Element Binding Research Articles

Czy powrót ustawowego bezprawia?

Constitutional crisis confronts legal practice with philosophical problems that normally may seem abstract or even purely academic. Among those, there is a question of material (content-dependent) criteria of legal validity, namely whether legal norms may actually have any content and remain binding elements of the law. It becomes palpable due to the legislative initiatives to decriminalize some violations of law committed by governmental officials. Such regulation deserves discussion in the light of theoretical conceptions of the claim to correctness (justice, righteousness) as a necessary feature of each act of enacting or applying the law. Arguably, even weaker conception of such claim, relying on the correctness relative to the public morality reflected in the fundamental values and principles of the positive law, is sufficient to challenge the potential presumption of validity of such decriminalizing provisions entailed by their possible formally accurate enactment

Genome-wide identification and characterization of SPX domain-containing proteins and their responses to phosphorus and/or nitrogen deficiency in apple (Malus domestica Borkh.)

Phosphate (Pi) and nitrate (Ni) deficiencies are the major constraints for apple, significantly impacting the fruit yield and quality. SPX domain-containing proteins play important roles in Pi homeostasis and Pi signaling pathway, as well as phosphorus (P) and nitrogen (N) nutrient balance in plants. However, SPX domain-containing proteins have not been analysed systematically in apple (Malus domestica Borkh.). In this study, a total of 25 MdSPXs were identified in M. domestica. According to phylogenetic relationships and C-terminal domains analyses, these MdSPXs were divided into four subfamilies: SPX (6), SPX-EXS (12), SPX-MFS (3), and SPX-RING (4).16 MdSPXs from these four subfamiles contain Phosphate starvation response 1-binding site (P1BS) in the promoter region. The expression profiles showed that 15 MdSPXs responded to P deficiency as well as N and P deficiencies, respectively, 13 MdSPXs responded to N deficiency in hydroponic ‘M26’ seedlings. The response of 11 common differentially expressed genes exhibited apparent tissue-specificity. Pi starvation response (PSR) involved in MdSPX1, MdSPX2, and MdSPX5 depended on the N availability. In the predicted protein-protein interaction network, MdSPXs interact with important components of the P signaling network such as MdPHO2, MdPHRs, MdSIZ1, and MdPHTs, etc., and components of the N signaling network such as with MdNRTs and MdNLP6, etc., in addition to several TFs such as bZIP TF MdTGA4, an important Ni-responsive TF. Overexpression of MdTGA4 changed the expression of MdSPX2, MdSPX4, MdPHO1;6, MdPHO1;7, MdPHO1;9 and MdMFS2 in the transgenic apple calluses, and these genes contain as-1, a special binding element of TGA, in the promoter region. These results preliminarily confirmed the key role of MdSPXs in Pi homeostasis and Pi signaling pathway as well as Ni signaling pathway, offered a valuable guide for integrating N-P interactive regulation pathways in apple, and provided important genetic resources for the future research on the efficient utilization of P and N nutrients in apple.

A glutathione S-transferase GhTT19 determines flower petal pigmentation via regulating anthocyanin accumulation in cotton.

Anthocyanin accumulations in the flowers can improve seed production of hybrid lines, and produce higher commodity value in cotton fibre. However, the genetic mechanism underlying the anthocyanin pigmentation in cotton petals is poorly understood. Here, we showed that the red petal phenotype was introgressed from Gossypium bickii through recombination with the segment containing the R3 bic region in the A07 chromosome of Gossypium hirsutum variety LR compared with the near-isogenic line of LW with white flower petals. The cyanidin-3-O-glucoside (Cy3G) was the major anthocyanin in red petals of cotton. A GhTT19 encoding a TT19-like GST was mapped to the R3 bic site associated with red petals via map-based cloning, but GhTT19 homologue gene from the D genome was not expressed in G. hirsutum. Intriguingly, allelic variations in the promoters between GhTT19LW and GhTT19LR , rather than genic regions, were found as genetic causal of petal colour variations. GhTT19-GFP was found localized in both the endoplasmic reticulum and tonoplast for facilitating anthocyanin transport. An additional MYB binding element found only in the promoter of GhTT19LR , but not in that of GhTT19LW , enhanced its transactivation by the MYB activator GhPAP1. The transgenic analysis confirmed the function of GhTT19 in regulating the red flower phenotype in cotton. The essential light signalling component GhHY5 bonded to and activated the promoter of GhPAP1, and the GhHY5-GhPAP1 module together regulated GhTT19 expression to mediate the light-activation of petal anthocyanin pigmentation in cotton. This study provides new insights into the molecular mechanisms for anthocyanin accumulation and may lay a foundation for faster genetic improvement of cotton.

