Aspergillus spp. as polyketide secondary metabolites. Aflatoxigenic fungi including A. flavus express the alternative oxidase (AOX), which introduces a branch in the cytochrome-based electron transfer chain by coupling ubiquinol oxidation directly with the reduction of O2 to H2O. AOX is closely associated with fungal pathogenesis, morphogenesis, stress signaling, and drug resistance and, as recently reported, affects the production of mycotoxins such as sterigmatocystin, the penultimate intermediate in aflatoxin B1 biosynthesis. Thus, AOX might be considered a target for controlling the propagation of and aflatoxin contamination by A. flavus. Hence, this review summarizes the current understanding of fungal AOX and the alternative respiration pathway and the development and potential applications of AOX inhibitors. This review indicates that AOX inhibitors, either alone or in combination with current antifungal agents, are potentially applicable for developing novel, effective antifungal strategies. However, considering the conservation of AOX in fungal and plant cells, a deeper understanding of fungal alternative respiration and fungal AOX structure is needed, along with effective fungal-specific AOX inhibitors.Aflatoxins are among the most hazardous natural cereal contaminants. These mycotoxins are produced by  Aspergillus spp., including A. flavus, as polyketide secondary metabolites. These opportunistic fungi are commonly detected as contaminants in cereal crops at both the pre- and post-harvest stages  is an integral monotopic membrane protein localized on the matrix side of the inner mitochondrial membrane . The enzthe spp. . AOX intrylation . AOX actrylation . Furtherynthesis . As AOX ynthesis . GenomicA. niger . AOX exhlus spp. .Although AOX is a promising target for the development of novel antifungal strategies, further studies are required to understand the physiological function of AOX and its association with fungal pathogenesis, morphogenesis, stress signaling, drug resistance, environment adaption, and secondary metabolism. Hence, this review addresses these issues by summarizing the current understanding of AOX and the alternative respiration pathway in pathogenic and agriculturally important fungi, and progress in studies on AOX inhibitors as antifungal agents.A. flavus is multifactorial and is closely connected with the cellular development, secondary metabolism, adaption to stress conditions, and interaction with host defense molecules  and sodium nitroprusside lead to fitness defects and the loss of viability in C. albicans  fungicides represent the most important group of fungicides developed on the basis of mitochondrial inhibition . QoI fun c genes . However c genes .C. albicans  regulating fungal development; (2) redirecting cellular metabolism; (3) sustaining ATP and NADPH supply; and (4) maintaining cellular ROS levels. Most fungal secondary metabolites, including aflatoxin, are produced after the fungus completes its initial growth phase and is beginning a stage of development, represented by sporulation . AOX is in vitro , suggestin vitro . AOX ovetabolism . It was tabolism . Studiesormation . Thus, Aormation . Howeverormation . To mainormation . AOX waslus spp. . It has lus spp. . The prein vivo.Although alternative respiration produces markedly lesser ATP and appears dispensable for virulence in some fungal pathogens , it mainTrypanosoma brucei is currently the only alternative oxidase protein structure available (M. perniciosa in vitro and alleviates witches’ broom disease in infected plants. It suggests that further studies on the structure and physiological activity of AOX in fungal cells and the structure-activity relationship of current AOX inhibitors will surely promote the development of effective fungal AOX inhibitors to control fungal propagation. Besides, fungal mitochondrial function is associated with other important cellular functions including ergosterol biosynthesis and cell wall maintenance (C. albicans, as combinatorial treatment with SHAM and fluconazole displayed synergistic antifungal activity (C. parapsilosis to caspofungin, a fungicide functioning by inhibiting β-D-glucan synthase (C. albicans lead to changes in cell wall organization; however, the authors reported decreased susceptibility to caspofungin, highlighting the requirement for a deeper understanding of the physiological functions of fungal AOX and fungal mitochondria. Nevertheless, these studies indicated that AOX inhibitors, though they may be not efficient antifungal agents when working alone, still can contribute to fungal inhibition by working with current antifungal agents.Alternative oxidase of human parasite vailable . AOX inhvailable . The devntenance , suggestactivity . The linsynthase . FurtherM. perniciosa spore germination and prevents the appearance of the symptoms of witches’ broom disease in infected plants without obvious effects on the plant itself. This study suggests the possibility of developing AOX inhibitors acting specifically on fungal cells. Further studies on the structure and physiological differences of AOX in fungal and plant cells will surely promote the development of novel fungal-specific AOX inhibitors. Another strategy to ease this concern is to use natural compounds of vegetal origin, such as flavone, quercetin, resveratrol, and curcumin, which can potentially inhibit fungal respiration. These natural compounds modulate mitochondrial function through different methods including the inhibition of mitochondrial enzymes, suppression of oxidative phosphorylation, and alteration of the mitochondrial redox balance (Pichia stipitis, and M. grisea, AOX induction by QoI fungicide was suppressed by flavonoid components (B. cinerea, flavone reportedly inhibited respiration in whole cells treated with potassium cyanide. It is reasonable to expect that some plant-based natural compounds may inhibit fungal AOX without affecting plant cells. Alternatively, plant-based inhibitors of the classical respiration complexes might function synergistically with synthetic AOX inhibitors, thus effectively inhibiting fungal respiration with minimal or no impact on plant cells.Another concern regarding the conservation of the respiratory machinery in pathogenic fungi and plants has also deterred the application of AOX inhibitors, since these inhibitors also act on plant AOX potentially and yield unexpected results. AOX is applicable in developmental plasticity and is associated with yield stability of food crops . During  balance . In Pichmponents . In B. cA. flavus. AOX inhibitors, either alone or along with current antifungal agents, are potentially applicable for developing novel effective antifungal strategies. However, the application of AOX inhibitors in food production is currently limited by the low efficiency and selectivity, and concerns raised from the conservation of AOX in fungal and plant cells. To overcome these limitations, a deeper understanding of fungal alternative respiration and fungal AOX structure, and screening of effective fungal-specific AOX inhibitors are required.In conclusion, fungal virulence composite AOX is potentially a suitable target owing to its association with pathogenesis, morphogenesis, environment adaption, fungicide resistance, cell wall regulation, lipid metabolism, and probably mycotoxin metabolism in studies on aflatoxin contamination and the propagation of FT, SL, and SW curated all references. FT drafted the manuscript. HC revised and finalized the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-1 and HTLV-2 are endemic to Brazil, and they have different effects in HIV/AIDS disease progression. HIV/HTLV-1 has been described as accelerating the progression to AIDS and death, while HIV/HTLV-2 slows the progression to AIDS. Provirus mutations of HTLV-1 were implicated in severe leukemia development and in problems in the diagnosis of HTLV-1; in contrast, provirus mutations of HTLV-2 had not been confirmed and associated with problems in HTLV-2 diagnosis or disease outcome. Nevertheless, data obtained here allowed us to recognize and understand the false-negative results in serologic and molecular tests applied for HTLV-1 and HTLV-2 diagnosis. Defective proviruses, as well as synonymous and nonsynonymous mutations, were associated with the diagnosis deficiencies. Additionally, since HIV-1 and HTLV-1 infect the same cells (CD4 positive), the production of HIV-1 pseudotypes with HTLV-1 envelope glycoprotein during HIV/HTLV-1 coinfection cannot be excluded. Defective provirus of HTLV-2 and Tax2c is speculated to influence progression to AIDS. env, and tax regions, we searched for defective type 1 particles that retain LTRs and lack internal sequences and type 2 particles that lack the 5′LTR region. In addition, using as references the prototypes ATK (HTLV-1) and Mo (HTLV-2), we searched for point mutations in the LTR and synonyms and nonsynonymous mutations and non-sense mutations in env and tax regions. Defective HTLV-1 and HTLV-2 provirus type 1 or 2 was detected in 31.8% of HIV/HTLV-1- and 32.0% of HIV/HTLV-2-coinfected individuals. Synonymous and nonsynonymous mutations were identified mostly in HTLV-2 and associated with lower levels of specific antibodies. No non-sense mutations that resulted in premature termination of Env and Tax proteins were detected. On the contrary, mutation in the stop codon of Tax2a produced a long protein characteristic of the HTLV-2c subtype. The clinical significance of these mutations in coinfected individuals remains to be defined, but they confirmed the lower sensitivity of serological and molecular diagnostic tests in HIV/HTLV-1/2 coinfections.Provirus mutations of human T-lymphotropic virus 1 (HTLV-1), mostly the lack of the 5′ long terminal repeat (LTR) genomic region, have been described and associated with severe adult T cell leukemia/lymphoma (ATLL), non-sense point mutations with low proviral load, and Western blotting indeterminate results. Until now, no information concerning provirus mutations of HTLV-2 and its consequences, as well as those of HTLV-1/2 in HIV-coinfected individuals, had been described. Therefore, we searched for these mutations in provirus samples of 44 HIV/HTLV-1- and 25 HIV/HTLV-2-coinfected individuals. Using protocols well established for amplification and sequencing of segments of the LTR, IMPORTANCE HTLV-1 and HTLV-2 are endemic to Brazil, and they have different effects in HIV/AIDS disease progression. HIV/HTLV-1 has been described as accelerating the progression to AIDS and death, while HIV/HTLV-2 slows the progression to AIDS. Provirus mutations of HTLV-1 were implicated in severe leukemia development and in problems in the diagnosis of HTLV-1; in contrast, provirus mutations of HTLV-2 had not been confirmed and associated with problems in HTLV-2 diagnosis or disease outcome. Nevertheless, data obtained here allowed us to recognize and understand the false-negative results in serologic and molecular tests applied for HTLV-1 and HTLV-2 diagnosis. Defective proviruses, as well as synonymous and nonsynonymous mutations, were associated with the diagnosis deficiencies. Additionally, since HIV-1 and HTLV-1 infect the same cells (CD4 positive), the production of HIV-1 pseudotypes with HTLV-1 envelope glycoprotein during HIV/HTLV-1 coinfection cannot be excluded. Defective provirus of