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Aptamers offer immense promise for targeted protein degradation. Aptamer–PROTAC (proteolysis-targeting chimera) conjugates present an advantageous strategy to improve delivery efficiency, solubility, and permeability while mitigating off-tissue distribution-induced on-target toxicity. Aptamer-based PROTACs provide a feasible and promising platform for the spatiotemporal degradation of undruggable proteins. Aptamer-based lysosome-targeting chimeras (LYTACs) constitute a cost-effective alternative for the degradation of membrane-bound and extracellular proteins. Targeted protein degradation (TPD) technologies, particularly proteolysis-targeting chimeras (PROTACs), have emerged as a significant advancement in drug discovery. However, several hurdles – such as the difficulty of identifying suitable ligands for traditionally undruggable proteins, poor solubility and impermeability, nonspecific biodistribution, and on-target off-tissue toxicity – present challenges to their clinical applications. Aptamers are promising ligands for broad-ranging molecular recognition. Utilizing aptamers in TPD has shown potential advantages in overcoming these challenges. Here, we provide an overview of recent developments in aptamer-based TPD, emphasizing their potential to achieve targeted delivery and their promise for the spatiotemporal degradation of undruggable proteins. We also discuss the challenges and future directions of aptamer-based TPD with the goal of facilitating their clinical applications. [ABSTRACT FROM AUTHOR]

Placenta specific protein 8 (Plac8) has been well studied in vertebrates, yet little is known in invertebrates. In this study, a novel Plac8 from the planarian Dugesia japonica was identified and its functions in immune responses and development were characterized. Our results show that Djplac8 was expressed in the pharynx, epidermis and intestine of intact adult planarian. The expression of Dj Plac8 increased significantly upon lipopolysaccharide (LPS) challenge, and inhibited the growth of the Gram-negative bacteria ( Escherichia coli and Pseudomonas aeruginosa ), suggesting the role of Plac8 in immune response. Spatial and temporal expression and distribution of DjPlac8 mRNA in regenerated planarians indicates that DjPlac8 was mainly expressed in the pharynx. In situ hybridization also revealed the elevated expression of the DjPlac8 gene in the embryonic pharynx, germ band and parenchyma cells, indicating an important role in embryonic development of D. japonica . When DjPlac8 was deactivated by RNA interference-mediated knockdown, the head of planarians underwent abnormal development. In summary, we identified Dj Plac8 as a novel multifunctional protein that plays essential roles in immune response and development of planarians. [ABSTRACT FROM AUTHOR]

Abstract: Freshwater planarians have traditionally been favored animal models in regenerative and developmental biology. Little is known about the immunological research in these animals. Here, we provide spectrophotometric, electrophoretic and histochemical evidence demonstrating the presence and characterization of phenoloxidase (EC 1.14.18.1; PO) activity in the planaria Dugenia japonica. PO activity was optimal at pH 7.5 and at a temperature of 45°C. ProPO in the humoral fluid was readily activated by trypsin and zymosan, and was susceptible to phenylthiourea. Two bands of PO activity in the humoral fluid with 150 and 300kDa by native PAGE and a single band with 150kDa after SDS-PAGE were resolved. Enzyme histochemical staining demonstrated the presence of PO in the epidermal cell and pharyngeal epithelium, which indicate that PO could serve as a protecting agent against environmental pathogens in D. japonica. [Copyright &y& Elsevier]

[Display omitted] • Betaine decreases the gene expression and activities of ACL, ACC, and FAS. • Betaine regulates the synthesis and composition of fatty acids in zebrafish. • Betaine induces mTOR signaling in the muscle of zebrafish. • Betaine inhibits the synthesis of fatty acids via mTOR signaling. Betaine has three active methyl groups, which determine its unique function in the metabolism of animals. In this study, the mechanism that betaine regulates the synthesis of fatty acids via mTOR signaling was investigated in zebrafish. It was found that betaine induced the protein expression of mTOR, P-4EBP1, and P-S6, but inhibited the gene expression of GSK-3β, ACC, ACL, and FAS in the muscle of zebrafish. In addition, betaine decreased the content of NEFA and TG as well as the activities of ACC, ACL, and FAS. The total content of PUFA was elevated, but the total content of SFA was decreased by betaine treatments. Moreover, the interference of GSK-3β gene induced mTOR signaling but decreased the gene expression of ACL, ACC, and FAS in muscle. In all, betaine could induce mTOR signaling, which may further participate in regulating the synthesis of fatty acids in the muscle of zebrafish. [ABSTRACT FROM AUTHOR]