Silibinin Overcomes EMT-Driven Lung Cancer Resistance to New-Generation ALK Inhibitors.

Epithelial-to-mesenchymal transition (EMT) may drive the escape of ALK-rearranged non-small-cell lung cancer (NSCLC) tumors from ALK-tyrosine kinase inhibitors (TKIs). We investigated whether first-generation ALK-TKI therapy-induced EMT promotes cross-resistance to new-generation ALK-TKIs and whether this could be circumvented by the flavonolignan silibinin, an EMT inhibitor. ALK-rearranged NSCLC cells acquiring a bona fide EMT phenotype upon chronic exposure to the first-generation ALK-TKI crizotinib exhibited increased resistance to second-generation brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK-TKIs, which was partially recapitulated upon chronic TGFβ stimulation, was less pronounced in ALK-rearranged NSCLC cells solely acquiring a partial/hybrid E/M transition state. Silibinin overcame EMT-induced resistance to brigatinib and lorlatinib and restored their efficacy involving the transforming growth factor-beta (TGFβ)/SMAD signaling pathway. Silibinin deactivated TGFβ-regulated SMAD2/3 phosphorylation and suppressed the transcriptional activation of genes under the control of SMAD binding elements. Computational modeling studies and kinase binding assays predicted a targeted inhibitory binding of silibinin to the ATP-binding pocket of TGFβ type-1 receptor 1 (TGFBR1) and TGFBR2 but solely at the two-digit micromolar range. A secretome profiling confirmed the ability of silibinin to normalize the augmented release of TGFβ into the extracellular fluid of ALK-TKIs-resistant NSCLC cells and reduce constitutive and inducible SMAD2/3 phosphorylation occurring in the presence of ALK-TKIs. In summary, the ab initio plasticity along the EMT spectrum may explain the propensity of ALK-rearranged NSCLC cells to acquire resistance to new-generation ALK-TKIs, a phenomenon that could be abrogated by the silibinin-driven attenuation of the TGFβ/SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells.

A 35-bp Conserved Region Is Crucial for Insl3 Promoter Activity in Mouse MA-10 Leydig Cells

The peptide hormone insulin-like 3 (INSL3) is produced almost exclusively by Leydig cells of the male gonad. INSL3 has several functions such as fetal testis descent and bone metabolism in adults. Insl3 gene expression in Leydig cells is not hormonally regulated but rather is constitutively expressed. The regulatory region of the Insl3 gene has been described in various species; moreover, functional studies have revealed that the Insl3 promoter is regulated by various transcription factors that include the nuclear receptors AR, NUR77, COUP-TFII, LRH1, and SF1, as well as the Krüppel-like factor KLF6. However, these transcription factors are also found in several tissues that do not express Insl3, indicating that other, yet unidentified factors, must be involved to drive Insl3 expression specifically in Leydig cells. Through a fine functional promoter analysis, we have identified a 35-bp region that is responsible for conferring 70% of the activity of the mouse Insl3 promoter in Leydig cells. All tri- and dinucleotide mutations introduced dramatically reduced Insl3 promoter activity, indicating that the entire 35-bp sequence is required. Nuclear proteins from MA-10 Leydig cells bound specifically to the 35-bp region. The 35-bp sequence contains GC- and GA-rich motifs as well as potential binding elements for members of the CREB, C/EBP, AP1, AP2, and NF-κB families. The Insl3 promoter was indeed activated 2-fold by NF-κB p50 but not by other transcription factors tested. These results help to further define the regulation of Insl3 gene transcription in Leydig cells.

