile GSK-3 is a promising target of mood stabilizers and other psychotropic drugs, it is unclear how its inhibition can encompass anti-manic, antidepressant, and antipsychotic effects. We propose a model in which mood stabilizers function through GSK-3 inhibition to re-sensitize cells to endogenous extracellular signals . ExtraceTherefore, pharmacological GSK-3 inhibition interrupts both autoregulatory feedback loops that drive GSK-3 to maintain an active state ,73. We hGSK-3 has well documented roles in neurodegenerative diseases, established through extensive mechanistic studies of GSK-3 in neurological models. Below, we provide a brief overview of the literature regarding GSK-3, neurodegenerative disease, and therapeutic insights.40 and Aβ42) accumulate in extracellular amyloid plaques. Plaque formation precedes tau pathology in AD and occurs through cleavage of the amyloid precursor protein (APP) into Aβ by β- and γ-secretases [42 peptide. Both APP and PS1 are GSK-3 substrates [40/42 in mouse brain and cell culture models [Gsk3a or Gsk3b in mice and observed no effect on Aβ levels [Alzheimer’s disease (AD) is the most common form of neurodegenerative disease with increasing prevalence in an aging population . Two patcretases . APP probstrates ,104,105 e models ,110,111.β levels . Howeverβ levels , so thatβ levels ,110,111,β levels ,114,115.As GSK-3 plays roles in both NFT formation and APP processing, GSK-3 has been a focus of extensive study for pharmacological interventions in AD. Inhibition with lithium and other agents in preclinical studies reduces Aβ mediated neurotoxicity, improves behavioral phenotypes, and rescues neuronal loss ,118,119.Parkinson’s disease (PD) is the second-most common neurodegenerative disorder and, like AD, tends to occur in older populations. PD is caused by neuronal loss in the substantia nigra, leading to dopamine deficiency, and intracellular accumulation of α-synuclein, called Lewy Bodies (LBs) . Tau patAPC or GSK3 therefore activates Wnt target genes, such as MYC (c-Myc) and CCND1 (cyclin D1), and promotes tumor growth, as described in greater detail below. GSK-3 also suppresses the epithelial-to-mesenchymal transition (EMT), a critical process in tumor invasion and metastasis  to phosphorylate and degrade β-catenin. Loss of iewed in ) by phosiewed in ,148,149.iewed in ,152,153,iewed in ). This aiewed in , which tSimilarly, GSK-3 phosphorylates and destabilizes c-Myc; inhibition of GSK-3 and stabilization of c-Myc can be oncogenic or pro-apoptotic depending on whether c-Myc protein accumulates at intermediate or high levels, respectively . UltimatGlioblastoma multiforme (GBM) is the most common malignant brain neoplasm in adults and carries a high mortality. PI3K/AKT signaling, which inhibits GSK-3 activity (reviewed in ), is criAPC, followed by a mutation in the oncogene KRAS, and culminates with a mutation of TP53 [APC leads to the robust activation of the Wnt pathway frequently observed in CRC [Colorectal cancer (CRC) is one of the most common cancers in adults. Our understanding of the pathophysiology of CRC has drastically improved survival since it has led to the use of widespread early screening. CRC commonly begins as a precancerous polyp, followed by sequential accumulation of somatic mutations that ultimately lead to malignant transformation; this adenoma-to-carcinoma sequence  classica of TP53 . APC is  of TP53 . Loss ofd in CRC ,180. APCd in CRC . APC enhd in CRC ,182, andd in CRC ,64,65. LApc in murine models of CRC triggered intestinal differentiation and tumor regression in spite of activating mutations in Kras or inactivating mutations in Tp53 [Apc expression and did not induce apoptosis. While the authors did not specifically comment on the activity of GSK-3 in this study, their findings underscore the dynamic nature of cell survival and differentiation in CRC; in this regard, a better understanding of how GSK-3 is involved at different stages of tumorigenesis in CRC may help evaluate its efficacy as a therapeutic target.Despite these compelling data that GSK-3 serves as a tumor suppressor in CRC, the exact role of GSK-3 in this malignancy remains controversial. For example, a conflicting report on the role of GSK-3 in KRAS dependent CRC demonstrated that GSK-3 inhibition decreased cell survival through an increase in β-catenin and c-Myc . Additio in Tp53 . HoweverKRAS mutations [Kras induction of IL-1α, which ultimately leads to NFκB activation [CCND1, BCL2, BCL2L1, and XIAP. Subsequent studies have confirmed that sensitivity of PDAC cells to GSK-3 inhibition is in part through loss of GSK-3 dependent NFκB signaling [Despite advances in therapies for other cancers, survival in pancreatic ductal adenocarcinoma (PDAC) remains poor, with incremental improvements through the use of escalating cytotoxic chemotherapy in addition to surgery when possible. Therefore, the possibility of targeted therapy has been of great interest. utations , which otivation . This intivation ). Thereftivation . Loss ofignaling ,201. Howignaling ,203,204 ignaling ,206,207.ignaling ,209,210.Clinically, these findings have prompted several trials of GSK-3 inhibitors in advanced solid tumors. LY2090314 is an ATP-competitive inhibitor of GSK-3 that demonstrated a tolerable safety profile in combination with pemetrexed and carboplatin for advanced/metastatic solid tumors, though a randomized-control trial will be needed to assess the efficacy of this protocol . A PhaseNormal hematopoiesis is characterized by a hierarchical evolution of hematopoietic precursors, which undergo several rounds of proliferation followed by either quiescence or lineage determination. GSK-3 has multiple roles in HSC maintenance, as demonstrated by the effects of GSK-3 inhibitors or loss of function in mouse hematopoietic cells. Inhibition of GSK-3 activates Wnt/β-catenin signaling to support HSC self-renewal but also activates mTOR complex 1 (mTORC1) signaling leading to HSC activation and an increase in multipotent progenitors at the expense of self-renewing HSCs . ConsistKip1 [Acute myeloid leukemia (AML), the most common acute leukemia in adults, results from clonal proliferation and maturation arrest of early myeloid progenitors. Progenitors with self-renewal capability have been defined functionally as leukemic stem cells (LSCs) or leukemia initiating cells (LICs) . Wnt/β-cKip1 . A subseKip1 . AlthougKip1 . MLL LSCKip1 ,222, modKip1 ,224, andKip1 . It shouKip1 .GSK3B, as demonstrated in a study of CML patients, in which CML cells from four of seven patients in blast crisis and one of four in chronic phase CML showed mis-splicing of GSK3B, yielding a form that did not interact with AXIN and was unable to phosphorylate β-catenin [Chronic myeloid leukemia (CML) LSCs also require active Wnt/β-catenin signaling . The Arm-catenin . HoweverThe role of GSK-3 in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and lymphomas remains poorly understood with primarily correlative pathophysiology driving studies of GSK-3 inhibition in these models. In other tumors, GSK-3 regulates FOXO proteins, PI3K/AKT signaling, NOTCH signaling, and NFAT signaling (reviewed in ), all ofIn regard to clinical trials, a phase I trial investigating lithium and tretinoin in non-promyelocytic AML demonstrated an acceptable safety profile with observed on-target effects, which led to AML cell differentiation . HoweverPotential antiviral activity of lithium has been investigated for over 40 years, as comprehensively reviewed recently . LithiumLithium was reported to have antiviral activity against herpes simplex in the 1970s. These studies included in vitro demonstration that lithium impairs HSV replication in cell culture ,241,242 Lithium has also been reported to have antiviral activity against diverse DNA and RNA viruses in vitro, including hepatitis C, adenoviruses, Dengue virus-2, pseudorabies and vaccinia viruses, parvoviruses, foot-and-mouth disease virus (FMDV), and feline calicivirus (FCV) . LimitedWhile lithium has direct antiviral activity (described in more detail below for coronaviruses), it may also function indirectly by enhancing host anti-inflammatory responses. The literature on lithium effects on the immune system is complex, with evidence for both pro-inflammatory and anti-inflammatory effects that may contribute to host responses to viral infections, as more fully reviewed in . The effPerhaps for these reasons, lithium salts have been tested for activity against a number of coronaviruses in cell culture models. LiCl impairs replication and infectivity of diverse coronaviruses, including infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and the β-coronavirus responsible for severe acute respiratory syndrome (SARS-CoV) ,255,256.50 for LiCl inhibition of GSK-3 [As the ICof GSK-3 ,20, as wof GSK-3 , and subof GSK-3 ,258,259.GSK3 mRNAs (GSK3A and GSK3B) impairs viral replication and infectivity; (5) inhibition of GSK-3 with Kenpaullone or LiCl impairs replication and infectivity of SARS-CoV and JHMV.Coronaviruses encode a nucleocapsid (N) protein that binds viral genomic RNA (gRNA) and is essential for viral replication, transcription, and virion assembly ,260,261.DDX1 or inhibition of GSK-3 impairs recruitment of DDX1, binding of N and DDX1 to viral mRNAs, and viral replication.Mechanistically, N phosphorylation plays multiple roles. Immunoprecipitation of N followed by mass spectroscopy shows that, in addition to GSK-3α and GSK-3β, N binds multiple RNA binding proteins involved in splicing and translation ,259,265.Taken together, these observations strongly support a critical role for GSK-3 in N protein function and in coronavirus infections. While the concentrations of lithium required to inhibit these functions of GSK-3 may be too high to treat infected patients, a role for other, clinically well tolerated GSK-3 inhibitors remains a potential option worth further exploration. As GSK-3 is a host kinase that may be required for multiple coronaviruses, targeting it may be effective against diverse pathogenic coronaviruses and this approach may be less susceptible to mutation and evolution of the viral genome. Clinically well tolerated GSK-3 inhibitors include Tideglusib (phase I–II) and Enzastaurin (phase I–III), a PKC inhibitor that also has activity against GSK-3 at a clinically well tolerated concentration ,268,269.Other protein kinases have also been proposed to phosphorylate the RS domain of N, including SR domain protein kinases (SRPK). Like GSK-3, SRPKs tend to processively phosphorylate multiple serines within a local domain. SRPK inhibitors have demonstrated efficacy against Hepatitis C, Sindbis virus, HIV, and cytomegalovirus ,271 and Despite the extensive literature on lithium effects, few direct targets have been identified, which is surprising given the simplicity of lithium and its mechanism of inhibition. Thus, the complexity of lithium effects has less to do with the number of targets and more do to with the wide-ranging functions of the known targets, especially GSK-3 and IMPase, which both play fundamental roles in diverse signaling pathways. Here we have focused on GSK-3, which has well over 100 substrates and functions in multiple signaling pathways, including RTK, GPCR, Wnt, and nuclear hormone signaling. GSK-3 manages to be a complicated enzyme even without the many inputs and outputs, as it employs several autofeedback circuits to regulate its own activity. These feedback circuits could play a key role in modulating sensitivity to diverse extracellular signals, for example in postsynaptic neurons. As these regulatory circuits are sensitive to lithium, they may also explain why lithium can ameliorate markedly distinct, pathological mood states without perturbing euthymic mood.GSK-3 sets itself apart from most signaling kinases by functioning constitutively and, in most cases, inhibiting rather than activating downstream effectors; thus, signaling pathways inhibit GSK-3 to turn on downstream signaling, a recurring double negative meme for GSK-3 signaling. It should not be surprising then that the role of GSK-3 in cancers is complex, with tumor-promoting and tumor-suppressive activities. Even in the well-characterized canonical Wnt signaling pathway, GSK-3 seems to be of two minds, as it is required for activation of the LRP5/6 co-receptor upstream of its more familiar role in suppressing Wnt signaling by driving degradation of β-catenin. With this in mind, it seems likely that the role of GSK-3 in psychiatric disorders and neurodegenerative disorders, as in cancers, will also be complex. We have presented here just an overview of an intricate regulatory system in these disorders, with much left to be uncovered.