Abstract Retinoic acid-inducible gene I (RIG-I), an RNA sensor with a conserved structure, activates the host interferon (IFN) system to produce IFNs and cytokines for eliminating pathogens upon recognizing PAMPs. However, the biological functions and the mechanism by which RIG-I regulates the innate immunity response in invertebrates are still unknown at present. Here we identified an atypical RIG-I in planarian Dugesia japonica. Sequence analysis, 3D structure modeling and phylogenetic analysis showed that this atypical protein was clustered into a single clade at the base of the tree in invertebrates, suggesting that Dj RIG-I is an ancient and unique protein of the RIG-I-like receptors (RLRs). In situ hybridization analysis revealed that the Dj RIG-I mRNAs were predominantly expressed in the pharynx and head of the adult and regenerative planarians. Stimulation with PAMPs induced the over-expression of DjRIG-I in planarians. The molecular simulation demonstrated that Dj RIG-I formed a large hole-structure for the docking of dsRNAs, and the pull-down assay confirmed the interaction between Dj RIG-I and viral analog poly(I:C). Importantly, some representative antiviral/antibacterial genes in the RIG-I-mediated IFN and P38 signaling pathway, TBK1 , IRF-3 , Mx , and P38, were significantly upregulated in planarians stimulated with PAMPs. Interference of the Dj RIG-I expression by RNAi, inhibited the PAMPs-induced over-expression, suggesting that Dj RIG-I is a key player for downstream signaling events. These results indicate that Dj RIG-I triggered the intracellular signaling cascades independent of the classical CARD domains and played an essential role in the virus/bacteria-induced innate immunity of planarian. Highlights • An atypical RIG-I-like gene (DjRIG-I) is isolated and characterized from planarian Dugesia japonica. • DjRIG-I mRNAs are significantly distributed throughout the body of the intact adult and regenerating planarians. • Stimulation with PAMPs induced the over-expression of DjRIG-I , which can also interact with viral analog poly(I:C). • Dj RIG-I participates in the virus- and bacteria-induced innate immunity of planarian. [ABSTRACT FROM AUTHOR]

Abstract The tumor necrosis factor (TNF) superfamily consists of a wide variety of inflammatory cytokine, including cell-bound and secreted proteins. These TNFs function through binding and activation of the TNF receptors for modulating TNF-associated intracellular signals. A set of mammalian TNF receptor-associated factors (TRAFs) that have emerged as the major signal transducers for the TNF receptor superfamily, play an important role in both adaptive and innate immunity. However, the existence of TRAFs and their biological functions in planarian are still unknown. In this study, a new member of TRAFs, DjTRAF2 , was identified in planarian Dugesia japonica. Phylogenetic analysis revealed that DjTRAF2 could be a new member of the invertebrate TRAF2 family. Sequence analysis showed that the open reading frame of DjTRAF2 had 1353 bp in length and encoded a putative protein of 450 amino acids with a predicted molecular mass of ~51.8 kDa and an isoelectric point of 7.052. Whole-mount in situ hybridization showed that DjTRAF2 was predominantly expressed in adult and regenerative pharynx, which is an important immune organ of planarian. Quantitative real-time PCR revealed that the transcriptional level of DjTRAF2 was significantly up-regulated after induced by pathogen-associated molecular patterns (polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan), suggesting that DjTRAF2 is involved in the immune response against pathogen invasion. Collectively, these results demonstrated that DjTRAF2 might play important roles in the innate immunity of planarian. Highlights • A member of TNF-receptor associated factors (TRAFs) from planarian D. japonica is identified and characterized as DjTRAF2. • DjTRAF2 is mainly distributed in regenerating and adult pharynx of planarians. • DjTRAF2 mRNAs are up-regulated after challenged by pathogen-associated molecular patterns. [ABSTRACT FROM AUTHOR]