HTLV-2 and Tax2c is speculated to influence progression to AIDS. Human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) were the first human retroviruses discovered by Gallo’s team in the early 1980s –8. The t–12–gag, pol, and env structural genes flanked by 5′ and 3′ long terminal repeat (LTR) sequences. In the 3′ portion of the genome is a pX region that encodes the Tax, Rex, p21, p12, p13, and p30 proteins as well as the antisense gene encoding the HTLV-1 basic leucine zipper factor (HBZ). Both Tax and HBZ are implicated in the development of ATLL, with Tax initiating cellular transformation and HBZ maintaining virus-induced cellular proliferation  indeterminate results –27. For –env, and tax segments employed in HTLV subtyping.At present, no data concerning provirus mutation and defective particles were described for HTLV-2 or HTLV-1/2 provirus mutations in HIV/HTLV-1 and HIV/HTLV-2 coinfection. Given that we carry out the surveillance of HTLV-1 and HTLV-2 subtypes in HIV/HTLV-1- and HIV/HTLV-2-coinfected individuals in São Paulo, Brazil, we decided to conduct the present study to add information concerning this subject by searching for mutations in the LTR, After the first screening by NCBI genotyping and REGA genotyping, all HTLV-1 sequences were classified as belonging to the Cosmopolitan subtype a of transcontinental subgroup A and sequences of HTLV-2 as HTLV-2a subtype, variant -2c. These classifications were confirmed by phylogenetic tree constructions (see 10.1128/mSphere.00923-20.1FIG S1env codifying region (B), and 972-bp nucleotide sequences of the tax codifying region (C), using the maximum likelihood approach with 1,000 bootstrap replications, repeated 10 times. The clades supported by bootstrap values of at least 70% are marked with a dot. Branches belonging to Transcontinental subgroup A of the Cosmopolitan subtype a (HTLV-1aA) are in dark blue; branches belonging to Japanese subgroup B of the Cosmopolitan subtype a (HTLV-1aB) are in yellow; branches of subgroup C of the Cosmopolitan subtype a (HTLV-1aC and GH78) are in light blue; branches of subgroup D of the Cosmopolitan subtype a  are in orange; the branch of subtype b  is in green; the branch of subtype c  is in purple; the branch of subtype d (HTLV-1d and pyg19) is in pink; and the branch of STLV, in black, was as an outgroup in panel B. Sequences of the present study are in red. Download FIG S1, DOCX file, 1.6 MB.HTLV-1 phylogenetic trees based on the 619-bp nucleotide sequence of the LTR region (A), 619-bp nucleotide sequences of the Copyright © 2020 Campos and Caterino-de-Araujo.2020Campos and Caterino-de-AraujoCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00923-20.2FIG S2env codifying region (B), and 894-bp nucleotide sequences of the tax codifying region (C), using the maximum likelihood approach with 1,000 bootstrap replications, repeated 10 times. The clades supported by bootstrap values of at least 70% are marked with a dot. Branches belonging to subtype a  are in yellow; branches belonging to subtype b  are in green; branches of subtype c  are in blue; and the branch of subtype d , in orange, was used as an outgroup. Sequences obtained in the present study are in red. Download FIG S2, DOCX file, 2.9 MB.HTLV-2 phylogenetic trees based on the 467-bp nucleotide sequence of the LTR region (A), 1,041-bp nucleotide sequences of the Copyright © 2020 Campos and Caterino-de-Araujo.2020Campos and Caterino-de-AraujoCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the env, and tax regions (30 [68.2%]), and defective particles of type 1 were detected in 20.4% (considering the lack of env and/or tax regions) and type 2 in 11.4% (considering the lack of the 5′LTR region) (env and/or tax region) and defective provirus type 2 in 16.0% of samples  . The sam samples . It is ienv and tax regions, as well as the number of defective proviruses classified as type 1 (DPV1) and type 2 (DPV2).env and tax regions are presented in tax region characteristic of the TaxA Brazilian genotype.Nucleotide substitutions, insertions, and deletions in the LTR of HTLV-1 (including the Tax-responsive elements [TRE1 and TRE2] and U3) and the synonymous and nonsynonymous mutations in env and tax regions, emphasizing that the signature of Tax2c (long Tax) was detected in 100.0% of sequences.env, and tax regions of HTLV-1 and HTLV-2 are presented in P < 0.0001 in all cases, except synonymous mutations in the tax region)  . Of note region) .pol] and nested PCR-restriction fragment length polymorphism [RFLP] [tax])  or molecular assays (qPCR  [tax]) . Differeenv, and tax sequences (positive or negative), although overall comparisons disclosed lower levels of antibodies in blood samples that had negative sequence results, statistically significant difference was detected only for the env region and when REM Gold ELISA was used for screening  and the variant, c, of the HTLV-2a subtype (HTLV-2c) circulating in São Paulo, confirming the HTLV subtypes more frequently detected in Brazil in individuals of African origin, Amerindians, and IDUs –33.–Concerning nucleotide substitutions in LTR regions, the results obtained showed nucleotide substitutions in TRE1 and TRE2, as previously described , 35. TheOther mutations were detected in the LTR genomic region, such as the C401T mutation, which confirms the molecular signature of the HTLV-2c subtype and mutations in the SER, ETS, poly(A), and U3RU5 regions, as reported by Barreto and coworkers in viral isolates from Salvador, Bahia, in the northeast region of Brazil . Interes38–env regions of HTLV-1 and HTLV-2, again, greater numbers of mutations were detected in HTLV-2. The amino acid change S183P in 100.0% of the HTLV-2c env sequences was also previously reported in Brazilian strains . Moreover, the unique lack of the env region detected in some samples in the present study allowed us to suppose pseudotype production. Tang and collaborators described pseudotyping of HIV-1 with HTLV-1 envelope glycoprotein during HIV-1/HTLV-1 coinfection, facilitating direct HIV-1 infection of female genital epithelial cells and implicating it in the sexual transmission of HIV in nonpermissive genital cells  and the transmembrane glycoprotein GD-21, common to both viruses, and just in this segment there is an amino acid change of S183P in HTLV-2 env sequences. We hypothesized that defective provirus type 1 particles or nonsynonymous mutations in the env region are responsible for the results obtained. On the other hand, when the same antibody levels were evaluated in relation to the detection of LTR, env, and tax proviral segments of HTLV-1 and HTLV-2, although minor levels of antibodies were detected in both EIAs in the overall analysis, only the REM Gold ELISA results reached statistical significance (association between the absence of env sequences and low levels of antibodies). We do not know which are the synthetic peptides or recombinant proteins present in this REM EIA, because this information is absent from the manufacturer’s instructions, but we hypothesized that when nonsynonymous point mutations and defective proviruses are circulating in such individuals, small amounts of antibodies are produced and, consequently, detected by this enzyme immunoassay. In fact, the lower sensitivity of this screening assay was previously described in HIV/HTLV-coinfected individuals in São Paulo, Brazil, when the comparative performance of Murex EIA and REM Gold ELISA was examined , gag (p19), and pX (Tax) proteins  detected in HIV/HTLV-1- and HIV/HTLV-2-coinfected individuals, which is similar to the overall rates described in ATL individuals infected with HTLV-1 –22. Howe–env, and tax regions used in HTLV subtyping. We employed degenerate primers and protocols optimized previously for detecting all Brazilian sequences described in the literature (gag gene of 66 bp (nucleotides 756 to 821). We believe these strategies may reduce the limitations of the study.Concerning the limitations of the present study, we take into account that we used a conventional DNA sequencing methodology (Sanger sequencing), which generates provirus DNA fragments (not the complete provirus) present only in the major clones. Although we consider that the best way to search for defective proviruses is using a high-throughput sequencing-by-synthesis instrument   or a recterature , 53, 54;The implication of the mutations and defective proviruses in HIV/HTLV-1- and HIV/HTLV-2-coinfected individuals detected here deserves more study, but we speculate that difficulties in HTLV-1 and HTLV-2 diagnosis as well negative results in molecular confirmatory assays in such individuals are, in part, attributable to these provirus mutations, corroborating results described elsewhere for HTLV-1-infected individuals .+ cell counts, high HIV viral load levels, and more X4 HIV strains were detected in such patients. In contrast, in HIV/HTLV-2-coinfected patients, we detected large numbers of CD4+ cells, low HIV viral load levels, and large numbers of R5 HIV strains . These data partially corroborate the studies that pointed to rapid progression to AIDS and death in HIV/HTLV-1-coinfected individuals and slow progression to AIDS in HIV/HTLV-2-coinfected individuals  could be due to the late starting of ART in such patients, when the recovery of CD4+ cells is not promising, but these are only speculations and merit other studies.In addition, we could not exclude the negative impact of defective particles in HIV/HTLV-1 coinfection, since low CD4ividuals –46. Of nIn conclusion, this is the first time that around 30% of provirus mutations were described in HTLV-2-infected individuals and in HIV/HTLV-1- and HIV/HTLV-2-coinfected individuals and is the first tentative description of HTLV-2 defective provirus classification. Indeed, the presence of point mutations and defective provirus was correlated with negative results in molecular assays and lower antibody production. Finally, the results obtained here could help us in the future to understand why HIV/HTLV-1 and HIV/HTLV-2 progress differently to AIDS and to HTLV-1/2-associated diseases.pol] and nested PCR-RFLP [tax]) were used as HTLV-1 and HTLV-2 confirmatory and discriminatory assays , 634 to 679 bp (env), and 996 to 1,074 bp (tax), and those of HTLV-2 provirus varied from 496 to 644 bp (LTR), 936 to 964 bp (env), and 1,089 to 1,134 bp (tax). Products available for sequence analysis were obtained from 44 HIV/HTLV-1- and 25 HIV/HTLV-2-coinfected individuals. From some patients in clinical and laboratory follow-up for 2 years, DNA samples were obtained at the beginning of study (first collection), 6 to 12 months later (second collection), and 13 to 24 months later (third collection). The sequences obtained from these patients are presented in DNA samples were extracted from peripheral blood leukocytes. The protocols for amplification and sequencing the LTR, terature , 53–55. 