Konektoru lietojums latviešu zinātniskajos rakstos

The means of creating the cohesion of the text have not been looked at in the research of Latvian scientific texts. Since it is important within the context of academic writing and language learning, the purpose of this article is to update the research on the linkage of propositions in Latvian scientific texts and, having studied the usage of various linguistic approaches, present the results of the feasibility study on the use of linguistic means, while also identifying issues that need further research. The research carried out is based on an analysis of the cohesion tools in two social and natural science articles, followed by quantitative research of the texts of six scientific disciplines (600 texts in total) in order to selectively examine the frequency of use of connectors and the differences in texts of different fields of science using AntConc software. The results of the study show that the binding elements that make up the cohesion of the text are a heterogeneous group in terms of belonging to different word classes, as already concluded in previous text syntax studies, as well as in relation to the use of grammatical forms (e.g. future form, participles). Part of the established binding elements of proposition (pronouns and nouns) relate mainly to the provision of referential identity, while others, on the other hand, are signs of an interactive metadiscourse and implement various semantic attitudes that allow them to be interpreted in this context. According to the results of the analysis on the frequency of use of connectors, there are differences between the texts of different fields of science. A further systemic study of these could form the basis for determining the specific characteristics of scientific reasoning by means of a quantitative method. Further systemic research of connectors could be partly based on the analysis of semantic attitudes carried out in syntax research (Latvian and other languages), the basis of text linguistics in cohesion research, as well as research on the interactive features of metadiscourse. At the same time, in connection with the analysis of semantics, text, discourse, and metadiscourse, it is necessary to focus on the development of the terminology of the topic in Latvian in the future. This, combined with a systemic continuation of research in the future, would have a practical role in the development of the classification of connectors, a catalogue, a lexicographic description, and teaching tools for academic writing.

Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern.

The arsenical resistance-3 (ACR3) family constitutes the most common pathway that confers high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3Bs ) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X-shaped structure. As the structural information available for these important arsenite-efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X-shaped of NhaA fold.

Aptamer-Small Molecule Conjugates As Potent and Reversible Surgical Anticoagulants Targeting Thrombin

Unfractionated heparin (UFH) is the first-line anticoagulant used in millions of heart surgeries annually. The high potency of UFH is attributed to its effective activation of the antithrombin-mediated anticoagulation pathway. However, the prolonged use of heparin in heart surgeries is often associated with bleeding disorders as it depletes both patient's coagulation and anticoagulation capacity. Moreover, UFH is immunogenic, which can trigger potentially life-threatening heparin-induced thrombocytopenia (HIT) when used repeatedly. Aptamers are short oligonucleotides isolated in vitro that bind to protein or small molecule targets with high affinity and specificity. Several aptamers have been created that act as anticoagulants as they can competitively block substrate binding to anticoagulation protease exosites. Compared to UFH, aptamers have advantages, including not activating antithrombin, reversibility by administration of a well-tolerated antisense oligonucleotide, and low immunogenicity. However, the anticoagulant potency of aptamers is often limited by their inability to effectively inhibit the catalytic sites of their protease targets. Here we provide a strategy to solve this potency problem by conjugating a protease active-site inhibitor to an exosite-binding aptamer via a polynucleotide linker. The resulting bivalent conjugate binds to both the active site and exosite of the target protease to completely inhibit protease function. Meanwhile, the synergy between two binding elements significantly improves the binding affinity compared to the small molecule inhibitor or the aptamer alone. As a proof of concept, we conjugated the thrombin active site inhibitor DAB to the 5’ terminus of anti-thrombin aptamer HD22 via a poly (7) adenosine linker (HD22-7A-DAB) and characterized its thrombin inhibition activity using a fluorogenic substrate cleavage assay. The results reveal that HD22-7A-DAB has a much higher thrombin inhibition activity (IC50 = 0.2 nM) than DAB (IC50 = 80 nM) or the unconjugated aptamer (IC50 > 1000 nM). The significantly higher inhibitory effect of HD22-7A-DAB compared to a 1:1 mixture of unconjugated DAB and HD22 also indicates that conjugation is essential to create an effective synergistic inhibitor. Notably, in the presence of 2 µM complementary DNA to HD22, the effect of HD22-7A-DAB conjugate was drastically diminished (IC50 > 1000 nM), which indicates that both aptamer and small molecule in ADIC can be reversed using an antidote oligonucleotide. HD22-7A-DAB also shows a UFH-rivaling anticoagulation effect and antidote-reversible behavior in a series of in vitro clotting assays, including thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thus the development of a thrombin-targeting small molecule aptamer conjugate inhibitor yields a potent and reversible anticoagulant with clinically relevant anticoagulant activity. In addition, these studies describe a generalizable strategy for rapidly developing aptamer-small molecule active site inhibitor conjugates for generate other potent and reversible inhibitors of the other coagulation proteases. These potent and reversible anticoagulant aptamer-active site drug conjugates have the potential to become UFH substituents in surgeries to reduce the risk of UFH-associated bleeding, UFH-insensitivity and HIT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IPH6501 Is a Novel NKp46-Targeting Tetraspecific Antibody-Based Natural Killer Cell Engager Therapeutic (ANKET) Armed with a Non-Alpha IL-2 Variant and Developed for the Treatment of CD20-Positive Malignancies