In this study, the mechanism that lithium (Li) promotes the production of reactive oxygen species (ROS) via the glycogen synthase kinase-3β (GSK-3β)/tuberous sclerosis complex 2 (TSC2)/target of rapamycin (TOR) signaling was investigated in the gill of zebrafish ( Danio rerio ). After the zebrafish were treated by 25 and 50 mg/L Li + , the mRNA expression of GSK-3β and TSC2 was inhibited, but the expression of TOR was induced in the gill of zebrafish. The levels of hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 ·- ), and hydroxy radical (·OH) as well as the activity of superoxide dismutase (SOD) were increased, while the activities of catalase (CAT), glutathione peroxidase (GSH-PX), and peroxidase (POD) were decreased by 25 and 50 mg/L Li + treatments. In the ZF4 cells, the mRNA expression of GSK-3β and TSC2 was inhibited, but TOR expression was induced by 1, 5, and 10 mmol/L Li + treatments. To further confirm that lithium promoted ROS production via GSK-3β inhibition, GSK-3β RNA was interfered. It was found that the interference of GSK-3β RNA induced the TSC2/TOR signaling. The levels of H 2 O 2 , O 2 ·- , and ·OH were increased, but the activities of CAT, GSH-PX, and POD were decreased by GSK-3β RNA interference. In addition, lithium decreased the mitochondrial membrane potential (MMP) with Rhodamine-123 assay, but increased the levels of ROS by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay. The present results indicated that lithium promoted the ROS production through the GSK-3β/TSC2/TOR signaling in the gill of zebrafish. [ABSTRACT FROM AUTHOR]

14-3-3 proteins are a family of highly conserved acidic proteins that regulate cellular processes. They act as a kind of important signaling molecules taking part in many crucial decisions throughout the development process. We have isolated and characterized two members of the 14-3-3 family, namely, Dj14-3-3 α and Dj14-3-3 ζ in the planarian Dugesia japonica . The Dj14-3-3 α and ζ genes encode polypeptides of 260 and 255 amino acids respectively. We have proved that the Dj14-3-3 α and ζ genes were especially expressed in the pharynx in adult and regenerating planarians by in situ hybridization and they were not involved in regeneration process. Besides, Dj14-3-3 α and ζ genes can compensate each other in planarians by RNA interference. The Dj14-3-3 α and ζ were significantly up-regulated expression when planarians were stimulated with the pathogen-associated molecular patterns including lipopolysaccharide (LPS), peptidoglycan (PGN), β-Glu and Poly (I:C), indicating that the Dj14-3-3 α and ζ may be involved in the immune responses. [ABSTRACT FROM AUTHOR]

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica ( D. japonica ), named DjPLSCR . The sequence analysis revealed that it contains an opening reading frame consisting of 726 bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~ 28.7 kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians. [ABSTRACT FROM AUTHOR]

Planarian, a representative of platyhelminthes, has strong regeneration ability and less complicated innate immune system. However, planarian immune system remains poorly understood. In this paper, a novel C-type lectin-like protein, namely, Dj CTL was identified and characterized in Dugesia japonica. Dj CTL was mainly expressed in the pharyngeal and epidermis and up-regulated upon the induction of lipopolysaccharide (LPS), peptidoglycan (PGN), Gram-positive and Gram-negative bacteria indicating that DjCTL may be involved in the immune responses. Recombination Dj CTL protein agglomerated rabbit red blood cells and interacted with LPS, PGN, mannose and galactose as well as both Gram-positive and Gram-negative bacteria, but it can only cause the agglutination of Gram-negative bacteria. Importantly, in the early periods of regeneration, DjCTL had a significantly high expression and was mainly expressed in early blastemas. RNA interference of DjCTL by dsRNA- DjCTL led to a slow wound healing during regeneration. These findings suggest that DjCTL participates in the innate immune response and plays an important role in early stages of regeneration. [ABSTRACT FROM AUTHOR]