10.1128/mSphere.00923-20.3TABLE S1env, and tax). Legend: a, primer nucleotide position is provided as aligned with the ATK prototype  and Mo prototype ; b, primers employed in nested PCR and sequencing reactions. Primers were employed to detect HTLV-1 and HTLV-2 provirus DNA in peripheral blood leucocytes of HIV-coinfected patients from the southeast (São Paulo) region of Brazil. Download Table S1, DOCX file, 0.02 MB.Primers employed in polymerase chain reaction assays , in which the nucleotide and amino acid substitutions were searched. HTLV-1 and HTLV-2 subtyping was primarily screened by NCBI genotyping (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) and REGA subtyping (http://www.bioafrica.net/rega-genotype/html/subtypinghtlv.html) websites. Neighbor-joining (NJ) and maximum-likelihood (ML) phylogenetic trees were constructed based on appropriate nucleotide substitution models determined by Modeltest v3.7  using PAUP v4b10 software. Bootstrapping was performed using the stepwise addition algorithm for 1,000 replicates. The Mel5 (HTLV-1c) sequence and Efe2 (HTLV-2b) were used as outgroups. The GenBank accession numbers of the prototypes as well the sequences obtained here and employed in the phylogenetic tree constructions are disclosed in Briefly, sequencing was performed using an ABI 3130 Genetic Analyzer . All of the sequencing chromatograms were assembled and edited with Sequencher 4.7 software. Multiple alignments were performed using the Clustal W multiple-sequence alignment tool from the BioEdit Sequence Alignment Editor, version 7.0.5.3, and software with a reference set available in the GenBank database  because of the loss of the stop codon in the tax gene position 8203, was used; consequently, an additional 25 amino acids at the C-terminal end of the Tax protein  and Mo (HTLV-2a) using BioEdit version 7.0.5.3 software and searched for nucleotide substitutions and amino acid changes (synonymous and nonsynonymous mutations). In the enotypes . In the  protein  were useOf interest, these prototypes were employed for subsequent analyses because they have been used to compose the HTLV-1/2 serologic immunoassays employed for screening, confirmation, and discrimination of HTLV-1 from HTLV-2 .It is worth noting that the ATK prototype is the complete nucleotide sequence of the HTLV-1 provirus genome integrated into leukemia cell DNA, derived from a Japanese adult T cell leukemia (ATL) patient . The Mo env, and tax segments after several amplifications and sequencing events, including testing different amounts of DNA inputs and primer pairs. The criterion to be considered an HTLV-1 defective type 1 provirus was the lack of the env and/or tax segment, and that for type 2 was the lack of a 5′LTR region , was employed for comparisons among groups using the Mann-Whitney U test (two groups). A The study was approved by the Ethics Committee for Research of IAL, CTC 62H-2015 and 21I/2016, under Ministry of Health protocol numbers CAAE 55837316.0.0000.0059 and 52493316.1.0000.0059. All of the procedures were performed in accordance with the principles established in the Declaration of Helsinki of 1975, as revised in 1983. The study was conducted anonymously.GenBank accession numbers for the prototypes and sequences obtained in this study can be found in supplemental


The Notch signaling pathway plays an essential role in a wide variety of biological processes including cell fate determination of vascular endothelial cells and the regulation of arterial differentiation and angiogenesis. The Notch pathway is also an essential regulator of tumor growth and survival by functioning as either an oncogene or a tumor suppressor in a context-dependent manner. Crosstalk between the Notch and other signaling pathways is also pivotal in tumor progression by promoting cancer cell growth, migration, invasion, metastasis, tumor angiogenesis, and the expansion of cancer stem cells (CSCs). In this review, we provide an overview and update of Notch signaling in endothelial cell fate determination and functioning, angiogenesis, and tumor progression, particularly in the development of CSCs and therapeutic resistance. We further summarize recent studies on how endothelial signaling crosstalk with the Notch pathway contributes to tumor angiogenesis and the development of CSCs, thereby providing insights into vascular biology within the tumor microenvironment and tumor progression. The Notch signaling pathway is highly conserved across all vertebrate species and orchestrates a diverse range of functions including cell growth, differentiation, and patterning. In mammals, this pathway features four transmembrane receptors (Notch 1–4) that interact with five canonical transmembrane ligands  . CanonicThe NICD–RBPJ interaction initiates transcription of Notch target genes to regulate cell fate determination, migration, differentiation, and proliferation under both normal and malignant conditions . ConsequIn contrast, non-canonical Notch signaling can be initiated by a non-canonical ligand and may not require cleavage of the Notch receptors. In some forms of non-canonical signaling, there is no involvement of RBPJ, which may reflect interactions with other signaling pathways upstream of the NICD–RBPJ complex . Given tde novo arteriogenesis), capillaries (vasculogenesis), and veins (venogenesis). These networks supply oxygen and nutrients to all tissues and remove wastes in the body in support of tissue growth and organ function under physiological and pathological conditions  signaling. Delta-like ligand 4 (DLL4) has been shown to be a critical Notch ligand for stimulating angiogenesis . Howeverogenesis .Moreover, Notch signaling may directly and/or indirectly regulate angiogenesis through crosstalk with VEGF receptors (VEFGRs) . For insThe ability of Notch signaling to dictate angiogenetic processes becomes important in the context of aberrant angiogenesis. Pathological angiogenesis plays an important role in abnormal vessel growth and contributes to the pathogenesis of such human diseases as diabetic retinopathy, ischemic cardiovascular diseases, inflammatory processes (rheumatoid arthritis), and cancer . In cancde novo arteriogenesis, the growth of functional collateral arteries from preexisting arterio-arteriolar anastomoses and de novo arterial development, in which smooth muscle cells (SMCs) cover ECs  and arteriolar ECs in the vascular system. CD36 downregulation has been linked to the upregulation of pro-angiogenic and arterial genes . Severalnditions . Howevernditions . This po the TME .Regulation of cell fate decisions is a hallmark characteristic of Notch signaling. In the vasculature, a primary effect of Notch signaling is the promotion of stalk/tip cell specification . During Crosstalk between VEGF and Notch signaling is fundamental to sprout formation, vascular EC maintenance, and the establishment of EC heterogeneity . When NoNotch signaling also plays a role to prevent excessive sprouting. Upon DLL4-mediated Notch activation, the induction of tip cells is repressed by a process called “lateral inhibition.” During lateral inhibition, VEGF signaling promotes the upregulation of DLL4 in tip cells, which in turn trans-activates Notch in the neighboring cells. Interestingly, a recent mathematical model of Notch-Jagged signaling revealed intriguing aspects about the existence of a third EC state. In addition to the two EC states , Notch signaling is also required for the proper formation of vascular branches. Recent studies have demonstrated that Notch regulates endothelial branching in response to other pathways, including BMP and SMAD1/5/8 signaling . SMAD6, in vivo experiments demonstrated that a VEGF-mediated positive signaling feedback can shape the timing, magnitude, and robustness of determining tip cell identity for angiogenic branching, which is terminated by DLL4-Notch signaling. Mechanistically, the induction of expression of tm4sf18  dictates the speed and robustness of EC selection for angiogenesis by stimulating VEGF signaling  by increasing the levels of Bcl2 , TNF-α, poptosis . The effin vitro and in vivo models. However, the toxicity of Notch inhibitors to normal cells should be considered. To minimize the side effects, cancer-specific targeting should be an optimal strategy.In the context of cancer, Notch signaling has been associated with multiple mechanisms of apoptosis. For example, the activation of Notch1 in colon cancer inhibits apoptosis by repressing p27 expression . Other sEndothelial-to-mesenchymal transition (EndoMT) is a process where ECs lose their endothelial-specific phenotype and progressively acquire mesenchymal properties such as formation of spindle-shaped elongated cell morphology, loss of cell–cell junctions and polarity, and increased cellular motility, loss of endothelial markers  cadherin), and the acquirement of mesenchymal features  .As major components of the vascular networks in the TME, ECs demonstrate plasticity and the potential to transdifferentiate into mesenchymal cells via EndoMT. EndoMT has been shown to participate in critical embryonic developmental processes and the pathogenesis in a variety of genetic and acquired human diseases including cancer-associated fibrosis , cardiacin vivo, particularly under pathological conditions such as tumor angiogenesis /EndoMT, the concept of partial EMT/EndoMT has emerged recently, which was described by ogenesis . The assNotch activation in conjunction with sprouting angiogenesis could be a crucial determinant of either partial or full EndoMT. However, other signaling pathways such as Wnt and TGF-β signaling pathway are also capable of promoting partial EndoMT. Mechanistic studies have demonstrated a role for both TGF-β and bone morphogenic protein (BMP) in conjunction with Notch signaling to regulate EndoMT in embryonic heart formation . In addiWhile the interaction between Notch and EndoMT is pivotal during tumor progression via a complex modulation of both the tumor and the surrounding stroma . Ghiabi These studies strongly support a role for Notch signaling in the development of multiple biological processes and diseases through regulation of EndoMT. Significantly, it is worth investigating whether and how Notch signaling contributes to the development of metastatic niche in tumor progression and therapeutic resistance through promoting EndoMT and/or partial EndoMT, either of which might play a critical role in the regulation of TME plasticity and CSC heterogeneity.Dysregulated Notch signaling has been documented in a variety of human cancers including melanoma , lymphomOncogenic Notch signaling activation is often linked to an increase in cellular proliferation, inhibition of apoptosis, suppression of cellular differentiation, and the induction of CSC features . In T-cein vitro and in vivo in a dose-dependent manner , medullary thyroid cancer (MTC), and skin cancer . In bladt manner , 2015. St manner .The crosstalk between Notch and other cell signaling pathways may explain some of the contrasting tumorigenic consequences that are observed in different types of cancer. Notch signaling is known to interact with multiple other oncogenic signaling pathways such as epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF), NF-κB, Akt, Hedgehog, mTOR, Ras, Wnt, estrogen receptor (ER), and androgen receptor (AR) signaling .The interaction of Notch1 with NFκB promotes tumor progression in colorectal adenocarcinoma through inhibition of apoptosis via the upregulation of the Bcl-xL . The comConversely, Notch1 functions as a tumor suppressor to transform high-grade adenomas into low-grade ones in murine models of colon cancer by epigenetically suppressing Wnt pathway target genes . In addiTo better understand the role of Notch signaling in tumor development, it is important to consider the innate function of