CD20 is expressed by >90% of B-cell non-Hodgkin's lymphomas (NHL). Several generations of CD20-targeting monoclonal antibodies including rituximab, ofatumumab, and obinutuzumab have been widely used for B-cell malignancy therapies. Despite the recent approvals of novel CD20-targeting agents, new alternatives and strategies are still required for patients, which are relapsing or refractory after several lines of treatment. High circulating NK cell numbers have been associated with better clinical responses to anti-CD20- targeting monoclonal antibodies, supporting the role of NK cells in efficacy of these treatments. IPH6501, a novel antibody-based NK cell engager therapeutics (ANKET), is a single tetraspecific molecule engaging two NK cell activating receptors NKp46 and CD16a (FcγRIIIa), the β chain (CD122) of the interleukin-2 receptor (IL-2R), and the CD20 antigen expressed on malignant B cells. The IL-2R binding element incorporated in IPH6501 is an IL-2 variant (IL-2v) designed with point mutations that abolish binding to the IL-2R-α chain (CD25), with the goal of limiting toxicity and interaction with Tregs. IL-2v incorporated into IPH6501 is directed towards NK cells through the binding with high affinity to NKp46 and CD16a, providing its ability to interact with IL-2R preferentially on NK cells and to promote their activation and proliferation at pM doses. IPH6501 was designed to induce NK cell mediated-cytotoxicity and cytokine secretion by co-engaging CD16a and NKp46. Only the binding of IPH6501 to CD20, bridging the NK cells to the target cells, was able to trigger the cytotoxic activity of NK cells. Non-saturating doses of IPH6501 on NK cells and on CD20+ cells were sufficient to promote maximal killing activity. Furthermore, IPH6501 promoted NK cell cytotoxicity against tumor cells expressing very low levels of CD20. IPH6501 also demonstrated superiority to control tumor cell growth in vitro, as compared to the CD20-targeting clinical benchmark antibodies rituximab and the Fc-optimized obinutuzumab. In vivo treatment with a mouse surrogate of IPH6501 induced peripheral NK cell proliferation and activation, and demonstrated potent antitumor efficacy in a xenograft mouse model using the aggressive human B-lymphoma CD20+ RAJI cell line engrafted subcutaneously. In non-human primate, a well-tolerated dose of IPH6501 led to peripheral NK cell expansion and to the depletion of CD20+ B cells in the circulation and within lymphoid tissues, with minimal systemic cytokine release. In conclusion, IPH6501 is a first-in-class CD20-targeting tetraspecific NK cell engager designed to promote NK cell proliferation and specific cytotoxicity against CD20+ cells. IPH6501 is thus a promising biologic designed to harness the anti-tumor functions of NK cells in CD20+ B cell malignancies. IND-enabling studies are ongoing, and IPH6501 is expected to enter FIH clinical study in 2023.

The 5-HT1A receptor biased agonist, NLX-204, shows rapid-acting antidepressant-like properties and neurochemical changes in two mouse models of depression