[Display omitted] • Deoxyribonuclease II in D. japonica are involved in the antimicrobial response. • Deoxyribonuclease II in D. japonica are involved in the wound-induced response. • Deoxyribonuclease II in D. japonica can clear young Gram-negative bacteria biofilm. • The construction of recombinant phages carrying Deoxyribonuclease II of D. japonica. Deoxyribonuclease II (DNase II) has been found to regulate inflammation, autoimmunity and apoptosis in vertebrates and invertebrates. The strong capacity of degrading DNA makes DNase II play an important role in the immune process. Planarian has become one of the model references due to its strong immune system, the environment they live makes planarians face the threat of microorganisms and injury, the strong immune system can protect planarians from the threat of bacterial and infection. In this study, we found that there was DNase in the lysis buffer of planarians, then we acquired the sequence of DjDN2s (Dugesia japonica DNase2s) and confirmed the Dj DN2s were conserved DNase IIs. The predicted structure showed the active sites and binding patterns of Dj DN2s. Whole-mount in situ hybridization results showed DjDN2s mainly expressed in immune organs. Quantitative real-time PCR revealed that the expression of DjDN2s upregulated in varying degrees when got hurt and challenged with bacteria, and the knockdown of DjDN2s led to the slower repair of wound. The recombinant phages which take Dj DN2 also had the ability to degrade DNA and clear young biofilm of Gram-negative bacteria. Collectively, DNase II of planarian might play a role in the antimicrobial response and wound-induced response. [ABSTRACT FROM AUTHOR]

β-Thymosin is a multifunctional peptide ubiquitously expressed in vertebrates and invertebrates. Many studies have found β-thymosin is critical for wound healing, angiogenesis, cardiac repair, hair regrowth, and anti-fibrosis in vertebrates, and plays an important role in antimicrobial immunity in invertebrates. However, whether β-thymosin participates in the regeneration of organisms is still poorly understood. In this study, we identified a β-thymosin gene in Dugesia japonica which played an important role in stem cell proliferation and neuron regeneration during the tissue repair process in D. japonica. Sequencing analysis showed that β-thymosin contained two conserved β-thymosin domains and two actin-binding motifs, and had a high similarity with other β-thymosins of invertebrates. In situ or fluorescence in situ hybridization analysis revealed that Djβ-thymosin was co-localized with DjPiWi in the neoblast cells of intact adult planarians and the blastema of regenerating planarians, suggesting Dj β-thymosin has a potential function of regeneration. Disruption Djβ-thymosin by RNA interference results in a slightly curled up head of planarian and stem cell proliferation defects. Additionally, we found that, upon amputation, Djβ-thymosin RNAi-treated animals had impaired regeneration ability, including impaired blastema formation, delayed eyespot formation, decreased brain area, and disrupted central CNS formation, implying Dj β-thymosin is an essential regulator of stem cell proliferation and neuron regeneration. • A β-thymosin gene is isolated and characterized from planarian Dugesia japonica. • Dj β-thymosin is involved in planarian central nervous system re-establishment as well as blastema and eyespot formation. • Dj β-thymosin is an essential regulator of stem cell proliferation and neuron regeneration. [ABSTRACT FROM AUTHOR]

The powerful regenerative ability of planarians has long been a concern of scientists, but recently, their efficient immune system has attracted more and more attention from researchers. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is related not only to antigen presentation but also to bacteria invasions. But the systematic studies are not yet to be conducted on the relationship between bacterial infection. Our study reveals for the first time that GILT of planarian (Dj GILT) plays an essential role in the clearance of Gram-negative bacteria by conducting H 2 O 2 concentration in planarians. In animals that DjGILT was silenced, it persisted for up to 9 days before all bacteria were cleared, compared with 6 days of the control group. When infected with E. coli and V. anguillarum , the level of H 2 O 2 was significantly increased in DjGILT -silenced planarians, and concomitantly, mRNA level of C-type lectin DjCTL , which modulates agglutination and clearance efficiency of invading bacteria, was decreased. Further study showed that the decrease of H 2 O 2 level led to a significant increase in DjCTL transcripts. Collectively, we proposed a mechanism model for the involvement of GILT gene in bacterial elimination. We have for the first time revealed the specific mechanism of GILT in innate immune response against bacterial infection. • A GILT gene, as an antioxidant, was cloned from Planarian, Dugesia japonica. • Dj GILT plays an essential role in the clearance of Gram-negative bacteria. • DjGILT mediated H 2 O 2 level affected DjCTL expression. [ABSTRACT FROM AUTHOR]