Notch in the cell or tissue type being studied. Just as the impact of Notch signaling varies among cancer types, it also varies among normal tissue types. For example, Notch signaling plays different roles during the normal development of different cell types in the pancreas. The Notch pathway plays a pivotal role in deciding the fate of progenitor cells as either exocrine cells  or endocrine cells (Islets of Langerhans) by lateral inhibition .In summary, Notch signaling has a significant functional role in a broad spectrum of human cancers. The oncogenic involvement of Notch signaling across different cancer types has emphasized its centralized role in the regulation of tumor formation, progression, and therapeutic resistance. The distinct role of Notch signaling in tumor progression emphasizes the need for further studies to better understand this pathway in a cellular- and tumor type-dependent manner. A deeper understanding of Notch signaling in various biological settings and pathological conditions may reveal key opportunities for effective therapies that target Notch in tumors. We will discuss the role of Notch signaling in tumor angiogenesis, cancer stem cells (CSCs), therapeutic resistance, and signaling crosstalk between CSCs and vascular ECs as well as the clinical potential of targeting the Notch signaling pathway in more detail.Tumor angiogenesis is characterized by excessive endothelial sprouting from preexisting blood vessels, which leads to overgrowth of randomly organized tumor vessels including venous and arteriolar vessels and capillaries . The groYin and Yang balance between pro-angiogenic and anti-angiogenic signaling to control overall angiogenic responses. During this dynamic angiogenic process, angiogenic receptors may serve as a pivotal axis to regulate the angiogenic functions of corresponding ligands. As an example, VEGFR2 is critical for upregulating VEGF signaling, while CD36 signaling may function as a critical negative regulator of angiogenesis (Stimulation of neoangiogenesis in the TME depends upon the induction of such pro-angiogenic factors as VEGF and basic fibroblast growth factor (bFGF) . The croogenesis .Several studies have highlighted the critical role of Notch signaling in the overall regulation of tumor angiogenesis . The NotIntriguingly, tumor vasculature becomes disorganized and overgrown upon pharmacologic inhibition of DLL4, ultimately reducing overall tumor growth due to dysfunctional tumor angiogenesis . Given tNotch signaling also regulates tumor angiogenesis and metastasis in a context-dependent manner. Notch signaling can upregulate Jag1 to antagonize the DLL4-dependent ‘stalk’ phenotype, thereby promoting EC sprouting and proliferation . ActivatAccumulating evidence in CSC research suggests that the principles of anti-cancer treatments should be revised with consideration for the intrinsic heterogeneity of cancer cells. CSCs are a subpopulation of cells within a tumor that are able to promote neoplastic initiation, growth, survival, and metastasis . CSCs arNotch signaling has emerged as a key pathway in CSCs. This signaling pathway may be involved in the regulation of survival, plasticity, and self-renewal of CSCs. Blockade of the Notch pathway using γ-secretase inhibitors can decrease CSC numbers, increase apoptosis, and limit tumorsphere formation efficiency, a measure of cancer stemness . Howeverin situ to invasive cancer and may worsen overall and recurrence-free survival , a protein expressed on the surface of breast CSCs, can lead to the maintenance of stemness through continuous self-renewal . Notch1 +/CD24–) phenotype reside at the invasive edge of the tumor, whereas a population of hybrid epithelial-mesenchymal (E/M) CSCs are localized within the tumor interior. Thus, a primary tumor may contain spatially distributed cells with varying extents of EMT. This spatial distribution of CSCs and EMT heterogeneity may be attributed to a TGF-β diffusion gradient and cell–cell communication through Notch-Jag1 signaling induced by inflammatory cytokines such as IL-6. Furthermore, knockdown of Jag1 in the hybrid E/M SUM149 TNBC cell line suggests a role for Notch-Jag1 signaling in the maintenance of the CSC phenotype , and smooth muscle cells (arteriolar and venous vessels) surrounding ECs . A seminin vitro . Gamma-secretase is the orchestrating enzyme of the crucial proteolytic activity that releases the NICD and is a key player in the canonical Notch signaling pathway. GSIs prevent cleavage of the NICD by blocking the proteolytic function of presenilin enzymes, thereby preventing Notch pathway activation . Therefoin vivo. In murine models of colorectal cancer, blocking Notch signaling with an anti-DLL4 antibody reduced the frequency of CSCs, delayed recurrence of tumors, and decreased metastasis , Due to its driving role in many types of cancer, significant attention has been paid in recent years toward the development of clinically valuable antagonists of Notch signaling. However, there are still many gaps to uncover in this field due to the heterogeneity and complexity of the molecular biology in human cancers including neuroendocrine tumors and breast cancers as well as in cardiovascular diseases. In terms of cancer treatment, a popular new strategy is the development of target-selective “smart” drugs based upon the foundation of characterized molecular mechanisms. The crosstalk between Notch signaling and many other oncogenic signaling pathways in carcinogenesis presents multiple opportunities for the design of new drugs aimed at blocking these interactions. However, this would require new studies to better understand the precise mechanisms by which Notch signaling pathway and molecular signaling critical to the Notch pathway promotes or inhibits tumor progression. In addition, developing innovative tools to more accurately predict their prognosis, and designing and testing personalized treatment strategies would be helpful to facilitate the optimal Notch targeting in cancer patients.Research in the past decade has focused upon eliminating tumor recurrence by targeting CSCs. Targeting Notch may be an optimal therapeutic strategy to disrupt the arteriolar niche and eradicate CSCs. However, targeting the Notch pathway may elicit cardiovascular toxicities because of its critical role in the maintenance of cardiovascular homeostasis by EC DLL4-Notch signaling . ThroughAA, AG-G, and RG wrote the manuscript. AG-G made the figures. JR, AB, and HC reviewed the manuscript. BR conceptualized and wrote the manuscript. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The dynamic coordination between kinases and phosphatases is crucial for cell homeostasis, in response to different stresses. The functional connection between oxidation and the intracellular signaling machinery still remains to be investigated. In the last decade, several studies have highlighted the role of reactive oxygen species (ROS) as modulators directly targeting kinases, phosphatases, and downstream modulators, or indirectly acting on cysteine residues on kinases/phosphatases resulting in protein conformational changes with modulation of intracellular signaling pathway(s). Translational studies have revealed the important link between oxidation and signal transduction pathways in hematological disorders. The intricate nature of intracellular signal transduction mechanisms, based on the generation of complex networks of different types of signaling proteins, revealed the novel and important role of phosphatases together with kinases in disease mechanisms. Thus, therapeutic approaches to abnormal signal transduction pathways should consider either inhibition of overactivated/accumulated kinases or homeostatic signaling resetting through the activation of phosphatases. This review discusses the progress in the knowledge of the interplay between oxidation and cell signaling, involving phosphatase/kinase systems in models of globally distributed hematological disorders. In the last decade, growing evidence has highlighted the importance of reactive oxygen species (ROS) interfacing intracellular signaling pathways to ensure cell survival ,2. This +/CD19+/CD23+ B lymphocytes proliferate in lymphoid tissues and bone marrow (BM), accumulating as mature quiescent cells ,10586,10In erythrocytes, the importance of intracellular signals against exogenous/endogenous oxidative stress is supported by evidence in mouse models genetically lacking either phosphatases such as PTPε or kinases such as Lyn or Fyn ,11,108. In erythrocytes, the imbalance of phosphatase–kinase activities might result in abnormal band 3 Tyr-phosphorylation state associated with reduced membrane mechanical stability ,112,113.Studies in G6PD deficient red cells exposed in vitro to diamide, β-thalassemic erythrocytes or sickle erythrocytes undergoing in vitro deoxygenation, have had documented an over-activation of Syk bypassing the canonical cascade and targeting clustered band 3 ,116,117.The proper function and survival of red cells in peripheral circulation requires the maintenance of optimal cell volume. This is regulated by the activity of various membrane ion transport pathways and channels, the large part of which is regulated by phosphorylation/dephosphorylation events ,19,118. A fine balancing between kinases and phosphatases is crucial in cell homeostasis. Oxidative stress might affect cell signaling pathways directly by targeting either kinases or phosphatases, or indirectly by inducing the activation or overactivation of cytoprotective systems such as in CLL cells. SFKs are involved in different cellular processes such as maturation, growth, and survival. The over-activation or the intracellular accumulation of active SFKs, such as Lyn, results in severe perturbation of the dynamic coordination between kinases and phosphatases, deeply affecting cell homeostasis and cell survival. The intricate nature of intracellular signal transduction mechanisms, based on the generation of complex networks of different types of signaling proteins, revealed the novel and important role of phosphatases in disease mechanisms such as in CLL. Thus, therapeutic approaches to abnormal signal transduction pathways should consider either inhibition of overactivated/accumulated kinases or “homeostatic” signaling resetting. This is no longer based on single-target inhibitory drugs, such as Lyn-specific inhibitors, but on up-modulation of the physiological molecular counter balancer of protein kinases, that is protein phosphatases. Such “homeostatic” approaches to intracellular signal transduction modulation allow inducing an equilibrated “resetting” of imbalanced signaling mechanisms, thus eliminating the typical side effects of traditional pharmacological approaches based on mono-target therapies. In addition, potentiation of endogenous antioxidant system(s) might represent an attractive additional therapeutic strategy to limit prolonged/chronic oxidation involved in the pathogenesis of incurable disorders such as CLL or invalidating diseases such as β-thalassemia or SCD. Thus, future studies should be designed to better understand the dynamic coordination between kinases and phosphatases and its connection with oxidation in hematological disorders. This will allow progression beyond the state of the art and the possible identification of new drug targets.