Sargassum thunbergii  Kuntze), inhibit adipogenesis in 3T3-L1 cells. However, the in vivo anti-obesity effects of these compounds have not been previously reported. Therefore, the objective of this study was to determine the effects of ST on weight loss, fat accumulation, as well as risk factors for type 2 diabetes and cardiovascular disease in high-fat diet (HFD)-induced obese mice. ST treatment significantly decreased body weight and fat accumulation in HFD-induced obese mice, while reducing insulin and factors related to cardiovascular diseases  in serum. ST-induced downregulation of PPARγ in white adipose tissue, and upregulation of the thermogenic genes, UCP-1 and UCP-3, in brown adipose tissue was also observed. In addition, oral administration of ST reduced the occurrence of fatty liver, as well as the amount of white adipose tissue in HFD mice. Cumulatively, these results suggest that ST exerts anti-obesity effects and may serve as a potential anti-obesity therapeutic agent.Obesity is a metabolic disease characterized by an increased risk of type 2 diabetes, hypertension, and cardiovascular disease. We have previously reported that compounds isolated from brown alga,  Globally, the number of obese individuals increased by more than three-fold between 1980 and 2014 with more than 600 million people considered to be obese . CurrentSargassum thunbergii (ST), which is an edible brown algae, is abundant within the subtidal regions of Korea, Japan, and China; it also reportedly has anti-oxidant, anti-allergy, and anti-inflammatory properties [Compound derivatives from natural substances have demonstrated significant protective effects against metabolic syndromes, including diabetes, hypertension, and obesity ,9,10. Spoperties ,17,18. Woperties . HoweverST was collected from the coast of Jeju Island, South Korea. Salt, sand, and epiphytes were removed using tap water. The freeze-dried seaweed samples were ground and passed through a 40–50 mesh by Pin-mill. Following this, 100 g of powdered ST was subjected to extraction with 70% ethanol solution at room temperature for 24 h. The extracts were subsequently concentrated and freeze-dried to obtain a 70% ethanoic extract of ST. The resulting powder was used for all subsequent studies.2O , vitamin mix V10001 , choline bitarrate , federal food, drug, and dcosmetic act (FD&C) Red Dye #40 , with a total caloric intake of 4507 kcal. The mice were randomly divided into six groups (five mice per group) as follows: Group 1, chow diet , 0.65% Ca, and 0.5% P); Group 2, HFD ; Group 3, HFD + positive control (Garcinia cambogia (Gar) extract (165 mg/kg) with HFD); Group 4, HFD + positive control (orlistat (10 mg/kg) with HFD); Group 5, HFD + ST extract (100 mg/kg); Group 6, HFD + ST extract (300 mg/kg). Each morning, ST extract and positive controls were administered orally for seven weeks. During the study duration, the body weights and survival rates of the mice were examined daily. After seven weeks, the mice were anesthetized, and blood samples were collected to determine the following biochemical parameters: total cholesterol, insulin, triglyceride, and leptin levels. A portion of the hepatic and white adipose tissues (WAT) were immediately fixed in 10% formalin , and then stained with hematoxylin and eosin (H&E) stain . The remaining hepatic tissues and WAT were frozen in liquid nitrogen and stored at −70 °C for biochemical assays. All experiments were performed in accordance with the experimental animal guidelines of Jeju National University Animal Center .Male C57BL/6 mice  were purchased from Jung Ang Lab Animal Inc. . Prior to use, the animals were acclimated to their environment at 22 °C with 55% humidity and a 12-h light/dark cycle for one week. The chow diet consisted of nitrogen-free extract (60.7%), proteins (15.2%), cellulose (4.1%), mineral ash (5.0%), moisture (12.1%), and lipids (2.9%) with a caloric intake of 2793 kcal. The HFD comprised casein, 30 mesh , L-cystein , corn starch , maltodextrin 10 , sucrose , soybean oil , lard , vitamin mix S10026 , dicalcium phosphate , calcium carbonate , potassium citrate, 1 Hg, 20 min, 4 °C). Commercial analysis kits were used to evaluate the content of serum triglyceride (TG), total cholesterol (TC), leptin, and insulin .Blood samples were collected from the mouse heart by cardiac puncture into an EDTA-rinsed syringe. The blood was centrifuged to collect serum  for 30 s and eosin Y solution  for 10 s, rinsed three times with distilled water, and mounted using xylene-based DPX mounting solution. H&E stained tissue slide images were observed via light microscopy and the size of adipose tissues was calculated using Image J software .TM, Taichung, Taiwan). Tissues were then homogenized using a bead beater (tacoTM Prep bead beater) for five cycles (40 s/cycle). The homogenized mixtures were then centrifuged . Supernatants were collected and the mRNA was extracted from the supernatant using phase separation and RNA precipitation. The isolated mRNA was reverse transcribed to complementary DNA (cDNA) using a cDNA reverse transcription kit . Subsequently, the Thermal Cycler Dice Real-Time System  was used to perform the reaction. cDNA amplification was conducted in optimal conditions (enzyme activation (95 °C for 10 s), followed by 40 cycles of denaturation (95 °C for 5 s) and extension (58 °C for 10 s)). The sequences for all primers were as follows: GAPDH: forward, 5′-TGTGTCCGTCGTGGATCTGA-3′, reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′; PPAR: forward, 5′-GTCACGGAACACGTGCAGC-3′, reverse, 5′-ACTCAGAAGTGGGCGAGGAC-3′; UCP1: forward, 5′- CAGAGAGTTTGTCCTCTGGTGC-3′, reverse, 5′- GAAAAATCAAGGGTCCTGCCCC-3′. UCP3: forward, 5′- GTTTTGCTGATCTCCTCACCTTT-3′, reverse, 5′- GAACTGCTTGACAGAGTCATAGAGG-3′.Adipogenic and thermogenic gene expression was analyzed via reverse transcription polymerase chain reaction (RT-PCR) techniques. mRNA was extracted from 30–40 mg mouse tissues using 1 mL Trizol reagent  placed in tubes containing 1mm of steel beads . Analysis of statistical significance was performed using statistical package for the social science software. Student’s Changes in body weight and fat accumulation in experimental mice.G. cambogia extract and orlistat have reportedly beneficial anti-obesity effects [To evaluate the anti-obesity effects of ST, we measured the body weights of mice once per week throughout the study. Considering that  effects ,21, theyFurther, we previously reported that indole derivatives isolated from ST inhibit adipogenesis in adipocytes. Therefore, we also measured changes in fat accumulation in the WAT of HFD-induced obese mice following ST treatment. We found that oral administration of ST at 100 or 300 mg/kg decreased fat accumulation in WAT , subcutaneous fat (Sub), mesenteric fat (Mes), and perirenal fat (Peri)) compared to that in the HFD group B. These G. cambogia) group reduced the triglyceride and TC levels in the serum compared to the HFD group. Similarly, treatment with ST (100 and 300 mg/kg) significantly decreased serum triglyceride and cholesterol levels compared with the HFD group. Furthermore, leptin levels were also markedly higher in the HFD group than in the chow diet group; while treatment with ST significantly decreased the serum level of leptin compared with the HFD group. These results indicate that ST treatment may be capable of improving metabolic disorders, such as cardiovascular disease and type 2.Excessive fat accumulation increases the risk of developing type 2 diabetes and cardiovascular disease in humans. Moreover, increased serum leptin levels are dependent on body fat accumulation. Meanwhile, HFD-induced obesity is characterized by an increased risk of type 2 diabetes with increased serum insulin levels. Obesity is a risk factor for cardiovascular diseases, and is associated with high levels of triglyceride and cholesterol in the serum . To detePark et al. reported that HFD markedly increases the size and accumulation of fat tissue in fatty liver. Moreover, obese and overweight individuals are at an increased risk of developing fatty liver with higher amounts of WAT . Fat accUcp1, Ucp3, and peroxisome proliferator-activated receptor gamma (Pparg) was assessed via RT-PCR. BAT plays an important role in the regulation of energy balance by thermogenesis-specific genes, including UCP-1 and UCP-3 [Ucp1 and Ucp3 in BAT was significantly increased in the HFD + ST (100 and 300 mg/kg) groups compared to the HFD group (G. cambogia extract and orlistat) increased Ucp1 and Ucp3 mRNA expression in BAT compared to the HFD group. These results suggest that ST significantly increased energy consumption via thermogenesis during energy homeostasis in BAT. In addition, Pparg mRNA expression (an adipogenesis gene) was significantly decreased in the WAT of the HFD + ST (100 and 300 mg/kg) treated groups compared to the HFD group. These results further suggest that ST may reduce the risk of obesity by upregulating the expression of Ucp1 and Ucp3 in BAT while downregulating Pparg in WAT.The mitochondrial uncoupling protein 1 (UCP-1), and uncoupling protein 3 (UCP-3) play important roles in regulating energy expenditure. Specifically, activation of UCP1 and UCP3 leads to regulation of thermogenesis in brown adipose tissues (BAT) and inhibits fat accumulation by increasing energy consumption. Hence, we assessed the effect of ST on adipogenesis- and thermogenesis-related gene expression. Specifically, the mRNA expression of nd UCP-3 ,25. ResuFD group . In addiObesity is characterized by increased fat accumulation throughout the body and can lead to various complications, including increased risk of type 2 diabetes and hyperglycemia. HFD can cause metabolic syndrome, including obesity, type 2 diabetes, and hypertension ,26,27,28Obesity is also known to induce high levels of hematological parameters related to cardiovascular diseases, such as triglycerides and cholesterols in human serum ,30. OralOur results indicated that ST might reduce the risk of obesity by upregulating lipolysis genes in BAT and downregulating the expression of lipogenesis genes in WAT. Taken together, these results suggest that ST may exert an anti-obesity effect by inhibiting fat accumulation in HFD-induced obese mice, and that it may be effective in functional food products for the treatment or prevention of obesity.