Activation of cortical serotonin 5-HT1A receptors may be a promising strategy to achieve rapid-acting antidepressant (RAAD) activity. NLX-204 is a selective 5-HT1A receptor biased agonist that, in naïve mice, robustly decreases immobility in the forced swim test (FST), and preferentially phosphorylates extracellular signal-regulated kinase (ERK1/2), involved in antidepressant activity. Here, we evaluated the properties of NLX-204 in two mouse models of depression. Male CD-1 mice were subjected to unpredictable chronic mild stress (UCMS) for 4-weeks or to repeated corticosterone (CORT, 20 mg/kg s.c./day) for 3-weeks before receiving acute administration of NLX-204 (2 mg/kg, p.o.). Depressive-like behavior was assessed in the FST, anhedonia-like behavior in the sucrose preference test (SPT) and locomotor activity was also recorded. Phosphorylation of ERK1/2 (pERK1/2) and cAMP response binding element (pCREB) were measured ex vivo in hippocampus and prefrontal cortex (PFC). UCMS or CORT treatment increased immobility in the FST, elicited a sucrose preference deficit, and decreased pERK1/2 and pCREB levels in PFC and hippocampus. NLX-204 reduced depressive-like behavior in the FST in CORT and UCMS mice, and normalized sucrose preference in CORT mice, suggesting anti-anhedonic activity. NLX-204 increased pERK1/2 levels in PFC of UCMS mice. NLX-204 also increased pCREB levels in PFC of CORT mice. These data suggest that NLX-204 has RAAD-like properties not only in naïve mice, but also in mice in a “depressive-like” state, and that these involve changes in PFC and hippocampal pERK1/2 and pCREB levels. These data provide additional evidence that activation of 5-HT1A receptors by selective biased agonists, such as NLX-204, may constitute a promising RAAD strategy.

Characterization and expression analysis of basic leucine zipper (bZIP) transcription factors responsive to chilling injury in peach fruit.

Peach (Prunus persica L.) is prone to chilling injury as exhibited by inhibition of the ethylene production, failure in softening, and the manifestation of internal browning. The basic leucine zipper (bZIP) transcription factors play an essential role in regulatory networks that control many processes associated with physiological, abiotic and biotic stress responses in fruits. Formerly, the underlying molecular and regulatory mechanism of (bZIP) transcription factors responsive to chilling injury in peach fruit is still elusive. In the current experiment, the solute peach 'Zhongyou Peach No. 13' was used as the test material and cold storage at low temperature (4°C). It was found that long-term low-temperature storage induced the production of ethylene, the hardness of the pulp decreased, and the low temperature also induced ABA accumulation. The changes of ABA and ethylene in peach fruits during low-temperature storage were clarified. Since the bZIP transcription factor is involved in the regulation of downstream pathways of ABA signals, 47 peach bZIP transcription factor family genes were identified through bioinformatics analysis. Further based on RT-qPCR analysis, 18 PpbZIP genes were discovered to be expressed in refrigerated peach fruits. Among them, the expression of PpbZIP23 and PpbZIP25 was significantly reduced during the refrigeration process, the promoter analysis of these genes found that this region contains the MYC/MYB/ABRES binding element, but not the DRES/CBFS element, indicating that the expression may be regulated by the ABA-dependent cold induction pathway, thereby responding to chilling injury in peach fruit. Over investigation will provide new insights for further postharvest protocols related to molecular changes during cold storage and will prove a better cope for chilling injury.

The Concept of Perfection in Lev Karsavin’s Religious Metaphysics

ABSTRACT This article examines the concept of the perfect, a key idea in Lev P. Karsavin’s metaphysics that largely determines his understanding of personhood and its ontological status. The associated concept of the perfect person develops throughout the entire philosophical period of the thinker’s work, from his Philosophy of History to his treatise “On Perfection,” written in the last year of his life in the Abez’ camps. In this article, I argue that the concept of perfection is the main structural element in Karsavin’s religious–philosophical system, making it ontologically full and complete. I believe the Christian idea of the perfection of man in God, philosophical variations of which Karsavin finds in Nicholas of Cusa’s system of total unity and Vladimir Solovyov’s idea of Godmanhood, is both the initial intellectual intuition and the ontological premise of the thinker’s metaphysical constructions. His religious–philosophical work has a continuity with Russian culture’s spiritual tradition, where patristic thought of man considers the ideal to be the spiritual transcendence of the person in his striving toward the Perfect God. This evangelic idea turns out to be the central binding element both of Karsavin’s Christian personology and of his metaphysics of history, which is resolved in a metaphysical vein.