Fucus vesiculosus and Saccharina latissimi) with or without the use of a microbial inoculant. The potential applications of seaweed silage as a feed ingredient in ruminant diets were considered. The results showed that, depending on the species, ensiling may be a suitable preservation method for brown seaweeds.In recent years, there has emerged a renewed interest in the inclusion of seaweed as an animal feed ingredient. Due to annual fluctuations in the availability and biochemical composition of seaweeds, effective preservation methods are needed. These are currently restricted to thermal processing methods. Ensiling is a commonly applied preservation technique for terrestrial forages intended for livestock feed but little is known about the characteristics of silage made from seaweeds. This study considered the potential of ensiling two brown seaweed species (Fucus vesiculosus (FV) and Saccharina latissimi (SL) with or without the use of a Lactobacillus plantarum (LAB) inoculant. The effect of ensiling on the stability of phlorotannin was also investigated using nuclear magnetic resonance (NMR). After harvesting, the seaweeds were wilted for 24 h and subsequently ensiled in laboratory-scaled silos for 90 days. SL silage showed a stronger fermentation pattern (pH < 4), dominated by lactic acid (50–60 g/kg Dry Matter (DM)), and a slightly higher acetic acid content compared to FV silages (p < 0.05). The fermentability of FV was limited (pH > 4.8) with low lactic acid production (<5 g/kg DM). The addition of the LAB inoculant showed no effect on the fermentation process but a modest effect on the chemical composition of both species was observed after the 90-day ensiling period. The results showed no losses in the nutrient content of FV after ensiling, however losses in the Crude Protein , ash (−36%), Neutral Detergent Fibre  and Acid Detergent Fibre  content of SL were observed. The ensiling process had a limited effect on the in vitro true dry matter digestibility and phenolic content of either species. Therefore, ensilage may be a suitable preservation method for the use of brown seaweeds as a ruminant feed; however, species-specific differences were observed.Ensiling could be an effective method to preserve seaweeds for animal feed applications, however, there is limited scientific knowledge in this area. Seaweeds are a promising ruminant feed ingredient, in part due to the content of phenolic compounds, which are receiving considerable interest as alternative antimicrobial agents in feed. The aim of the study was to compare the effect of ensiling on the nutritional composition and fermentation characteristics of two brown seaweed species,  Lactobacillus plantarum (LAB), are commercially available and have been successfully used for increasing the fermentation quality of the ensiled forage [Global seaweed production doubled from 14.7 in 2005 to 30.4 million tonnes in 2015 and continues to grow at an annual rate of about 6% . Phenolid forage . Howeverd forage ,22.Fucus vesiculosus and Saccharina latissimi) with or without the use of a LAB inoculant. The stability of phlorotannins, before and after ensiling, was also investigated to assess the potential of seaweed in ruminant nutrition as a functional feed ingredient.The aim of the study was to compare the effect of ensiling on the nutritional composition and fermentation characteristics of two brown seaweed species (Fucus vesiculosus (FV) and Saccharina latissimi (SL) were randomly collected by hand from a sheltered beach in Bangor, County Down, Northern Ireland , either from the intertidal (FV) or subtidal area (SL), during low tide in July 2017. The areas were divided into three equal areas, and approximately 1-kg portions (fresh weight) of each species were collected from each area. Seaweed samples were identified by a marine phycologist and stored in sample bags to be transported to the laboratory. The seaweeds were washed thoroughly with cold fresh water to remove sand and particulate species prior to further processing. Two species of brown seaweed, 9 CFU/kg FW) of Lactobacillus plantarum (strain NCIMB 3014) previously dissolved in deionized water and thoroughly mixed by hand, according to the manufacturer’s instructions. The remaining seaweeds (SLu/FVu) were treated in the same way without the dissolution of the LAB culture in deionized water, and thus underwent natural fermentation. The seaweeds were vacuum-packed in sterile bags (dimensions 20 × 30 cm) using a vacuum packing machine  to remove ≥ 95% of the air from the bags. The silos were then stored in the dark for 90 days. After the storage time, three bags from each treatment were randomly selected for fermentation analysis and the remaining bags were frozen at −20 °C, and later freeze-dried and milled through a 1-mm sieve for further chemical and in vitro digestibility analysis.The silage preparation was carried out according to the method of Wout et al. . The wasn = 3) were prepared by mixing 30 g of silage from each bag in 150 mL of deionized water. The pH of the filtrate was determined using a pH electrode  after shaking the solution at 4 °C overnight and filtering through Whatman No. 6 filter paper. Ammonia-N (NH3-N) was determined using a calibrated ammonia ion-selective electrode . Volatile fatty zcids (VFA) and alcohols were measured following ethanol/water extraction using gas chromatography analysis  equipped with a Zebron ZB-FFAP  Capillary column , and nitrogen as the carrier gas. The instrument was coupled to a flame ionization detector. Silage extracts , was used as certified reference material; a variability of ± 20% (of recovery) was considered acceptable. All chemical analyses were carried out in triplicate.Pre- and post-ensiled seaweed samples were analysed for dry matter (DM) (AOAC 930.15) and ash (AOAC 942.05) according to AOAC official methods . Neutralg . Following the addition of Folin– Ciocalteu (1N) reagent and aqueous sodium carbonate (20% w/v), the solution was stored in the dark for 60 min and the absorbance was read at 725 nm . Phloroglucinol  was used as an external standard (1.0 to 50 µg/mL), and results were expressed as g of phloroglucinol equivalents per kg DM (g PGE/kg DM). Pre- and post-ensiled seaweed samples were analysed for total phenolic content (TPC) using the Folin–Ciocalteu method, adapted from Li et al. . Briefly13C-NMR spectroscopy was carried out on a Bruker DPX600 NMR  spectrometer. Chemical shifts were reported in parts per million (ppm).The stability of phenolic compounds was observed using nuclear magnetic resonance (NMR) by investigating the molecular structure of the compounds before and after ensiling. The number of scans was set at 256 and the chemical shifts were referenced by setting the TMS peak to 0 ppm. The seaweed extracts were prepared using 0.3 ± 0.05 g of freeze-dried seaweed material in a deuterated acetone–water mix . After 30 min at room temperature, the acetone was removed by rotary evaporation and the resultant solid was re-dissolved in 0.1 mL deuterium oxide . II incubation method, followed by NDF digestion in an ANKOM 200 Fibre Analyzer , as described previously by Holden [2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.1 g of CaCl2·H2O and 0.5 g of urea in 1 L of deionized water) and solution B (15 g of Na2CO3 and 1 g of Na2S.9H2O in 1 L of deionized water). A final buffer solution was made by mixing solutions A and B (5:1 ratio) before adjusting the pH to 6.8. Rumen contents were collected from three post-slaughter bovine rumens from an abattoir in N. Ireland. The rumen contents were then isothermally transported to the laboratory, homogenised, filtered through four layers of cheesecloth and purged with CO2. The rumen fluid was added to the digestion jar containing the pre-warmed (39 °C) buffer solution (v/v 1:5). After a 48-h incubation period, the bags were rinsed four times with distilled water, and the residues were subjected to digestion for 60 min in neutral detergent solution and dried at 60 °C for 48 h. The in vitro true dry matter digestibility (IVTDMD) was calculated as the difference between the dry matter incubated and the residue after neutral detergent treatment divided by the dry matter incubated. A total of two in vitro incubation runs were carried out and each time the sample was incubated in triplicate.In vitro true dry matter digestibility was determined using the Daisyy Holden . Ground y Holden . Prior tStatistical analysis was performed using JMP version 14.0 . The statistical model used for the analysis of variance (ANOVA) was:p < 0.05) compared to SL0. On the other hand, SL0 was characterized by higher (p < 0.01) CP, ash, aNDF and IVTDMD compared to FV0. In terms of the mineral composition (p < 0.001) in Mn (114 mg/kg DM) and S (14 g/kg DM) compared to SL0 .position , SL0 wasp > 0.05) between the species, as the CP of SL reduced from 60 to 41 g/kg DM after 90 days. There was a large reduction in the aNDF and ADF content of SLu compared to SL0. Furthermore, the ash content of SL reduced from 242 to 156 g/kg DM after ensiling. p < 0.05) in the following trace minerals were observed between SL0 and SLu samples, respectively: Mn (33 to 17 mg/kg DM), Fe (161 to 90 mg/kg DM), Zn (25 to 18 mg/kg DM) and Br (862 to 720 mg/kg DM). Ensiling had no effect on the high iodine content of SL, which remained >2000 g/kg DM after 90 days. Furthermore, the IVTDMD of SL remained high (>90%) after ensiling. In general, the ensiling process had a limited effect on the chemical composition or IVTDMD of FV; the differences observed among the species before and after ensiling seemed to be related to changes in the SL values. The addition of the inoculant showed a modest effect on the chemical composition of both species after the 90-day ensiling period (p < 0.01) (45.7 vs. 41.1 g/kg DM), whereas a quantitatively modest increase (p < 0.05) in the WSC content was also observed for the same treatment. Regarding FV, FVi showed a higher (p < 0.05) TPC than FVu.g period . The CP p < 0.05). Butyric acid and valeric acid were absent in all silage samples tested. The ethanol content was also higher in SL compared to FV silages. The use of the LAB inoculant showed no effect (p > 0.05) on any of the end-point silage fermentation parameters analysed. p > 0.05; 13C-NMR spectra displayed in 13C-NMR in brown seaweeds has previously been reported by Ford et al. [13C-NMR spectra showed a reduced signal after ensiling FV, compared to FV0 (blue spectrum). This was particularly evident when the inoculant was applied to the ensiling process, which is also reflected in The TPC of FV0 was more than 20 times greater than that of SL0. Moreover, ensiling did not alter the TPC content of either species  than the recommended levels for optimal silage quality (>25% FW) . A shortAscophyllum nodosum silage. The limited lactic acid fermentation of FV was probably due to the high phenolic content. The TPC of FV0 was 17 g/kg DM, although contents of > 20 g/kg DM have been reported elsewhere [F. vesiculosus, are produced as a defence mechanism, and as such have potent antimicrobial properties [This is the first study to address the potential of ensiling FV for livestock feed. The fermentation of FV was limited compared to SL. The pH of FV silage failed to reach the optimal silage range (3.8– 4.2), as evidenced by a lack of organic acid production. These findings are in line with Herrmann et al. , who foulsewhere ,37. SL ilsewhere . It is llsewhere ,37,38. Poperties . These moperties ,18. The Lactobacillus plantarum inoculant, at a