ANK2), originally identified as a key cytoskeletal-associated protein in the brain, is highly expressed in the heart and plays critical roles in cardiac physiology and cell biology. In the heart, ankyrin-B plays key roles in the targeting and localization of key ion channels and transporters, structural proteins, and signaling molecules. The role of ankyrin-B in normal cardiac function is illustrated in animal models lacking ankyrin-B expression, which display significant electrical and structural phenotypes and life-threatening arrhythmias. Further, ankyrin-B dysfunction has been associated with cardiac phenotypes in humans (now referred to as “ankyrin-B syndrome”) including sinus node dysfunction, heart rate variability, atrial fibrillation, conduction block, arrhythmogenic cardiomyopathy, structural remodeling, and sudden cardiac death. Here, we review the diverse roles of ankyrin-B in the vertebrate heart with a significant focus on ankyrin-B-linked cell- and molecular-pathways and disease.Ankyrin-B (encoded by  ANK1, ANK2, and ANK3 [ANK1 encodes ankyrin-R (AnkR), ANK2 encodes ankyrin-B (AnkB), and ANK3 encodes ankyrin-G (AnkG)  .ANK1 have been linked to ~50% of hereditary spherocytosis cases [+/K+ ATPase (NKA) [ANK1 also encodes four small ankyrin-1 isoforms  that are highly expressed in striated muscle. These isoforms bind obscurin and stabilize the sarcoplasmic reticulum in striated muscle [Ankyrin-R is primarily expressed in erythrocytes and was the first ankyrin identified as an adaptor protein , linkingis cases , a complis cases  and resuis cases . AnkR reis cases , Na+/K+ se (NKA) , and these (NKA) . Althougse (NKA) . ANK1 ald muscle .ANK2 encodes multiple isoforms that may contribute to disease. AnkB will be reviewed in detail below.Ankyrin-B was first identified in the brain  and has V1.6, βIV spectrin, and L1CAMs [V1.5 and CaMKII to the cardiomyocyte intercalated disc [V1.5 expression and localization are disrupted, and voltage-gated NaV channel activity (INa) is significantly decreased. These animals experience a reduction in heart rate, impaired atrioventricular conduction, increased PR intervals, and increased QRS intervals [SCN5A variant in the AnkG-binding motif of NaV1.5 has been associated with Brugada syndrome and arrhythmia [ANK3 encodes multiple isoforms of AnkG. Giant AnkG is a 480-kD protein required for proper AIS and node of Ranvier assembly due to the clustering of NaV channels [Ankyrin-G plays an important role across multiple excitable tissues. In the brain, AnkG links integral membrane proteins with the actin/spectrin-based membrane skeleton at axon initial segments (AIS) including Nad L1CAMs ,22,33. Ited disc ,14,34. Irhythmia . This sachannels . Human vchannels . The rolV1.5 [+/Ca2+ exchanger (NCX) and Na+/K+-ATPase (NKA) [ANK2 variants who presented with arrhythmogenic right ventricular cardiomyopathy (ARVC) [AnkB and AnkG are ubiquitously expressed, but their functions are distinct. Although AnkG plays a crucial role in the brain, variants in AnkG have been connected to Brugada syndrome  and, morV1.5 , while AV1.5 . HoweverV1.5 . Cardiomse (NKA) . Furthery (ARVC) . Importay (ARVC)  and heary (ARVC) . Ankyriny (ARVC) . FurtherANK1 and ANK3, ANK2 generates multiple gene products. The diversity of known and potential gene products is large and includes splice products defined as small, canonical, and Giant AnkB isoforms [Similar to isoforms . In factAnkB-188 and AnkB-212 are two ankyrin-B isoforms present in the heart . AnkB-18+ channel (Kir6.2) [V1.3) [Canonical AnkB is an adaptor protein that acts as a pivotal regulator in the localization and organization of ion channels, structural proteins, signaling molecules, and adaptor proteins ,41. Defi(Kir6.2) , voltage) [V1.3) , and βII) [V1.3) , causing) [V1.3) .+/− myocytes display reduced expression and improper localization of AnkB-binding partners, as well as strong electrical phenotypes including extrasystoles, early afterdepolarizations (EADs), and delayed afterdepolarizations (DADs) following adrenergic stimulation. The mechanism underlying AnkB-associated arrhythmias is attributed to improper calcium handling via disrupted organization of AnkB-binding partners [+/− cardiomyocytes disturbs Ca2+ and Na+ currents to predispose the cell to action potential prolongation [Animal models have been fundamental in illustrating the role of AnkB in the heart. AnkBpartners . Throughongation .ANK repeats, which are defined by their repeated alpha-helical structure [ANK repeats are not specific to the three canonical ankyrin proteins and are present across a range of functionally diverse proteins including SHANK, BARD1, and ANKRD. The AnkB MBD regulates the localization of ion channels and transporters  that is comprised of a death domain (DD) and C-terminal domain (CTD) . The MBDtructure . ANK repsporters .N-ZU5C-UPA tandem structure. The ZU5N domain binds directly with the spectrin cytoskeleton to aid in the structural integrity of the myocyte [C region of the AnkB-SBD associates with the B56α subunit of protein phosphatase 2A (PP2A), an interaction that localizes PP2A with a primary target, RyR2 [ANK2 p.Q1283H variant within ZU5c was recently associated with arrhythmia, potentially due to loss of PP2A activity and altered phosphorylation of RyR2 [The SBD is a highly conserved region of most ankyrin proteins, where the AnkB SBD is composed of a ZU5 myocyte . Criticaet, RyR2 ,13. The  of RyR2 .The AnkB DD, composed of six helices that resemble the death domain of apoptotic-related proteins, is proposed to play a role in the auto-inhibitory functions of AnkB through the binding of the DD to the UPA region of SBD . AdditioANK repeat 1 within the MBD and the three amino acid sequence EED within the CTD are shown to facilitate inter-domain binding [Finally, the AnkB CTD is characterized by its elongated structure and its high composition of charged amino acids . It is h binding . AbolishANK2. A p.E1425G variant was discovered in a French family suffering from sinus bradycardia, atrial fibrillation, and sudden cardiac death and was the first to be implicated in AnkB syndrome [AnkB variants have been identified in all four domains of the AnkB protein and are linked to a spectrum of cardiovascular phenotypes. AnkB is classically associated with human arrhythmia syndromes, many of which demonstrate incomplete penetrance and variable expressivity ,50,51,52syndrome ,53.2+ overload, and possible disruption of pacemaking activity [Although the p.E1425G variant is localized to the AnkB regulatory domain, loss-of-function variants in all four ankyrin domains are now associated with AnkB syndrome ,54. Notaactivity ,36.N region of the SBD that directly binds βII-spectrin, disrupting this interaction. Importantly, AnkB co-immunoprecipitates with a larger complex composed of βII-spectrin, NKA, and NCX [C region disrupts PP2A activity via loss of B56α targeting, which increases RyR2 phosphorylation and disrupts the calcium dynamics associated with excitation-contraction (EC) coupling [Variants within the SBD pose distinct mechanisms of disease. Several AnkB variants including the p.R990Q , p.A1000 and NCX . Althoug and NCX . Notablycoupling . NotablyANK2 have also been associated with sinus node disease (SND) [ANK2 loss of function mutations, ANK2 transection in chromosome 4, leading to ANK2 haploinsufficiency, was associated with ankyrin-B syndrome [Variants in se (SND)  and mostse (SND) . In factse (SND) . Similarsyndrome .ANK2 have also been associated with structural heart disease. Lopes et al. found that individuals with ANK2 variants had a greater maximum wall thickness in the left ventricle, in hypertrophic cardiomyopathy [Although AnkB is commonly associated with arrhythmias, variants in myopathy . AnkB hamyopathy . Followimyopathy .ANK2 variants have been identified in individuals with arrhythmogenic right ventricular cardiomyopathy (ARVC) [Recently, y (ARVC) , a diseay (ARVC) . The Anky (ARVC) . A largey (ARVC) . In ordey (ARVC) . Althougy (ARVC) , providiANK2 variants are associated with cardiovascular phenotypes including sinus node disease, atrial fibrillation, heart rate variability, catecholaminergic polymorphic ventricular tachycardia (CPVT), ACM, cardiomyopathy, syncope, and sudden cardiac death. We strongly predict that disease penetrance and severity will ultimately be predicated by the interaction between genetic  and environmental factors. A key future area of research is to understand the impact of secondary variants and acquired/environmental factors  on ankyrin-B stability and disease.Despite their variability, the majority of treatment options for AnkB-associated conditions continue to mitigate the clinical symptoms of disease as opposed to their molecular causes. Currently, SND, ARVC, and AnkB syndrome necessitate symptom-dependent approaches ranging from β-blocker administration to pacemaker implantation ,37,56. OThe clinical manifestations of AnkB-attributed pathologies are also intriguingly patient specific. For example, cases of ARVC and AnkB syndrome, which comprise disparate structural and electrical phenotypes, have both been linked to the AnkB E1425G variant . This geIn summary, AnkB is critical for the expression, targeting, and regulation of multiple proteins involved in cardiac excitability, structure, and signaling. AnkB is a pivotal regulator of ion channels and transporters , structural proteins , and calcium regulatory proteins  . Togethe


Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.The retinal pigment epithelial (RPE) cell monolayer forms the outer blood–retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines  and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs  across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow P The retinal pigment epithelium (RPE) located in the posterior eye between the neural retina and choroidal circulation is essential for vision . The RPEPermeation across the RPE cell layer needs to be investigated when the ocular pharmacokinetics of new chemical entities is evaluated. The rabbit has been the most commonly used species in ocular pharmacokinetic studies ,6; howevRPE cells cultured on filter supports provide an alternative to isolated tissue sheets in permeation experiments. Several RPE cell models are available, with each having their own strengths and weaknesses. Human primary RPE cells are widely used in retinal biology studies, but they are rarely used in drug discovery. Due to the poor availability of adult human RPE cells, human fetal RPE cells (hfRPE) have been the most commonly used human primary RPE cell model. However, drug permeation across the primary RPE cells has not been reported.The continuously growing human RPE cell line, ARPE19 , is the We have recently established two novel RPE cell models: ARPE19mel  and LEPIIn addition to clinical cell replacement therapy applications , the RPE7.4 values ranging from −5.1 to 1.92) across ARPE19, ARPE19mel, LEPI, hfRPE, and hESC-RPE cell monolayers. Importantly, in this study, we compared the obtained apparent permeation coefficients (Papp) to the values recently obtained in our laboratory in an ex vivo bovine RPE-choroid model [In summary, several RPE cell models are widely used in different fields of retinal research, but their barrier properties focusing on drug permeation have not been systematically compared within the same laboratory. In this paper, we present the permeation characteristics of nine small molecular-weight drugs with varying lipophilicities  and LEPI cells [2 polyester filters with a 0.4 µm pore size  at 160,000 cells/cm2, and cultured for 4 weeks in DMEM-F12 supplemented with 1% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine, before the experiments.PI cells  were maiARPE19mel cells. We have recently published a detailed description and characterization of re-pigmented ARPE19mel cells [2 1420 Multilabel counter, Perkin Elmer, MA, USA) utilizing isolated porcine RPE melanin standards (0.00–2.00 µg melanin/µL) [el cells . In the el cells  were addel cells . The melanin/µL) ,27. SimihfRPE cells. The hfRPE cells were purchased from ScienCell . The cells were expanded as reported previously [2) onto 1.12 cm2 Corning Transwell polyester inserts  at passage 3. The cells were cultured in EpiCM medium for 2 weeks before the permeation experiments . We have previously provided a detailed characterization of these cells [eviously , and seese cells .2, with the medium being replaced three times a week.All the cells were cultured at +37 °C in 5% COhESC-RPE cells. The hESC-RPE cells were differentiated from two genetically distinct hESC lines: Regea08/017 and Regea08/023. The derivation and characterization of the lines have been reported previously in [® Costar® Ultra-Low attachment plates  in RPEbasic medium containing KnockOut™ Dulbecco’s modified Eagle’s Medium supplemented with 15% KnockOut™ Serum Replacement, 2 mM GlutaMAX™, 1% MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, and 50 U/mL penicillin-streptomycin . Embryoid body (EB) formation was induced by overnight induction with 10 μM blebbistatin . For the following 2 days, EBs were allowed to undergo spontaneous differentiation in RPEbasic medium and on day 4, the EBs were plated down to 0.75 μg/cm2 LN-521 and 10 μg/cm2 human placental collagen type IV  coating in RPEbasic medium. The medium was subsequently changed three times each week. After 30–45 days of differentiation, pigmented foci were selected with a scalpel, dissociated with Tryple, and re-plated in culture wells coated with LN-521 and col IV (RPE passage 1). Forty-five days later, the hESC-RPE was again re-plated (RPE passage 2) and then a further 9 days later, the hESC-RPE cells were frozen and subsequently thawed to final culture passage 3 in Corning® Matrigel® Matrix-coated  PET inserts with a 1 µm pore size (cat. 83.3932.101 Sarstedt), with 250,000 cells/cm2. The hESC-RPE cells were cultured for 62 days prior to the permeation studies. The success of hESC-RPE cell differentiation was confirmed in detailed experiments; the RPE-specific properties of the differentiated cells are presented in ously in . Human ETampere University has the approval of the National Supervisory Authority for Welfare and Health  to conduct research on human embryos. Tampere University also has supportive statements from the Ethical Committee of the Pirkanmaa Hospital District to derive, culture, and differentiate hESC lines (Skottman/R05116). No new hESC lines were derived in this study.2 filters and 0.35 mL in the basolateral and 0.1 mL in the apical compartment in the case of 0.3 cm2 filters.The permeation characteristics of aztreonam, ciprofloxacin, fluconazole, ganciclovir, ketorolac, methotrexate, quinidine, and voriconazole  were evaapp in cm/s) was determined with Equation (1) below:0 (ng/cm3) is the exposure concentration in the donor compartment, and A is the area (cm2) of the cell monolayer. The equation was valid if sink conditions were maintained, i.e., the receiver compartment concentration was below 10% of the concentration in the donor compartment.The apparent permeation coefficient :The drug concentrations were determined with a liquid chromatograph  and a triple-quadrupole mass spectrometer  utilizing electrospray ionization in the positive ionization mode. We have previously described the method development and validation in detail ,30, and app values, efflux ratios, and flux profiles are presented in detail in The Papp values was observed in LEPI (0.4–29 × 10−6 cm/s) and hESC-RPE (0.4–32 × 10−6 cm/s) cells, similar to what was previously found in the bovine RPE-choroid that displayed Papp values for these drugs ranging from 1.4 to 69.2 × 10−6 cm/s , had Papp values in a similar range in all cells  and voriconazole (logD7.4 1.21), were similar in all cell models (7.4 −1.61) displayed 20-fold differences in its Papp values , ciprofloxacin (3.9), ganciclovir (2.7), ketorolac (3.1), and methotrexate (3.0) across LEPI cells, i.e., evidence for a preference for the apical-to-basolateral (outward) direction .Compounds with a high affinity for melanin, i.e., ciprofloxacin and quinidine, displayed lag times of 100 and 200 min, respectively, in their permeation across hESC-RPE cells in the inward direction A,B. In tapp values. An overview of the cell model properties is presented in We performed a quantitative and systematic comparison of RPE cell model barrier functions by investigating drug flux across the cell monolayers of ARPE19, ARPE19mel, hfRPE, LEPI, and hESC-RPE cells. Our results clearly indicate that the hESC-RPE and LEPI cells restrict the drug permeation to a similar extent to that encountered in the ex vivo RPE model (bovine RPE-choroid), whereas ARPE19, ARPE19mel, and hfRPE cells display a leaky barrier, as indicated by the rapid drug flux and high Papp values displayed a wide range: there was a 5-fold difference between the highly permeating lipophilic voriconazole and the slowly permeating hydrophilic aztreonam [app values of hESC-RPE and LEPI cells, whereas the range of hfRPE, ARPE19, and ARPE19mel cells was narrow , the cell line displayed a 7.6 times higher P-choroid . However-choroid , suggest-choroid ,32,33; h-choroid . We obse-choroid ,34. In m-choroid . Simple -choroid .app values had a wide range and the cells restricted drug flux more efficiently than bovine RPE-choroid  ,23,24. T-choroid . The ARPapp values in ARPE19 models and hfRPE cells  displayed the highest permeation rates across bovine RPE-choroid, hESC-RPE, and LEPI cells, with similar PPE cells . This inapp of methotrexate was 11.4-fold higher in the basolateral-to-apical direction across bovine RPE-choroid compared to hESC-RPE and LEPI, and the corresponding difference in ganciclovir Papp values was 6.6–7.4-fold, respectively. The poor permeation of ganciclovir and methotrexate can be explained by their physicochemical properties: hydrophilic compounds are not able to cross the cell monolayer transcellularly and tight junctions restrict the paracellular permeation. However, low permeation in both directions might partly be a consequence of active efflux on both sides of the RPE, and the minor directional differences can be caused by slight variations in the active transport rates. Ganciclovir is a substrate of MATE1 and methotrexate is a substrate of several transporter proteins, including MRP1, MRP4, and MRP5. Similarly, hydrophilic ketorolac is a substrate for OAT2 and both species differences between humans and cows and possible differences among the protein expression in human cell models can cause variation in the permeation, even between tight RPE models. All of the listed transporters have been detected at the protein level in RPE cell cultures [7.4) and both intravitreal clearance [Directional permeation can be an indication of active transport, and efflux ratios outside the range of 0.5–2 point to the involvement of active transport . We obsecultures ,37. A recultures . Passivelearance . In addilearance . Our datapp values, but the linear phase in the drug flux across pigmented cells can be delayed in the case of melanin-bound compounds [app values are not dependent on the extent of pigmentation (Differences in pigmentation are not expected to affect the compounds’ Pompounds . This isompounds , and theompounds , which compounds ,9. Sinceompounds , are simentation A,B. As pPermeation across the RPE is an important parameter in ocular pharmacokinetics and thus needs to be screened during early drug development. In addition, realistic permeation values are crucial in in silico pharmacokinetic models. In this paper, we showed that hESC-RPE and LEPI cells display appropriate barrier properties against drug flux and are therefore valuable in vitro models in permeation experiments. Similar to Caco-2 cells and intestinal tissue , these RIn conclusion, our results reveal clear differences in the barrier function of RPE cell models. While useful in drug uptake studies, ARPE19-based models and hfRPE cells do not possess an appropriate barrier to restrict drug flux across the monolayer, whereas hESC-RPE and LEPI cells form a tight barrier comparable to bovine RPE-choroid. This difference was seen most clearly with hydrophilic compounds. The advantage of hESC-RPE cells is that they display lag-times with pigment-binding compounds due to the heavy pigmentation of these cells; however, their maintenance and differentiation are demanding and time-consuming. LEPI cells, on the other hand, are easily maintained and can be rapidly expanded and differentiated, making them an advantageous model for the outer blood–retinal barrier in assays requiring large numbers of cells, although they lack pigments. Overall, LEPI and hESC-RPE are clearly suitable outer blood–retinal barrier models for evaluating the permeation of small molecular-weight compounds.