PMON29 A Comparative Analysis of Musashi-Dependent Control of Mouse and Human Pituitary mRNA Translation

Abstract Specialized cell lineages within the anterior pituitary produce specific hormones which, in turn, regulate diverse biological processes ranging from growth, metabolism, and reproduction to stress. Interestingly, these specialized cell lineages exhibit a high degree of cellular plasticity, readily altering their differentiation state to produce different hormones in response to organismal demand. Underlying molecular mechanisms controlling cellular plasticity in the pituitary are not fully defined but appear to involve post-transcriptional regulation exerted by the Musashi family of sequence-specific RNA binding proteins. Musashi is a bifunctional regulator of target mRNAs and can exert either repression or activation of translation in a context-dependent manner. Our recent work has implicated a role for Musashi-dependent repression of key pituitary mRNAs in the mouse, including a lineage-specific transcription factor (Pou1f1), the gonadotropin releasing hormone receptor (Gnrhr), and two anterior pituitary hormones (Prl and Tshb). In this study, we look to determine the potential relevance of Musashi-dependent mRNA translational control to the human pituitary. Reflecting our findings in the adult mouse, a re-analysis of published human fetal pituitary single cell RNA sequencing data revealed Musashi1 (MSI1) and Musashi2 (MSI2) expression in both stem/progenitor cell populations as well as in all hormone-producing cell lineages of the anterior pituitary. We further report that the human homologs of identified Musashi-target mRNAs in the mouse pituitary also possess Musashi binding elements (MBEs) in their 3' untranslated regions (UTRs). In the case of the human Pou1f1 3' UTR, we were able to verify Musashi-dependent mRNA translational repression in reporter assays as seen with the murine Pou1f1 3' UTR. Interestingly, a germline Pou1f1 mutation identified in human patients, disrupts a consensus MBE in the human Pou1f1 3' UTR and abolishes Musashi-dependent repression. We developed a bioinformatic pipeline to characterize pathogenic human mutations catalogued in the Geno2MP database for disruption or creation of consensus MBEs in pituitary-specific mRNA 3' UTRs. Our findings suggest an evolutionarily conserved role for Musashi in the post-transcriptional regulation of pituitary mRNAs in both mice and humans and identify several human mutations which may perturb Musashi regulation of human pituitary function. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF01 | PMON46 Regulation of the Pituitary Cell Lineage Regulator Prop1 by the Stem Cell Determinant Musashi

Abstract The pituitary gland controls responses to changing physiological requirements during growth, pregnancy, puberty, and stress. Synthesis and secretion of appropriate pituitary hormones in response to changing organismal demands is mediated through a remarkable capacity for cellular plasticity of the distinct hormone-producing cells. The mechanisms governing pituitary cellular plasticity are not fully understood. We hypothesize that plasticity in the adult tissue is mediated through similar mechanisms to those that occur during embryonic and postnatal pituitary development. The Prophet of Pit1(Prop1), a paired-like homeodomain transcription factor, is the earliest pituitary-specific transcription factor and is highly expressed in pituitary progenitor cells where it controls lineage commitment to the distinct hormone-producing cell types. Our recent work has identified post-transcriptional regulation of pituitary gene expression in the adult tissue via the stem cell determinant, mRNA binding protein, Musashi. The full-length mouse Prop1 (mProp1) mRNA 3' untranslated region (UTR) contains 24 consensus binding elements for Musashi (MBEs) and demonstrates Musashi1-dependent translational activation in vitro. Specifically, the terminal mProp1 3' UTR MBE directs 40% of Musashi1-mediated translation activation, and mutational disruption of this terminal MBE abolishes all Musashi1-dependent translational activation. Immunoprecipitation of Musashi-mRNA complexes from adult mouse pituitaries confirmed Musashi1-mProp1 mRNA association in adult tissue. The gene sequence data for the human Prop1 (hPROP1) mRNA 3' UTR is incomplete in the Ensembl and RefSeq databases. Using published RNA sequencing datasets, we identified an expressed full-length hPROP1 mRNA 3' UTR of 6996 nucleotides with 54 predicted consensus MBEs, suggesting that the human PROP1 mRNA, like the murine Prop1 mRNA, may be subject to Musashi-dependent translational control. Our findings are consistent with the hypothesis that the Musashi protein exerts evolutionarily conserved control of Prop1 mRNA translation to facilitate plasticity of anterior pituitary function. Presentation: Saturday, June 11, 2022 1:31 p.m. - 1:36 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The RNA-Binding Protein HuR Is Integral to the Function of Nociceptors in Mice and Humans.

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.

Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β-catenin/MDR1 signaling to sustain chemoresistance.