rate of 109 CFU/kg fresh seaweed, had a limited effect on the fermentation quality of both species. This demonstrates that the seaweeds ensiled under natural conditions underwent a similar fermentation process compared to those provided with a microbial stimulant. This provides further evidence that seaweeds harbor endogenous enzymes required for the hydrolysis of complex carbohydrates, as suggested by Sandbakken et al. [L. plantarum is a slow lactic acid producer at pH > 5, and therefore the inability of the pH to drop below 4.9 in FV silage probably limited the effect of the inoculant on silage quality. Furthermore, the low initial WSC content of both species (< 60 g/kg DM), and the inability of L. plantarum to degrade algal cell walls, together with the high content of Cl, I and other minerals, might have limited the inoculant’s efficiency [L. plantarum inoculant reduced the CP content of SL silages. Overall, the results showed that a favourable lactic acid fermentation was achieved, even without the use of the microbial inoculant. Further work is needed to explore the changes that occur within the microbial communities during the ensilage of seaweeds and the potential beneficial effects of different inoculant types on silage quality.The effects of any microbial inoculant on silage quality are dependent on a range of factors, including the original forage composition, epiphytic microflora, ensiling conditions and the type of inoculant used . Accordin et al. . L. planficiency ,42. Regaficiency . These fficiency , who fouSaccorhiza polysaccharides (10 g/kg DM), fermentation still occurred. The authors concluded that this was probably due to the use of alternative microbial energy sources. In fact, in the present study, the ensiling process decreased the aNDF and ADF fractions of SL from 384 to 86 g/kg DM and from 145 to 60 g/kg DM, respectively. A similar finding was observed during the ensilage of the red seaweed, Gracilaria vermiculophylla [The initial content and availability of WSC can be a limiting factor during the ensiling process . In the lophylla . The stolophylla . Laminarlophylla , represelophylla , and arelophylla . The strlophylla . Therefolophylla ,45. The lophylla ,47 and clophylla . Our finlophylla . In addilophylla ,49.13C-NMR was used to assess the differences in the linkage profiles of the purified phenolic extracts before and after ensiling. The 13C-NMR spectra in L. plantarum inoculant, the signals from the phenolic compounds were relatively diminished, which indicated that there was a degradation of the phenolic compounds. This is also reflected in the FC assay data, although it is noteworthy that the FC assay is indirect and less specific compared to the NMR method [Moreover, R method ,50. NoneR method . Hence, 3N content of SL silage was between 50 and 70 g/kg CP, which is in agreement with this study, which found the NH3N of FV and SL to be 53 g/kg CP and 63 g/kg CP, respectively. Whilst the ensiling process did not incur any losses in the CP content of FV, a decrease from 60 g/kg DM to <46 g/kg DM CP was observed for SL. This might be explained by the lower phenolic content of SL compared to FV. The protein-binding capacity of phenolic compounds in seaweeds has been demonstrated previously [3N content between FV and SL silages. Instead, other products of protein degradation may have been generated [In terrestrial-based forages, protein represents 75–90% of the total nitrogen content. After ensiling, much of the nitrogen is transformed into soluble non-protein nitrogen, which is rapidly degraded by microbes in the rumen, thus implying a loss in protein use efficiency. Ammonia-N is commonly used as an indicator for the extent of proteolytic activity during the ensiling process . Previoueviously  which coenerated , which wAscophyllum nodosum; this could have implications for milk safety from a human nutrition perspective [The ash contents of FV0 (133 g/kg DM) and SL0 (242 g/kg DM) were comparable to levels reported previously for the same species ,53. The spective . Based ospective , the incGracilaria vermiculophylla silages which negatively affected the dry matter digestibility. However, in the current study, the ensiling process had no effect on the IVTDMD of SL. The reason for this is unclear, but it suggests that seaweed components that were not measured by detergent fibre analysis were utilized by the rumen microbes during the in vitro incubation period. The digestibility of seaweeds is expected to be highly variable and dependent on the species, the carbohydrate profile and composition , as well as on the adaptation of the animal to the feed [Previous studies have noted the differences in the digestibility of various seaweed species in the ruminant diet due to biochemical differences in their composition ,58. In athe feed . In vivothe feed ,18 and hthe feed ,61. Indethe feed . This wathe feed , which wLactobacillus plantarum inoculant had a minor effect on silage quality, although proteolysis was partially mitigated by its addition in SL silages. The high I content, particularly regarding SL, is a major concern for the use of seaweed silage as a ruminant feed. Finally, ensiling had a minor effect on the phlorotannin content of brown seaweeds but a better understanding of their biological activity post-ensiling is needed to improve our appreciation of their contribution to the nutritive value of seaweed silage. Further questions regarding optimal dietary inclusion rates and the potential effects on animal productivity, including milk and meat quality, need to be addressed before the use of seaweed silage as a ruminant feed can be implemented.The present study found species-related differences in the silage characteristics of brown seaweeds. SL silage showed a homofermentative pattern, with a suitable pH decrease dominated by lactic acid production. Whilst the TPC and IVTDMD of SL were unaffected by the ensiling process, losses in CP, ash, NDF and ADF were observed. In contrast, FV underwent a restricted lactic acid bacteria fermentation despite the use of an inoculant, with few changes in the chemical composition post-ensiling. The


S-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a GS loss-of-function (S127L) and a GS gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the Gq/11 pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations.The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin–melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via G MC4R gene were identified in obese patients [A complex hypothalamic network of orexigenic and anorexigenic signals regulates body weight and energy expenditure. One of the key players in this network is a G protein coupled receptor (GPCR), the melanocortin 4 receptor (MC4R), which is mainly expressed in the paraventricular nucleus (PVN) of the hypothalamus. The MC4R receives input from the leptin–melanocortin pathway and thereby integrates peripheral and central metabolic signals. Activation of the MC4R by the proopiomelanocortin (POMC)-derived melanocyte stimulation hormones alpha and beta  counteracts the signal of orexigenic peptides such as neuropeptide Y (NPY), Agouti-related peptide (AgRP) or ghrelin, and therefore deceases appetite ,2,3. Itspatients .S, which leads to an increase in intracellular cyclic adenosine-monophosphate (cAMP) [i/o, Gq/11 and G12/13 [4, d-Phe7)-α-MSH (NDP-α-MSH) [The MC4R signals mainly via activation of the stimulating G protein Ge (cAMP) . In recee (cAMP) ,8,9, andd G12/13 ,11,12,13d G12/13 . The recd G12/13  and is ad G12/13 . The endd G12/13 ,17 or asd G12/13 ,18. In aP-α-MSH) .S signaling. However, other MC4R mutations were described as wild type (WT)-like and were, therefore, believed to be irrelevant for the obese patient phenotype.So far, many mutations identified in obese patients have been functionally characterized in overexpressing in vitro systems, and a number of these mutations showed a loss-of-function in GMC4R mutations have been identified that exhibit biased signaling, which is defined as the selective activation or inactivation of one pathway over another [MC4R mutations in the mitogen-activated protein kinase (MAPK) pathway. Interestingly, in some of the tested mutations no functional defect in GS signaling had been described before [S signaling but with a markedly affected ability to recruit β-arrestins [In recent years, several  another . Tao andd before . Recentlrrestins ,23.S-signaling properties in order to fully characterize MC4R mutations. The aim of the current study was, therefore, the comprehensive characterization of three prototypical MC4R mutations in their ability to activate all four major G protein families, plus ERK activation, β-arrestin2 recruitment and ligand-dependent internalization. As a proof of principle study, we focused on a very common MC4R variant, V103I, and two MC4R missense mutations: S127L and H158R.These observations indicate that it may be necessary to study more than just the receptor’s GS signaling [MC4R mutation, S127L, has been frequently reported as a partial loss-of-function in GS signaling, which has an increased constitutive activity and is located in TMD 3 [MC4R mutation, H158R, is a constitutively active gain-of-function mutation in GS signaling that has a decreased ability to form homodimers [MC4R variant, which is defined by a minor allele frequency (MAF) above one percent in the normal population, whereas the other two are considered to be rare with a lower MAF, we will continue referring to all of them with the term “mutations”.The most common single nucleotide polymorphism (SNP) in Europe, V103I, is located in the transmembrane domain (TMD) 2, and has been mostly described as WT-like in Gignaling ,27,28,29ignaling ,35,36,37in TMD 3 ,43,44,45modimers ,47. It iS activation pattern, these three mutations are vastly different. However, these findings do not strictly align with the phenotype. This suggests that other GPCR signaling pathways might be important for weight regulation, and therefore these were investigated in the present study.With regard to the GMC4R mutations in vitro for all known/so far reported signaling pathways to explore the role of GS and non-GS pathways in weight regulation. Therefore, in addition to cAMP measurements to assess GS activation in the absence and presence of 100 nM AgRP, we measured Gi/o activation via determination of second messenger (cAMP) modulation by adenylyl cyclase (AC) in the absence or presence of pertussis toxin (PTX) pretreatment. In the presence of PTX, which locks the Gαi/o subunit in its GDP-bound inactivated state, the G protein is no longer able to inhibit the AC to form cAMP [i/o signaling to the total amount of intracellular cAMP, we will refer to these measurements as Gi/o coupling in order to simplify the results part. We used reporter gene assays measuring phospholipase C (PLC) activity through nuclear factor of activated T-cell (NFAT) response for Gq/11, RhoA activation via serum-response factor (SRF) for G12/13, and ERK1/2 phosphorylation via the serum response element (SRE). Recruitment of β-arrestin2 by the receptor after the ligand challenge was determined by protein interaction studies and total and cell surface expression were analyzed using a protein complementation assay. In addition, ligand-induced internalization was visualized by staining the receptor with a fluorophore-coupled ligand.In this study, we characterize the three described orm cAMP . AlthougCell surface and total expression of all three variants were determined. This