CDKN2a locus, is one of the most frequently mutated or deleted tumor suppressors in human cancer. The safeguard roles of ARF in tumorigenesis are mainly mediated via the MDM2-p53 axis, which plays a prominent role in tumor suppression. Under normal conditions, low p53 expression is stringently regulated by its target gene, MDM2 E3 ligase, which induces p53 degradation in a ubiquitin-proteasome-dependent manner. Oncogenic signals induced by MYC, RAS, and E2Fs trap MDM2 in the inhibited state by inducing ARF expression as a safeguard measure, thereby activating the tumor-suppressive function of p53. In addition to the MDM2-p53 axis, ARF can also interact with diverse proteins and regulate various cellular functions, such as cellular senescence, apoptosis, and anoikis, in a p53-independent manner. As the evidence indicating ARF as a key tumor suppressor has been accumulated, there is growing evidence that ARF is sophisticatedly fine-tuned by the diverse factors through transcriptional and post-translational regulatory mechanisms. In this review, we mainly focused on how cancer cells employ transcriptional and post-translational regulatory mechanisms to manipulate ARF activities to circumvent the tumor-suppressive function of ARF. We further discussed the clinical implications of ARF in human cancer.Tumorigenesis can be induced by various stresses that cause aberrant DNA mutations and unhindered cell proliferation. Under such conditions, normal cells autonomously induce defense mechanisms, thereby stimulating tumor suppressor activation. ARF, encoded by the  CDKN2a) locus—frequently mutated or deleted in human cancer—encodes two different tumor suppressors, INK4a (referred to as p16INK4a) and ARF (referred to as p14ARF in humans and p19ARF in mice) [The cyclin-dependent kinase inhibitor 2A (in mice) ,4  via direct interaction, thereby blocking direct physical interaction of MDM2 with p53 in the cytosol and nucleus. As MDM2 is the E3 ligase for p53, ARF-induced MDM2 sequestration and inhibition prevents ubiquitination and proteasome-dependent degradation of p53, resulting in p53 stabilization ,10. p53 chondria . Althougull mice . Structuull mice ,24,25.However, based on these observations, it was deduced that ARF formed a complex with numerous proteins to neutralize its charge and stabilize its spatial conformation. The importance of ARF in protection from aberrant tumor cell development has been further understood due to recent knowledge regarding the transcriptional and post-translational regulation of ARF by the newly revealed ARF regulators. This review focused on the newly extended regulatory network of ARF and its therapeutic implications in cancer.The paradoxical nature of oncogene-induced ARF expression has drawn attention to the search for ARF transcriptional factors . While lp53-null and wild type mouse embryonic fibroblasts (MEFs), suggesting that ARF might have p53-independent roles. Indeed, Arf-null cells showed resistance to MYC-induced apoptosis, indicating that ARF induction by MYC expression could be an important checkpoint in preventing aberrant cell proliferation through the activation of the fail-safe program [E2F3b-deficient cells, leading to p53 activation and triggering of G0/G1 cell cycle arrest [MYC is an oncoprotein that regulates cell proliferation, differentiation, inflammation, and metabolism by regulating the transcription of various target genes . To avoi program . As E2F  program ,45. The  program . The E2F program . On the  program . E2F3b dDmp1-null MEFs show a delay in ARF accumulation when cells are sub-cultured and grow faster than wild type MEFs. H-RasV12 expression results in tumorigenic phenotypes, and the delay in replicative senescence of Dmp1-null MEFs have been observed, suggesting that DMP1 functions as a tumor suppressor by increasing ARF expression [DMP1, also referred to as cyclin D-binding Myb-like protein, can also induce cell growth arrest by inducing ARF expression ,48. Threpression . In contpression . DMP1β, pression .FoxO is a subtype of the forkhead-box transcription factor, and has been identified as an ARF transcription factor . LymphomArf-deficient embryos, Tgfβ2-deficient embryos showed eyes with a hyperplasia phenotype and low ARF expression and acidic β-galactosidase on embryonic day 13.5, implying that TGFβ2 might function as a positive regulator of ARF transcription during embryogenesis [ARF locus in a TGFβ2-dependent manner, and inducing histone acetylation and RNA pol II recruitment. This in turn promotes ARF promoter remodeling and activation [Although ARF transcriptional regulation under conditions of oncogenic stresses has drawn significant attention, ARF regulation is an important process in embryogenesis ,51,52. Sogenesis . Smad 2 tivation . Unlike tivation . In mutaBmi-1-deficient cells showed cellular senescence phenotypes, including decreased cell proliferation, defective S-phase cell cycle, cytoplasmic enlargement, unresponsiveness to growth factors, and increased acidic β-galactosidase, indicating that Bmi-1 deficiency was closely related to cellular senescence [Bmi-1-deficient cells, ARF and INK4a were upregulated. In the Bmi-1-deficient mouse model, a significantly small size and an ataxia phenotype were observed, and these phenotypes could be restored by intercrossing the BMI-1-deficient mice with Ink4a/Arf locus-deficient mice [The polycomb group transcription factor, BMI-1, was identified as an oncogene that cooperates with MYC in mouse lymphomas ,54,55,56nescence . While tent mice .Cbx7-deficient cells showed severe growth arrest and high ARF and INK4a levels, thereby indicating that CBX7 and senescence suppressed each other. CBX7-induced suppression of ARF and INK4a expression occurs in a BMI-1-independent manner, suggesting that CBX7 may repress ARF and INK4a transcription by interacting with another polycomb-repressive complex-1 (PRC1) subset, but not with BMI-1.Another polycomb group protein, CBX7, has been identified as a transcriptional repressor of ARF . CBX7 exPolycomb-repressive complex-2 (PRC2) represses ARF transcription by associating with Twist-1 . Twist-1Bmi-1-deficient cells was reduced upon TBX2 expression without any change in INK4a, suggesting that TBX2 specifically inhibits ARF expression.Through senescence bypass library screening, TBX2 has been identified as a transcriptional suppressor of ARF . TBX2 prEGFR, a membrane-bound receptor tyrosine kinase, serves as a mediator of proliferative signaling by activating diverse downstream components . SuppresThe control of ARF expression at the transcriptional level has been sufficiently described; accumulating evidence regarding the post-translational regulation of ARF suggests that oncogenic stress-mediated ARF induction may be associated with molecular mechanisms that regulate protein levels and functions. In particular, the involvement of phosphorylation, ubiquitination, and proteasome- and lysosome-dependent pathways in the post-translational regulation of ARF has recently been observed.Phosphorylation is one of the well-known post-translational modifications involved in numerous cellular signaling pathways. Serine, threonine, or tyrosine residues of proteins are commonly phosphorylated by kinases, which are enzymes that add the phosphate group onto the target amino acids via esterification reactions . PhosphoDespite the important roles of phosphorylation in protein activity regulation, only one phosphorylation site has been identified in ARF . Inoue eUbiquitination is an important cellular process in which small molecules called ubiquitins are added to the target proteins; it occurs in three steps, requiring three enzymes, i.e., E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Ubiquitin forms a thioester bond with E1 in an ATP-dependent manner, resulting in the initial activation of ubiquitin. Then, ubiquitin is transferred to E2. E2-conjugated ubiquitins are transferred to the target proteins by E3 ligases; this is the rate-determining step of ubiquitination . More thAs human ARF does not contain any lysine residue, studies on ARF ubiquitination mainly focused on exploring the possibility of N-terminal ubiquitination of ARF . In humaV12 in cells, increased TRADD expression, followed by ARF stabilization is observed, resulting in the promotion of OIS. Nucleostemin (NS) is a GTPase that is localized in the nucleolus and is involved in overall protein synthesis [ULF, also referred to as thyroid hormone receptor interactor 12, has been identified as the first E3 ligase for ARF . ULF depynthesis . NS stabynthesis . GLTSCR2ARF) in response to serum stimulation, thereby promoting cell proliferation via growth factor signaling [ARF levels and suppressed cell proliferation. β-TrCP2-induced p19ARF ubiquitination and degradation require phosphorylation at serine 75. This is performed by ribosomal protein S6 kinase beta-1 (S6K1), which is activated in response to growth factor- or nutrient-dependent mammalian target of rapamycin (mTOR) signaling. As only p19ARF—but not p14ARF or other species—contains the β-TrCP2 and S6K1 degron, the regulation of p19ARF via the mTOR-S6K1-β-TrCP2 axis was restricted in the mouse model.MKRN1 is an E3 ligase that regulates diverse cellular signaling pathways by leading to the ubiquitin-mediated proteasome-dependent degradation of target proteins. Ko et al. (2012) reported that MKRN1 ubiquitinates ARF, thereby leading to the proteasome-dependent degradation of ARF . MKRN1 dignaling . β-TrCP2Recently, deubiquitinating enzymes (DUBs), E3 ligase counter enzymes that remove ubiquitin molecules from the target proteins by cleaving the thioester bonds of ubiquitins, have been reported to regulate ARF. USP10 has been identified as the first direct DUB for ARF . USP10 iSubstrates containing the HSC70 canonical binding motif are recognized by a chaperone-co-chaperone complex, and are then bound by lysosome associated membrane glycoprotein 2 (LAMP2A) . InteracRecently, Han et al. (2017) reported that HSP90, a molecular chaperone, regulated ARF turnover via CMA . Treatme2-kinase (JNK) pathway-dependent manner [NPM  is a multifunctional protein that controls a variety of cellular phenomena, including proliferation, genomic stability maintenance, cell death, rRNA processing, and ribosome biogenesis . It is at manner . c-Jun it manner . Under nt manner . AKT intIn addition to NPM, several other ARF binding partners have been reported as regulators of ARF localization and stability. TBP-1 has been identified as an ARF stabilizer . TBP-1, ARF-null mice generated by specifically targeting exon 1β in the CDKN2a locus spontaneously develop numerous tumors, including sarcomas, lymphomas, and lung carcinomas, resulting in death within one year [ARF-knockout mice using carcinogens, such as dimethylbenz(a)anthracene (DMBA) or X-ray, promotes tumor progression, leading to a short life span of six months with drastic tumor development. Epigenetic modifications on ARF promoters have been identified in patients with a wide spectrum of tumors. As the ARF promoter contains a CpG island, ARF silencing by hypermethylation of these promoter