Chemoresistance in colorectal cancer initiating cells (CICs) involves the sustained activation of multiple drug resistance (MDR) and WNT/β-catenin signaling pathways, as well as of alternatively spliced-isoforms of CD44 containing variable exon-6 (CD44v6). In spite of its importance, mechanisms underlying the sustained activity of WNT/β-catenin signaling have remained elusive. The presence of binding elements of the β-catenin-interacting transcription factor TCF4 in the MDR1 and CD44 promoters suggests that crosstalk between WNT/β-catenin/TCF4-activation and the expression of the CD44v6 isoform mediated by FOLFOX, a first-line chemotherapeutic agent for colorectal cancer, could be a fundamental mechanism of FOLFOX resistance. Our results identify that FOLFOX treatment induced WNT3A secretion, which stimulated a positive feedback loop coupling β-catenin signaling and CD44v6 splicing. In conjunction with FOLFOX induced WNT3A signal, specific CD44v6 variants produced by alternative splicing subsequently enhance the late wave of WNT/β-catenin activation to facilitate cell cycle progression. Moreover, we revealed that FOLFOX-mediated sustained WNT signal requires the formation of a CD44v6-LRP6-signalosome in caveolin microdomains, which leads to increased FOLFOX efflux. FOLFOX-resistance in colorectal CICs occurs in the absence of tumor-suppressor disabled-2 (DAB2), an inhibitor of WNT/β-catenin signaling. Conversely, in sensitive cells, DAB2 inhibition of WNT-signaling requires interaction with a clathrin containing CD44v6-LRP6-signalosome. Furthermore, full-length CD44v6, once internalized through the caveolin-signalosome, is translocated to the nucleus where in complex with TCF4, it binds to β-catenin/TCF4-regulated MDR1, or to CD44 promoters, which leads to FOLFOX-resistance and CD44v6 transcription through transcriptional-reprogramming. These findings provide evidence that targeting CD44v6-mediated LRP6/β-catenin-signaling and drug efflux may represent a novel approach to overcome FOLFOX resistance and inhibit tumor progression in colorectal CICs. Thus, sustained drug resistance in colorectal CICs is mediated by overexpression of CD44v6, which is both a functional biomarker and a therapeutic target in colorectal cancer.

CREB3L1 and CREB3L2 control Golgi remodelling during decidualization of endometrial stromal cells.

Upon progesterone stimulation, Endometrial Stromal Cells (EnSCs) undergo a differentiation program into secretory cells (decidualization) to release in abundance factors crucial for embryo implantation. We previously demonstrated that decidualization requires massive reshaping of the secretory pathway and, in particular, of the Golgi complex. To decipher the underlying mechanisms, we performed a time-course transcriptomic analysis of in vitro decidualizing EnSC. Pathway analysis shows that Gene Ontology terms associated with vesicular trafficking and early secretory pathway compartments are the most represented among those enriched for upregulated genes. Among these, we identified a cluster of co-regulated genes that share CREB3L1 and CREB3L2 binding elements in their promoter regions. Indeed, both CREB3L1 and CREB3L2 transcription factors are up-regulated during decidualization. Simultaneous downregulation of CREB3L1 and CREB3L2 impairs Golgi enlargement, and causes dramatic changes in decidualizing EnSC, including Golgi fragmentation, collagen accumulation in dilated Endoplasmic Reticulum cisternae, and overall decreased protein secretion. Thus, both CREB3L1 and CREB3L2 are required for Golgi reshaping and efficient protein secretion, and, as such, for successful decidualization.

Cutaneous burn injury induces neuroinflammation and reactive astrocyte activation in the hippocampus of aged mice