provides information on protein folding and how the mutation influences the trafficking of the receptor to the plasma membrane, which may influence signaling. Introduction of these three mutations into the MC4R did not result in any significant changes in cell surface expression and total expression .MC4R mutations, we used confocal imaging of labeled MC4R as well as β-arrestin2 recruitment as an indication of receptor desensitization. In our experience, MC4R C-terminally tagged with GFP is exhibiting signaling deficits, hence we chose another labeling approach. We used TAMRA-labeled NDP-α-MSH in order to stain receptors expressed at the cell surface and quantified the amount of labeled receptors in endosomes after 30 min of stimulation using confocal images  peptidic ligand. Therefore, an easy way to communicate biased signaling was used to present the results in a more comprehensive way, by plotting the bias calculated as described in the method section for each MC4R mutation. The values derived from concentration–response experiments  and the potency of the ligand at the receptor mutation (EC50). Values were normalized to the reference, MC4R WT, which is plotted as 1.0 for each pathway. Values above 1.0 represent an increase in signaling capacity in this pathway in comparison to the WT, whereas values below 1.0 represent a decrease in activity. Mutations with a WT-like behavior have bias values close to 1.0, meaning that there is in fact no bias. Efficacy, potency and bias values are stated in A complete characterization of all known pathways for three receptor mutations for three different agonists generates a substantial amount of data . The intMC4R mutations are usually performed with the endogenous agonist α-MSH in most published studies. We characterized MC4R mutations V103I, S127L and H158R in all mentioned pathways with this ligand and identified that all three mutations showed clear signaling biases towards particular pathways  [S signaling in the presence of AgRP implies a loss-of-antagonist effect for the mutation. Notably, H158R showed a similar strong bias towards Gq/11 as the V103I mutant with a rise in PLC activation. In other tested pathways, the mutation closely resembled the WT activity.We found the already-reported gain-of-function in Ge (Emax) . InteresThe second endogenous agonist for MC4R, β-MSH has been neglected in most MC4R studies so far, but is proven to be important for weight regulation ,48,49 anq/11 coupling, its capacity to activate the GS pathway is reduced (though not significantly), which is also reflected in its response to the antagonist AgRP. In addition, it showed a strong positive bias towards Gi/o coupling, RhoA activation via G12/13 and increased ERK phosphorylation. S127L on the other hand again presented full loss-of-function in all tested pathways and even its response to AgRP was impaired. The responses of H158R to β-MSH were comparable to its α-MSH responses with increased GS and Gq/11 signaling and WT-like activity in G12/13 and β-arrestin2 recruitment, though it also showed augmented ERK phosphorylation. Again, AgRP loses its antagonistic effect for this mutation. In contrast to α-MSH, H158R showed a strong bias towards Gi/o signaling when stimulated with β-MSH.Here, the V103I mutation presents itself with a distinctly different signaling behavior compared to α-MSH stimulation. Although it still exhibits an increased GMC4R mutations. Since it is widely used, we included this ligand in our study  How does a mutation influence the total receptor expression in the cell? (2) What is the impact of the mutations on cell surface expression? (3) Is there any signaling bias involving all known signaling pathways and endogenous ligands for this specific GPCR? (4) Does a mutation create any bias of ligand-induced G protein activation versus β-arrestin recruitment? (5) Does an altered response to the antagonist (in case of a known endogenous antagonist) exist? This extensive study can help to elucidate the role of each of these mechanisms in the physiological and potential pathophysiological role of a receptor and its mutations.50 should only be calculated if the maximal response is greater than 25% of the maximal WT activity. In addition, the use of different read-out systems for signaling pathways makes a comparison between different studies difficult, therefore a clear statement is necessary on how those data were determined.Furthermore, it will be of importance to establish benchmarks and guidelines concerning the way in which in vitro data of the functional characterization should be presented. Here, it would be relevant to present either raw data or calculated values normalized to WT basal activity  to ensure comparability of assay data. We recommend with respect to MC4R-related in vitro assays that ECS signaling [Additionally, the choice of cell system may affect the results and should be considered carefully. Endogenous cell systems are always considered the best choice; however, for many studies those are not available. Chinese hamster ovarian (CHO) and African green monkey (COS) cell lines are easy to transfect and are able to produce high amounts of second messengers such as cAMP, which gives them an important role in GPCR research. For neuroendocrine receptors, hypothalamic rodent cell lines, such as GT1-7 or mHypoE-N39 and mHypoE-N41, are suitable due to their close physiological background, but with current options, transfection has been shown to be difficult. For all the mentioned cell lines, their non-human background complicates any actual interpretations on the physiological roles of human receptor function. Human cell lines such as human embryonic kidney cells (HEK293) are easy to transfect and suitable for a variety of functional assays and are well established in GPCR research. HEK293 cells are proven to not express MCRs  and do nignaling , is margignaling . This neFor a long time, ERK phosphorylation was considered as a G protein-independent and β-arrestin-dependent signaling pathway. Very recently, Grundmann et al. reported data that hint towards the need for G protein presence for ERK phosphorylation, whereas β-arrestins seem to be less involved . TherefoMC4R mutations had a very variable outcome depending on their genetic background. Here, polygenetic scores (PGS) were calculated for each individual, which measure the cumulative effect of common alleles and their effects on the person’s risk of disease. So, MC4R mutations carriers with favorable PGS had an average BMI, which was approximately equal to the population mean, while those with an unfavorable PGS were more strongly affected [MC4R mutations.A recent genome-wide association study (GWAS) has reported that patients with pathogenic affected . TherefoMC4R mutations showed no significant change in our chosen cell system in the basal state. Therefore, we can conclude that the results from the functional characterization reflect the actual signaling capacity of the mutations rather than their expression on the cell surface. This is in line with published data for V103I [Our investigations regarding total and cell surface expression of the or V103I  and someor V103I ,40,58. Hor V103I .In our study, the capacity to form endosomes after stimulation with NDP-α-MSH of V103I was disturbed, which aligns with recently published data . This inS127L has been shown to become internalized similarly to the WT when stimulated with α-MSH, determined by ELISA studies . We evenS pathway [S [S coupling that was reported for H158R [The MC4R has been shown to be constitutively active in the G pathway , and twothway [S ,59, whicthway [S ,53,60. Wor H158R .MC4R mutations, the results were not significant. H158R, on the other hand, was found to have reduced basal ERK phosphorylation compared to MC4R WT [Since only a few studies addressed other pathways for MC4R, information about basal activity is scarce. S127L has been investigated for ERK phosphorylation and was found to have an increased basal activity in this pathway . Althoug MC4R WT , which wMC4R mutations in vitro. Its effect has been described for all three studied mutations in various details. GS signaling determined in this study is in accordance with the majority of already published data for all three mutations. Stimulation by α-MSH in presence of 100 nM AgRP resulted in impaired antagonistic potency for S127L, but an increase for H158R. For MC4R S127L, AgRP had a reduced antagonistic potency, which is in line with published data [The endogenous ligand α-MSH is commonly used to investigate hed data . One stuhed data . We did q/11 signaling for two of our mutations, V103I and H158R, which have never been investigated in this pathway before. Over the years, evidence has accumulated that suggests a role of PLC activation in the anorexigenic effect of MC4R, and the activation of Gq/11 signaling through MC4R has been documented in several studies [MC4R mutations are found in obese patients with normal function in GS, but impaired NFAT activation as read-out system of Gq/11 activation could be rescued in vitro with a synthetic ligand biased towards PLC activation [q/11 pathway, as a strong bias towards PLC activation of V103I might play a part in its suggested protective role against obesity [q/11 coupling as well [The most eminent observed effect was the increase in G studies ,61,62,63tivation . This hi obesity ,32,33,34 as well . The obs/o and Gq/11 proteins [Similarly, ERK phosphorylation has been suggested to be important for MC4R function in vitro and in vivo ,8,9, thoproteins . For V10proteins . Though S signaling of β-MSH in the presence of 100 nM AgRP is not as strongly affected compared to the one of α-MSH. Interestingly, the stimulation of V103I with β-MSH had a tendency towards low GS signaling, which is conflicting with recently published results [S, as already published [The endogenous ligands α- and β-MSH share a core amino acid sequence (His-Phe-Arg-Trp) but differ in length and protein stability. They are both involved in the anorexigenic effect of MC4R signaling ,22,48,49 results . The S12ublished . There iMC4R mutations V103I and H158R have a diverse signaling profile when stimulated with β-MSH compared to α-MSH. They still exhibit a strong bias towards PLC, though other signaling pathways are enhanced as well, such as Gi/o and G12/13 coupling. In addition, H158R was able to increase ERK phosphorylation in comparison to the WT. This might be the result of the strong bias towards Gq/11 and Gi/o coupling that increased the activation of MAPK. As mentioned before, V103I was published to exhibit an increased ERK phosphorylation [For the other tested pathways, the two rylation . Again, S signaling [MC4R V103I our results in GS signaling were in line with most publications that used NDP-α-MSH [50 [50 compared to the WT combined with a decreased Emax [50 and a diminished maximal response [NDP-α-MSH is an improved synthetic form of α-MSH that has been shown to be more potent in Gignaling . Again, DP-α-MSH ,66. For -MSH [50 ,42 or a sed Emax , or bothresponse . In our 12/13 and MAPK was increased for V103I. The latter has already been reported recently [q/11 or Gi/o signaling, since these pathways are not increased. This hints towards an unknown mechanism independent of G proteins. For S127L, only one study investigated the ability to activate a non-GS pathway. Here, the authors found that it could increase pERK levels when stimulated with NDP-α-MSH. Although it was only tested for one concentration of agonist and no conclusion about bias