sequences has frequently been reported in various human cancers, such as breast, bladder, colon, liver, gastric, lung, oral, prostate, and brain cancer [CDKN2a locus has also been frequently reported in numerous cancers, including breast, bladder, liver, lung, oral, prostate, and kidney cancer [Over the last three decades, many mechanistic and clinical observations have indicated a close relationship between ARF and tumor progression in animal and human cancer models. one year ,93,94. Tn cancer ,109,110.y cancer ,112. Fury cancer ,115,116.CDKN2a locus have been well characterized in diverse cancers; the uncoupling of ARF mRNA and protein expression has also been observed in lung cancer [CDKN2a locus is found in non-small cell lung cancer (NSCLC), implying that post-translational regulation of ARF may be involved in cancer development (CDKN2a locus aberration. High HSP90 and CHIP expression, with reduced ARF expression, has also been identified in NSCLC [Genetic and epigenetic modifications in the g cancer ,118. Lowelopment . In maliin NSCLC ; high HSin NSCLC . USP10 ain NSCLC ,120.Since its discovery, ARF regulation has been extensively studied because of its importance in the determination of cell fate in response to oncogenic signals. ARF induces cellular senescence, cell cycle arrest, and apoptosis via p53-dependent or -independent pathways in response to oncogenic stress, which activates the fail-safe program ,4. When Numerous transcription factors have been identified as transcriptional regulators of ARF, and their clinical implications in cancer have been reported. However, the uncoupling of ARF mRNA and protein expression in human cancer remains unclear. For a decade, the molecular mechanisms underlying post-translational regulation of ARF have been suggested as key events in maintaining the balance of ARF expression in response to various signaling pathways. Most post-translational regulatory mechanisms control the nucleolar, nuclear, and cytoplasmic localizations of ARF, leading to stabilization/destabilization. In particular, PKCα-induced ARF phosphorylation leads to cytoplasmic localization and a change in the functional role of ARF . PhosphoCDKN2a locus. However, these modifications do not explain why several cancer patients show low ARF expression without CDKN2a locus aberration. As described above, diverse factors are involved in the post-translational regulation of ARF, demonstrating meaningful clinical implications in cancer patients. Targeting the fine-tuned post-translational regulation of ARF would enable the development of independent prognostic markers and therapeutic strategies for various cancers.Many cancer patients show genetic and epigenetic modifications in the


Enterobacteriaceae, carrying the transferable colistin resistance gene mcr-1, has recently provoked a global health concern. This report presents the first detection of a hydrogen sulfide (H2S)-producing Escherichia coli variant isolated from a human in China, with multidrug resistance (MDR) properties, including colistin resistance by the mcr-1 gene, which could have great implications for the treatment of human infections.Colistin is considered to be a ‘last-resort’ antimicrobial for the treatment of multidrug-resistant Gram-negative bacterial infections. Identification of  Escherichia coli is a significant cause of diseases in animals and humans worldwide [2S) test, are important for identification of the Enterobacteriaceae species. The production of H2S, however, is not a typical characteristic of E. coli, though the H2S-producing variants of E. coli have also been reported previously [2S through orthologous enzymes, and recent studies have implicated H2S as a significant signaling molecule by protecting the bacteria from antibiotic-induced damage [2S can also prevent oxidative damage through stimulation of superoxide dismutase (SOD) and catalase activities [2S can also control the expression of Staphylococcus aureus virulence genes [2S-producing E. coli isolated from the fecal sample from a clinically healthy patient in China.orldwide , resultieviously . Bacterid damage . H2S cantivities . Recent ce genes . In thisSalmonella in the human fecal samples; we suspected this sample as Salmonella, and found this isolate was a lactose fermenter and H2S producer, according to a previous protocol [Salmonella or E. coli, we plated the sample on eosin methylene blue agar, and then confirmed the results with PCR identification and whole genome sequencing. Together, this is one isolate of interest, H2S-producing E. coli isolated from a 32-year old female from Guangxi province, China, during occupational health examination in 2015.An active epidemiological surveillance study for foodborne pathogens was conducted towards healthy and diarrheal patients in Guangxi province, China. The initial aim was to screen protocol . To confhttps://cge.cbs.dtu.dk/services/). We used the virulence factor database (VFDB) to obtain the virulence genes in this H2S-positive E. coli isolate. We performed antimicrobial susceptibility testing of the E. coli isolate using the broth microdilution method, as per the Clinical and Laboratory Standards Institute (CLSI) criteria [The isolate was sequenced using the MiSeq platform , utilizing either 500 or 600 cycles of paired-end reads. The de novo assembly, using SPAdes 3.6, resulted in a genome size of 493,599 bp with GC content of 52.1%. The genome was annotated using the Rapid Annotation using Subsystem Technology (RAST) annotation server, and 1730 coding sequences (CDS) were identified. Detection of resistance genes and multilocus sequence typing (MLST) was accomplished at the Center for Genomic Epidemiology (CGE) (2S-producing E. coli belonged to sequence type (ST) 10, serotype O10:H19, fimH25-fumC11 type. The typical virulence genes found in this E. coli isolate are shown in 2S-positive E. coli isolate for susceptibility to different antibiotics revealed that this H2S-positive variant was resistant to aminoglycosides, β-Lactams, polymyxins, fluoroquinolones, phenicols, sulfonamides, tetracyclines, and trimethoprim. Genome analysis revealed that this isolate also carried 3-mercaptopyruvate sulfurtransferase (sseA), indicating for the H2S production [sseA and the mcr gene were on the chromosome. 2S as a microbial defense mechanism, revealing resistance against different clinically relevant antibiotics, including the ‘last-line’ therapeutic drug colistin, and also suggests the need of bacterial H2S inhibition in the treatment of infections caused by E. coli. The first extensive study of H2S-positive E. coli strains was found in Denmark [2S-generating enzymes (sseA in E. coli), especially, as mentioned, provided defense against antimicrobial compounds only in aerobic conditions [2S is a universal defense mechanism found in bacteria as well as in mammals [E. coli strain is one of the predominant STs in the world [We found Hoduction , and mulnditions . The int mammals . Moreovehe world .aadA1, aadA2, trimethoprim resistance gene dfrA12, β-Lactams resistance gene blaTEM-1B, polymyxins resistance gene mcr-1, fluoroquinolones resistance genes oqxA, oqxB, phenicols resistance genes floR, cmlA1, sulfonamides resistance gene sul3, and tetracyclines resistance gene tet(A) in the H2S-positive E. coli isolate. The isolate was susceptible to carbapenems and cephalosporins (2S-producing variant of Escherichia coli isolated from a urinary tract infection (UTI) was also found to be resistant to different clinically relevant antibiotics. A previous study by Bailey et al. [dfrA12, sul3, tet(A), and cmlA1, including other AR genes in E. coli of healthy adults. It is interesting that E. coli of healthy humans represented a significant reservoir for several AR genes, as found in our study. The presence of aadA1, aadA2, and dfrA12 genes were also reported previously in E. coli isolated from clinical samples in Malaysia [E. coli carrying the mcr-1-gene in China in 2016 [mcr gene and their variants has been reported in the E.coli across different continents. The bacterial cell membrane is the initial site of action for colistin. Colistin binds to lipopolysaccharide (LPSs) and phospholipids in the outer cell membrane of Gram-negative bacteria [mcr-1 gene has raised concerns during the last few years [E. coli. However, the extensive use of fluoroquinolones has led to increasing fluoroquinolone resistance. The genes for multidrug efflux pump OqxAB, which are active on fluoroquinolones, were found for the first time in clinical isolates on a plasmid in E. coli in the USA in 2009 [oqxA and oqxB, including other genes in clinical isolates of E. coli, obtained from UTIs in Azerbaijan and Iran [blaTEM-1B and tet(A) were found, including other AR genes in E. coli isolated from a patient in Lebanon, and linked to a bloodstream infection. Interestingly, previous studies reported that among sul1, sul2, and sul3 genes responsible for sulfonamide resistance, both sul1 and sul2 are highly prevalent, and sul3 has rarely been found [sul3, could be an interesting characteristic of this H2S-producing E. coli strain. Antibiotic resistance genes found in this study were also reported previously in E. coli isolated from humans in various studies in Australia [We found aminoglycosides resistance genes osporins . The stuMalaysia . Since t in 2016 , the exiew years ,14. Fluo in 2009 . A recenen found ,18. Therustralia , Argentiustralia , Croatiaustralia , Sweden ustralia , Spain [ustralia , Boliviaustralia , Algeriaustralia , Nigeriaustralia , and Litustralia .2S-producing colistin-resistant E. coli carrying the mcr-1 gene, which also possesses the rare sulfonamide resistance gene sul3. The emergence and spread of the colistin resistance gene mcr-1 in E. coli has attracted considerable attention worldwide. As endogenous H2S reduces the efficacy of many clinically used antimicrobials, the inhibition of this gas should be considered an effective therapy against a wide range of bacteria, including pathogenic E. coli. Continuous surveillance and molecular characterization of H2S-producing mcr-carrying E. coli is needed to shed light upon all of the transmission pathways. It is important to strengthen the hygiene practices in the hospital to reduce the environmental contamination by H2S-producing MDR E. coli. Our results require future extensive study and follow-up evaluations in order to understand the trends of the AR gene’s epidemiology in H2S-producing E. coli, in clinical settings and in the community, with time, and ultimately anticipate the detection of bacteria that can possibly cause serious public health concerns. In the future, it would be an interesting study to determine the H2S production by E. coli, in both aerobic and anaerobic conditions, to understand its contribution to antibiotic resistance. As a representative case, the H2S-producing E. coli isolate with AR genes observed in the study emphasizes the importance of rational use of antibiotics in future clinical practices.This is the first report to describe HRaw sequencing reads have been deposited in the NCBI BioProject database under accession number PRJNA576077.