BackgroundBy 2050, one in six people globally will be 65 or older. Confusion and delirium are significant complications after burn injury, especially in the elderly population. The etiology is still unknown, however complications may be driven by pro-inflammatory activation of astrocytes within the hippocampus (HPC) after burn injury. Reduced levels of phosphorylated cyclic-AMP response binding element (pCREB), caused by elevated systemic pro-inflammatory cytokines, could lead to cognitive decline and memory impairment. MethodsTo examine the effects of remote injury on neuroinflammation in advanced age, young and aged mice were subjected to a 15 % total body surface area scald burn or sham injury, and euthanized after 24 h. Expression of ccl2 and tnfa were measured by qPCR in the whole brain and HPC. Astrocyte activation was measured by immunofluorescence within the HPC. pCREB was measured by immunohistochemistry in the dentate gyrus. ResultsWe saw an 80-fold increase in ccl2 and a 30-fold elevation in tnfa after injury in the whole brain of aged mice compared to young groups and aged sham mice (p < 0.05 and p < 0.05, respectively). Additionally, there was a 30-fold increase in ccl2 within isolated HPC of aged injured mice when compared to sham injured animals (p < 0.05). When investigating specific HPC regions, immunofluorescence staining showed a >20 % rise in glial fibrillary acidic protein (GFAP) positive astrocytes within the cornu ammonis 3 (CA3) of aged injured mice when compared to all other groups (p < 0.05). Lastly, we observed a >20 % decrease in pCREB staining by immunohistochemistry in the dentate gyrus of aged mice compared to young regardless of injury (p < 0.05). ConclusionsThese novel data suggest that remote injury in aged, but not young, mice is associated with neuroinflammation and astrocyte activation within the HPC. These factors, paired with an age related reduction in pCREB, could help explain the increased cognitive decline seen in burn patients of advanced age. To our knowledge, we are the first group to examine the impact of advanced age combined with burn injury on inflammation and astrocyte activation within the brain.

Targeted editing of OsSWEET13, a bacterial leaf blight susceptible gene in rice using CRISPR tool

Bacterial leaf blight (BLB) of rice is considered to be a disease of economic importance as the disease causes severe yield losses in all rice growing regions. Transcription activator like effector (TALE) molecules are produced by the pathogen, Xanthomonas oryzae pv. oryzae (Xoo) bind to the effector binding element (EBE) of the promoter of SWEET gene and activates transcription of SWEET genes, making the plant susceptible to the disease.&nbsp; Innate resistance to Xoo in certain rice genotypes is due to mutations in EBE present in the upstream regulatory region of SWEET genes. CRISPR–mediated targeted modification of susceptibility gene/promoter is an effective approach to develop BLB resistance in rice. The present study was an attempt to repress TALE triggered signalling via introducing indels in the EBE of OsSWEET13 gene in a local popular rice genotype, CO51 employing CRISPR/Cas9 mediated genome editing tool with a view to imparting BLB resistance. Agrobacterium-mediated transformation using immature embryos followed by regeneration resulted in four independent transformation events. Five plants representing three events were found to have one nucleotide deletion in the target sequence. These deletion mutations in EBE could potentially interfere with the binding of the corresponding TALE to confer resistance against certain strains of BLB.&nbsp; Keywords: Rice, Bacterial Leaf Blight, SWEET13 gene, Effector Binding Element, CRISPR/Cas9.

Cd2+ tolerance and removal mechanisms of Serratia marcescens KMR-3

Heavy metal contamination is a global issue, with cadmium (Cd2+) and its treatment becoming major environmental challenge that could be solved by microbial restoration, an eco-friendly technique. Serratia marcescens KMR-3 exhibits high tolerance and removal rate of Cd2+ (≤500 mg/L). Here, we aimed to explore mechanisms underlying tolerance to and removal of Cd2+ by KMR-3. Scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectrometry were conducted to analyze characteristics of the KMR-3 biofilm and Cd2+ combined forms. The results revealed varying degrees of cell adhesion, membrane thickening, and shrinkage on the surface of the bacteria. The binding elements, electronic binding energy, and functional groups on the surface of the bacteria exhibited changes. Furthermore, the biofilm amount following treatment with Cd2+ was 1.5–3 times higher than that in the controls, treatment with Cd2+ substantially enhanced biofilm generation and increased Cd2+ adsorption. Cd2+ adsorption by its own secondary metabolite prodigiosin produced by KMR-3 was enhanced by 19.5 % compared with that observed without prodigiosin. Through transcriptome sequencing and RT-qPCR, we observed that Znu protein-chelating system regulated gene expression (znuA, znuB, and znuC), and the efflux mechanism of the P-type ATPase regulated the expression of genes (zntA, zntB, and zntR), which were significantly enhanced. Through the combined action of various strategies, KMR-3 demonstrated a high tolerance and removal ability of Cd2+, providing a theoretical basis to treat Cd2+ pollution.