could be stated, the response was significantly lower than the MC4R WT [The synthetic ligand diminished the bias effect in the PLC of V103I and H158R, although for the MC4R WT the synthetic agonist was more potent in all pathways investigated. Interestingly, bias towards Grecently . Here, i MC4R WT . In our  MC4R WT , which iMC4R mutations that determined the activation of all four G protein families in addition to ERK activation, β-arrestin2 recruitment, receptor expression and internalization. We could demonstrate that the signaling capacity of MC4R WT depends on its endogenous ligand. Our summarized data  supplemented with 5% fetal bovine serum  and 1% non-essential amino acids  at 37 °C and humidified air containing 5% CO2. For cAMP measurements, reporter gene assays, total and cell surface expression , 1.5 × 104 cells per well were seeded in poly-l-lysine-coated  96-well plates and incubated for 24h. The HiBiT assays were performed in white opaque, poly-l-lysine-coated 96-well plates , and the other assays in translucent well plates . For Bioluminescence resonance energy transfer (BRET) assays, HEK293 cells were seeded in 6-well plates for transfection (8 × 105 cells/well) and later reseeded in white opaque 96-well plates for measurement (2.2 × 104 cells/well).Human embryonic kidney 293 (HEK293) cell line was purchased from ATCC, authenticated by single nucleotide polymorphism (SNP) analysis and regularly tested for mycoplasma contamination. Cells were cultivated in N-terminally tagged with the hemagglutinin (5′ YPYDVPDYA 3′) epitope (HA). For β-arrestin recruitment BRET assays, the cDNA of rARRB2  was cloned into the expression vector pFN31A , resulting in the N-terminal tag with NanoLuc (NL). MC4R and cholinergic muscarinic receptor 3  were C-terminally tagged with the protein tag Halotag (HT) using the pFC14A expression vector  when they serve as a mock control. For MC4R, the linker between receptor and protein tag was changed into ADPPVV using site-directed mutagenesis for improved assay performance. For total and cell surface expression, MC4R was cloned into pBiT3.1-N , resulting in an N-terminal tagged receptor with the HiBiT protein tag. The mutations V103I, S127L and H158R were incorporated into all expression vectors using site-directed mutagenesis. Mutagenesis primer can be found in MC4R cDNA was amplified from genomic DNA, cloned into eukaryotic expression vector pcDps and The compounds α-MSH, β-MSH, NDP-α-MSH, PTX and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich , and AgRP from Phoenix Pharmaceuticals .HEK293 cells were transfected 24 h after seeding. For cAMP measurements and reporter gene assays, cells were transfected with 45 ng plasmid DNA and 0.45 µL Metafectene . In case of reporter gene assays, the transfection included an additional 45 ng of reporter DNA per well in MEM without supplements. For the HiBiT assay, transfection was performed as described previously .®  transfection reagent according to the manufacturer’s instructions. HEK293 cells, seeded in 6-well plates  containing 24 mm #1 glass coverslips , were transfected with 0.4 µg DNA per well.In order to stain the receptor with fluorophore-tagged ligand, MC4R WT and the three mutations were transfected in HEK293 cells using EffecteneFor BRET assays, HEK293 cells in 6 wells were transfected using 8 µL FuGene HD  and 2.4 µg DNA in Opti-MEM (Gibco) 4–6 h after seeding. The BRET partners were co-transfected with the NL:HT ratio of 1:10 (200 ng:2 µg). A total of 200 ng of pGEM-3Zf (+) was added as carrier DNA.® HiBiT detection system . Here, HEK293 cells express the HiBiT-tagged receptor in low amounts and measurement was performed according to the manufacturer’s protocol (rapid measurements protocol). In short, 48 h after transfection, media was changed to 50 µL Opti-MEM without phenol red per well and injected with 50 µL of either HiBiT Extracellular substrate  for determination of cell surface expression or HiBiT Lytic substrate  for total expression using a plate reader . Afterwards, the plate was shaken orbitally for 3 min at 300 cycles per minute, incubated for 10 min at room temperature and then luminescence was measured. Cells transfected with empty pcDNA3 were used as background control and were subtracted from the sample emissions.The number of receptors expressed on the cell membrane as well as total cell expression were determined using the NanoGloα-MSH for 30 min and then washed with HBSS. Cells adhering to 24 mm #1 glass coverslips were mounted into Attofluor™ steel chambers  and imaged in HBSS  using a SP8  inverted confocal microscope. The excitation wavelength was 561 nm (power at the sample of a few microwatts), and emitted fluorescence from TAMRA was collected in the 580–650 nm range using a Leica HyD Detector. The scan rate was 100 Hz, with a pixel size of the order of 50 nm.A total of 48 h after transfection and prior to imaging, cells were incubated with 100 nM TAMRA-NDP-Differential interference contrast (DIC) images were collected on a photo multiplier (PMT) placed on top of the condenser of the microscope.C-terminally tagged with HT and β-arrestin2 was N-terminally tagged with NL. In case of a recruitment of β-arrestin2 to the receptor, which indicates the initiation of receptor internalization, radiation-free energy transfer occurs between energy donor and energy acceptor.The recruitment of β-arrestin2 to MC4R was determined by a novel BRET technique, the NanoBRET™ , which utilizes an improved luciferase  as an energy donor and a protein tag  that can bind to the energy acceptor (HT Ligand 618). The MC4R was 5 cells/mL in Opti-MEM without phenol red supplemented with 4% FBS. Cells were divided into two pools, adding either the acceptor NanoBRET™ Ligand 618 (1 µL/mL cells of 0.1 mM solution) or the same amount of DMSO as background control. After reseeding 90 µL cell suspension/well into a white 96-well plates, incubation was continued for 4–6 h.A total of 16–20 h after transfection in 6-well plates, cells were detached, and cell number was adjusted to 2.2 × 10For BRET measurements, cells were challenged with 10 µL of either α-MSH, β-MSH or NDP-α-MSH (10 µM to 1 nM) for 5 min at 37 °C. For measurements, 25 µL of NanoBRET™ substrate was injected using a plate reader (Mithras LB 940). Donor and acceptor emission were measured at 460 nm and 610 nm, respectively, and the BRET ratio was calculated by dividing acceptor emission by donor emission:The ratio was corrected for background bleed through by subtracting of the background ratio (no acceptor DMSO control) and conversion to milliBRET units (mBU):To calculate the difference between stimulated and basal interaction, the NET BRET was acquired using the formula:S and Gi coupling to the MC4R were determined using the AlphaScreen™ assay  performed according to the manufacturer’s protocol and described elsewhere [S coupling. Determination of Gi coupling was investigated by pretreatment with 50 µg/mL PTX 18 h before stimulation. All stimulations were performed at 37 °C and 5% CO2. Incubation was stopped by removing the compounds and lysing the cells in lysis buffer containing IBMX for 1.5–2 h at 4 °C on a shaking platform.Glsewhere . In brieThe determination of cAMP using the AlphaScreen™ assay is a competitive immune assay based on antibody-coupled acceptor beads that bind cAMP and streptavidin-coupled donor beads that bind to a biotinylated cAMP probe. Excitement of the donor bead leads to the conversion of ambient oxygen to a singlet state. In close proximity of donor and acceptor beads through binding the biotinylated probe, the acceptor reacts with the singlet molecule and emits light. Absolute amounts of cAMP are determined by the simultaneous measurement of a cAMP standard. The plate reader (Mithras 940 LB)) was used for the read out, at 565 nm.2. The stimulation was stopped by discarding the media and cell lysis was induced by addition of 1× passive lysis buffer  and horizontal shaking for 15 min at room temperature.Activation of PLC, RhoA and ERK1/2 phosphorylation was determined using luciferase-based reporter gene assays that utilize responsive elements in the promotor region of the gene encoding a firefly luciferase. For PLC activation, the NFAT responsive element  was co-transfected with the receptor mutations. Similarly, co-transfections with SRF responsive element  for RhoA activation and Serum response element  for ERK1/2 phosphorylation were performed. A total of 48h post transfection, cells were challenged with α-MSH, β-MSH or NDP-α-MSH (10 mM to 1 nM) in MEM without supplements for 6 h at 37 °C and 5% COMeasurement of luciferase activity provided information about the activation of the respective second messenger by transferring 10 µL lysate into a white opaque 96-well plate. Injection of 40 µL firefly luciferase substrate  and measurement of luminescence was performed with a plate reader (Mithras LB 940).p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.Statistical analysis was performed using two-way ANOVA, followed by Dunnett’s post hoc test in GraphPad Prism 6. Statistical significance was set at * 50 values). For mutations that had EC50 values outside of concentration tested, it was set to the appropriate highest concentration tested. Signaling bias analysis was performed as described by Kenakin (2017) [test referring to the tested receptor mutation and ref being reference (MC4R WT). Emax is the maximal response (efficiency) of the mutation, which was determined from the concentration–response curves. Where no Emax could be determined from incomplete curves , the response of the maximal concentration was substituted as has been described by Winpenny et al. (2016) [Concentration–response curves of each experiment were analyzed by fitting a non-linear regression model for sigmoidal response in Graph Pad Prism 6  to determine potency (ECn (2017)  using th. (2016) .max/EC50) was calculated using the equation:test is the standard error the tested receptor mutation and SEMref is the standard error of the reference (MC4R WT).Standard error (SEM) of Δlog(Emax/EC50):Bias was calculated creating the antilog of the Δlog (EThe standard error of the bias factor was determined by the equation:


Giardia duodenalis  with characteristic pear/tear drop shape, bilaterally symmetric nuclei and flagella  stricture (IgG4 related cholangitis). CBD brushing incidentally revealed trophozoites of a Figure .G. duodenalis .Giardiasis is one of the commonest intestinal infections affecting individuals globally. The worldwide prevalence ranges from 1% to 65% while countries like India report prevalence as high as 46.5% to 97.4%.Giardia duodenalis has two morphologic forms: cysts and trophozoites. Cysts are excreted in stool and are infectious forms while excystation produces trophozoites in proximal small bowel. It is transmitted through faeco‐oral route. Cysts are resistant to chlorine treatment and may survive in cold water for several months.G. duodenalis may be detected in CBD brushingsPatient may present with self‐limiting infestation, nonspecific symptoms like diarrhea and abdominal cramps or chronic malabsorption and stunted growth. Diagnosis can be made by identification of organisms by stool microscopy or endoscopic biopsy. Other tests include nucleic acid amplification or antigen detection test. Rarely None.Conceptualization: Abha ThakurData Curation: Abha Thakur, Haimanti SarinWriting ‐ original draft preparation: Abha ThakurWriting ‐ review & editing: Haimanti SarinBoth authors have read, edited and reviewed the manuscript.