CRABS CLAW couldpotentially be involved in mediating nectary development. A significant numberof simple sequence repeat and single nucleotide polymorphism markers werepredicted in the Capsicum annuum sequences. The new resultsprovide valuable genetic information about flower development in pepper.The measurement of gene expression can provide important information about genefunction and the molecular basis for developmental processes. We analyzed thetranscriptomes at three different developmental stages of pepper flower. In the cDNA libraries for B1, B2, and B3: 82718, 77061,and 91491 unigenes were assembled, respectively. A total of 34,445 unigenesequences and 128 pathways were annotated by KEGG pathway analysis. Severalgenes associated with nectar biosynthesis and nectary development wereidentified, and 8,955, 12,182, and 23,667 DEGs were identified in the B2 vs B1,B3 vs B1, and B3 vs. B2 comparisons. DEGs were involved in various metabolicprocesses, including flower development, nectar biosynthesis, and nectarydevelopment. According to the RNA-seq data, all 13 selected DEGs showed similarexpression patterns after q-PCR analysis. Sucrose-phosphatase,galactinol-sucrose galactosyltransferase, and sucrose synthase played veryimportant roles in nectar biosynthesis, and  BLADE-ON-PETIOLE (BOP)1, BOP2, and CRABS CLAW (CRC)have been isolated and confirmed to be associated with the development of thenectary , SHATTERPROOF1/2,APETALA2/3 (AP2/3),PISTILLATA (PI), andSEPALLATA1/2/3 (SEP1/2/3), have demonstratedroles in regulating CRC expression  originated in Central and South America.It is one of the most important economic vegetables in the world and is consumed asa fruit or is processed into other products  permit thatthe whole transcriptome can be sequenced, and they are convenient and rapid methodsthat can be used to investigate gene expression at the whole-genome level and defineputative gene function . Some DEGsinvolved in nectar biosynthesis and nectary development were selected and analyzedby quantitative real-time PCR (qPCR). Our study provides valuable geneticinformation for the elucidation of pepper flower nectar biosynthesis and nectarydevelopment.The pepper cultivar 9704B was grown in experimental fields at the College ofHorticulture and Landscape, Yunnan Agricultural University, China. Nectarydevelopment was divided into three stages .These stages were based on the cytology results reported by Total RNA was isolated using the CTAB reagent method (Invitrogen). RNA from theB1, B2, and B3 stages of flower development was used to construct the sequencelibraries. A NanoDrop 1000 spectrophotometer and an Agilent 2100 Bioanalyzerwere used to verify RNA quality and quantity prior to further processing. Thetotal RNA was treated with DNase I prior to library construction. MagneticOligo(dT) beads were used to purify the poly-(A) mRNA. Adaptor-ligated fragmentswere generated according to manufacturer's instructions for T4 DNA polymerase,T4 polynucleotide kinase, Klenow 3' to 5' exo-polymerase, and T4 DNA ligase.When the adaptor-ligated fragments were separated on a 1.0% agarose gel, thedesired range of cDNA fragments (200 ± 25 bp) were excised from the gel and thenthe cDNA fragments were enriched and amplified using PCR. The cDNA library wassubjected to Solexa sequencing using an Illumina HiSeq2000 sequencing platform.De novo transcriptomeassembly of these short reads was performed using the SOAP denovo assembling program, which first combined reads with a certainlength of overlap to form longer fragments without Ncalled contigs. The readswere mapped back to the contigs. Paired-end reads were used to check the contigsthat had come from the same transcript. Secondly, using "N" to represent unknownsequences, SOAP de novo connected the contigs to produce thescaffolds. Paired-end reads were then used again to fill in the gaps between thescaffolds. These were designated as unigenes.The raw data processing and functional annotations were performed according to-5) to search theunigene sequences against many protein databases, including the non-redundantdatabase (Nr), SwissProt, the Kyoto Encyclopedia of Genes and Genomes (KEGG),and the Orthologoue Group of proteins (COG)  were used to analyze the metabolicpathways (6C / (N × L /103), where RPKM(A) is the expression of unigene A, C is thenumber of fragments uniquely aligned to unigene A, N is the total number offragments uniquely aligned to all the unigenes, and L is the base number in thecoding region (CDS) of unigene A. The p-value corresponding tothe differential transcript expression in two samples was determined accordingto p-values in a numberof tests. We used FDR ≤ 0.001 and the absolute value of log2 ratio ≥ 1 asthresholds to consider whether the gene expression difference wassignificant.The KEGG database and related software applications(http://www.genome.jp/kegg/kegg4.html) were used to analyze the metabolicpathways  markers were detected using the MISA Perlscript (http://pgrc.ipkgatersleben.de/misa/). Mono-, di-, tri-, tetra-, penta-and hexa-nucleotide sequences with minimum repeat numbers of 10, 6, 5, 5, 5, and5, respectively, were used as the search criteria. SOAPaligner software (Release2.21.08-13-2009) was used to mine for the single nucleotide polymorphism (SNP)markers. The thresholds for SNP identification were carried out according to-ΔΔCtmethod.Three biological replicates were generated for each developmental stage , with technical triplicatesfor each sample. An actin gene was chosen as internal control for normalization,and relative gene expression was calculated using the 2By Illumina sequencing 57,826,112, 59,057,626, and 59,459,964 raw reads weregenerated for the B1, B2, and B3 development stages, respectively. Afterfiltering of low-complexity reads, low-quality reads, and repetitive reads, morethan 53 million 49nt clean reads were obtained for the B1, B2, and B3 cDNAlibraries, respectively .de novo assembly of thehigh-quality reads. The number of contigs and information about the pepperflower contigs is shown in The Trinity program was used for -5) was used to obtain 59,285significant BLAST hits. The size distribution for the CDSs is shown in The BLASTX program  . In eachThe annotated sequences were mapped to the reference canonical pathways containedin the KEGG database. In total, 34,445 unigenes were assigned to 128 KEGGpathways. The most highly represented category was "Metabolic pathways" with7658 (22.23%) members. The "Biosynthesis of secondary metabolites" and"Plant-pathogen interaction" pathways were also well represented, with 4110(11.93%) members and 1969 (5.72%) members, respectively.vs. B1, B3 vs. B1, and B3vs. B2) were compared. For each different stage, weidentified thousands of DEGs, indicating that there had been substantialchanges. The B2 vs. B1 comparison showed 8,955 DEGs, with 2666up-regulated and 6289 down-regulated  members. The"Biosynthesis of secondary metabolites", "RNA transport", and the"Plant-pathogen interaction" pathways were also well represented, with 495(12.54%) members, 255 (6.97%) members, and 239 (6.53%) members, respectively members. The "Biosynthesis ofsecondary metabolites" and "Plant-pathogen interaction" pathways were also wellrepresented, with 693 (14.75%) members and 370 (7.87%) members, respectively members. The "Biosynthesis ofsecondary metabolites" and "Plant-pathogen interaction" pathways were also wellrepresented, with 1069 (13.39%) members and 583 (7.3%) members, respectively (fourunigenes), alkaline alpha-galactosidase(CaAGA) (four unigenes), sucrose-phosphatase 1(CaSPP1) (three unigenes), and sucrose synthase7-like (CaSUS7) (six unigenes). The last unigenewas galactinol-sucrose galactosyltransferase 5-like(CaGSG5). The B2 vs. B1 librariescomparison showed that seven unigenes were up-regulated and eight weredown-regulated , eight unigenes were part of putativebeta-fructosidase (βFRA), one unigene belonged to agamous-like MADS box proteinAGL5, and the other unigenes belonged to the L-ascorbateoxidase (AO). The B2 vs. B1 library comparisonshowed that 20 unigenes were up-regulated and eight were down-regulated ,CL7013.Contig2 , Unigene21647, and CL3588.Contig1 and CL2403.Contig1 were up-regulated in their expression at the flowerdevelopment stage (B3). The CL2191.Contig2  and CL4219. Contig2  displayed similarexpression patterns, and were up-regulated at the nectary forming stage (B2)(Some known age (B2).A total of 11,654 SSRs were identified in 97,475 unigenes. Mono-nucleotide SSRsrepresented the largest fraction , followed by tri-nucleotides, di-nucleotides , hexa-nucleotide tandem repeats, penta-nucleotides , and tetra-nucleotides . Additiode novo assembly in the B1, B2, and B3 libraries,respectively. The gene or protein names and descriptions were assessed, andtheir putative conserved domains, gene ontology terms, and potential metabolicpathways were annotated. This study will help to improve our understanding ofthe processes involved in regulating flower development, nectar biosynthesis,and nectary development in pepper flowers. More contigs and unigenes werereported compared to previous transcriptomic studies in other plants, such astomato  were highlyhomologous to Nicotiana tabacum (XM_016585183), Solanumpennellii (XM_015210288), S. tuberosum(XM_006348118), Ipomoea nil (XM_019321835), Betavulgaris (XM_010676936), Phoenix dactylifera(XM_008806164), Jatropha curcas (XM_012220325), andSesamum indicum (XM_011088670)   which stCaSUS, we identified a number of other genes thatwere up-regulated at the flower development and were involved in simple sugarmetabolism. These were CaGSG5 (Unigene14855), 4CaS6PP , 3 CaSPP1 , 4 CaGSG1 . Significantly, the In addition to YABBYtranscription factor gene families in Arabidopsis and they aredeterminants of abaxial cell fate in the lateral floral organs  encodes atranscription factor associated with the regulation of carpel and nectarydevelopment (CRC is currently the only known gene to be absolutelyrequired for nectary development (CaCRCs thatwere highly homologous to N. tabacum (AY854799), S.tuberosum (XM_006348604), Malus x domestica(XM_008391173), Prunus mume (XM_008245598), Gossypiumraimondii (XM_012625767), Theobroma cacao(XM_018114782), and A. thaliana At1g69180  expressionprofiles . There welopment .CRC is elopment . This stQ446412). Both we


APBA3 gene, was identified with OR values for association with POF of 13.33 and 4.628 in the discovery and replication sets, respectively. No rs55941146 minor allele homozygotes were present in either cases or controls. The APBA3 protein binds FIH-1 that inhibits hypoxia inducible factor-1α (HIF-1α). HIF-1α contributes to granulosa cell proliferation, which is crucial for ovarian follicle growth, by regulating cell proliferation factors and follicle stimulating hormone-mediated autophagy. Our data demonstrate that APBA3 is a candidate novel causal gene for POF.Premature ovarian failure (POF) is a complex disease of which the etiology is influenced by numerous genetic variations. Several POF candidate genes have been reported. However, no causal genes with high odds ratio (OR) have yet been discovered. This study included 564 females of Korean ethnicity, comprising 60 patients with POF and 182 controls in the discovery set and 105 patients with POF and 217 controls in the replication set. We conducted genome-wide association analysis to search for novel candidate genes predicted to influence POF development using Axiom Precision Medicine Research Arrays and additive model logistic regression analysis. One statistically significant single nucleotide polymorphism (SNP), rs55941146, which encodes a missense alteration  in the  Premature ovarian failure (POF) is an idiopathic, complex disease in which menopause occurs before the age of 40 years . The exaThe ovarian follicle is important to oocyte growth and folliculogenesis is a crucial mechanism contributing to female fertility ,9,10. ThThere have been numerous reports of associations between single nucleotide polymorphisms (SNPs) and POF onset ,20,21. TSubjects comprised a total of 564 females of Korean ethnicity. Sixty patients with POF and 182 controls in the discovery set, and 120 patients with POF and 218 controls in the replication set. Patient genomic DNA samples were genotyped using an PMRA. Kinship analysis detected 30,876 associations among 242 individuals in the discovery set, and there were no first and second degree relationships. Kinship analysis was also conducted in the replication set and a total of 322 individuals were left. Our results indicated that none of the study participants were related.n = 346,668). All individuals passed the individual-level missingness threshold of <0.1. Application of Hardy-Weinberg equilibrium (HWE) threshold of >1.0 × 10−5 to the control group resulted in the exclusion of 7937 markers. A marker-level missingness threshold of >0.001 excluded 210,158 markers because of low genotyping rates, and the minor allele frequency threshold (<0.05) further excluded 255,378 markers. Finally, only autosomal markers were included in our analysis. Hence, 20,046 markers mapped to the X and Y chromosomes or mitochondrial DNA were excluded. Consequently, a total of 625,170 markers were removed, while 277,022 remained. After additive model association logistic regression analysis, 45 SNPs were identified as significantly associated with POF in the discovery set. An identical analysis was then conducted using the replication set, resulting in validation of only one of the 45 SNPs from the discovery set with a significant p-value (2.996 × 10−7) followed by a Bonferroni correction (APBA3 coding region (A > C) on chromosome 19.Raw data generated by genotyping of the discovery set using Axiom PMRA chips were first filtered by removing markers with missing annotation and deletion/insertion markers  generated using the Sorting Intolerant From Tolerant (SIFT) and PolyPhen-2 programs, respectively, indicated that the rs55941146 variant is predicted to have a deleterious effect on the APBA3 amino acid sequence, and is likely damaging to the APBA3 three-dimensional structure.The rs55941146 variant is a missense SNP (A > C) in the APBA3 gene encodes amyloid-beta precursor protein binding family A member 3, which is also referred to as mammalian uncoordinated 18-1 (MUNC 18-1) interacting protein 3 (MINT3). APBA3 functions both to modulate processing of the amyloid-beta precursor protein (APP) by binding to its C-terminal domain and regulating factor-inhibiting hypoxia inducible factor-1 (FIH-1) via its N-terminal domain [The l domain ,23. FIH-l domain . FIH-1 il domain ,26. Bothl domain . Hence, l domain . Therefol domain , leadingl domain . Under nl domain .Gonadotrophins, including FSH, have established roles in stimulating follicle growth and preventing the GC apoptosis associated with follicle atresia ,30,31. TOvarian follicle atresia, in which immature follicles degenerate, is an important stage of follicle growth , triggerHIF-1α is important, not only as a factor downstream of FSH-mediated autophagy, but also an inducer of proliferation factors. Knockdown of HIF-1α induces downregulation of proliferation markers, such as cyclin D2 (CCND2) and proliferating cell nuclear antigen (PCNA) . FurtherThe majority of candidate genes identified by GWAS as associated with various diseases have OR values < 1.5, and such genes with relatively low OR values have a weak impact in increasing disease risk . CompareAPBA3 SNP (rs55941146) may influence FSH-mediated autophagy and transcription factor induction by association with HIF-1α in granulosa cells. This would be expected to induce down-regulation of autophagy, and of various transcription factors in pre-antral and antral follicles stages, under hypoxic conditions. Variation in APBA3, which regulates the FIH-1/HIF-1 pathway, may lead to impaired FIH-1 downregulation during hypoxia and consequent inappropriate inactivation of HIF-1. Reduction in the HIF-1α level can lead to suppression of normal levels of autophagy and increased transcriptional activity. Therefore, our data suggest that APBA3 is associated with POF in the Korean female population and represents a new candidate gene for this condition.In conclusion, we identified a variant with a high OR for association with POF, relative to previously reported candidate genes, which is predicted to have a detrimental impact on the amino acid sequence and tertiary structure of the APBA3 protein. This For the discovery set, 242 women were, retrospectively, selected for inclusion in this study from a total of 367 individuals who visited Korea University Anam Medical Center from 2016 to 2019 and Bundang CHA Hospital until 2010. For the replication set, 322 women were, retrospectively, selected for inclusion in the analysis from 338 individuals who gathered by CHA University until 2004. Samples used in this GWAS study was authorized by the Institutional Review Board of Korea University Anam Medical Center (2016AN0216) and the Institutional Review Board of Bundang CHA Hospital (2010123). The samples used in the replication study were authorized by the Institutional Review Board of CHA University (1044308-201310-BR-002-01).n = 902,380) were initially evaluated in samples from the 242 individuals in the discovery set. SNP data pre-processing was performed using the Affymetrix Power Tool to generate dish quality control (DQC) values to ensure samples were of sufficient quality for analysis. The dish quality control (DQC) threshold value was 0.82, where samples with DQC values < 0.82 were excluded from further genotype analysis. Quality control of the remaining samples was by sex check to identify differences between clinically determined sex and sex predicted based on genotype data. In the following step, samples with call rate values less than the threshold (97%) were removed. Finally, abnormal rates of heterozygosity and plate quality control values were evaluated and relationship tests were performed.DNA was extracted from peripheral blood samples from the 580 individuals recruited to the study. SNP array analysis was conducted using Axiom Precision Medicine Research Array (PMRA) 2.0 chips. SNPs (n = 60) and controls (n = 182) in the discovery set, and patients (n = 105) and controls (n = 217) in the replication set, were included, comprising a total of 564 samples. Data were analyzed using descriptive statistics and additive model logistic regression analysis. For the additive model association analysis, thresholds were as follows: individual-level missingness <0.1, marker-level missingness <0.001, and minor allele frequency >0.05. HWE was applied only in controls, using an empirical p-value threshold of >1.0 × 10−5. Finally, 166,866 SNPs were included in the analysis.Korean patients with POF  [To account for multiple comparisons, MA, USA) , LocusZoMA, USA) , and qqmMA, USA) . KinshipMA, USA) .https://sift.bii.a-star.edu.sg/, 180820) was used to analyze the functional consequences of the SNPs identified and protein biological function and PolyPhen-2 v2.2.2.  was used to predict effects of variants on both the amino acid sequence and protein tertiary structure.SIFT v6.2.1 (