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Current Issues in Molecular Biology May 2024Melanocytes, located in the epidermis' basal layer, are responsible for melanin pigment production, crucial for skin coloration and protection against UV...
Melanocytes, located in the epidermis' basal layer, are responsible for melanin pigment production, crucial for skin coloration and protection against UV radiation-induced damage. Melanin synthesis is intricately regulated by various factors, including the Wnt signaling pathway, particularly mediated by the microphthalmia-associated transcription factor (MITF). While MITF is recognized as a key regulator of pigmentation, its regulation by the Wnt pathway remains poorly understood. This study investigates the role of Sfrp5pepD, a peptide antagonist of the Wnt signaling pathway, in modulating melanogenesis and its potential therapeutic implications for pigmentary disorders. To tackle this issue, we investigated smaller peptides frequently utilized in cosmetics or pharmaceuticals. Nevertheless, there is a significant scarcity of reports on peptides associated with melanin-related signal modulation or inhibiting melanin production. Results indicate that Sfrp5pepD effectively inhibits Wnt signaling by disrupting the interaction between Axin-1 and β-catenin, thus impeding downstream melanogenic processes. Additionally, Sfrp5pepD suppresses the interaction between MITF and β-catenin, inhibiting their nuclear translocation and downregulating melanogenic enzyme expression, ultimately reducing melanin production. These inhibitory effects are validated in cell culture models suggesting potential clinical applications for hyperpigmentation disorders. Overall, this study elucidates the intricate interplay between Wnt signaling and melanogenesis, highlighting Sfrp5pepD as a promising therapeutic agent for pigmentary disorders. Sfrp5pepD, with a molecular weight of less than 500 Da, is anticipated to penetrate the skin unlike SFRPs. This suggests a strong potential for their use as cosmetics or transdermal absorption agents. Additional investigation into its mechanisms and clinical significance is necessary to enhance its effectiveness in addressing melanin-related skin conditions.
PubMed: 38920996
DOI: 10.3390/cimb46060324 -
Nature Communications Jun 2024Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including...
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.
Topics: Animals; Opioid Peptides; Nociceptin; Receptors, Opioid; Neurons; Humans; Mice; Male; Ventral Tegmental Area; Nociceptin Receptor; HEK293 Cells; Brain; Mice, Inbred C57BL; Ligands; Biosensing Techniques
PubMed: 38918403
DOI: 10.1038/s41467-024-49712-0 -
PloS One 2024SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at...
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.
Topics: Escherichia coli Proteins; Peptidylprolyl Isomerase; Escherichia coli; Protein Binding; Molecular Chaperones; Protein Sorting Signals; Hydrophobic and Hydrophilic Interactions; Calorimetry; Arginine; Amino Acid Sequence
PubMed: 38917203
DOI: 10.1371/journal.pone.0305823 -
BioRxiv : the Preprint Server For... Jun 2024The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive...
The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as acceptor. We have optimized the assay into 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.
PubMed: 38915662
DOI: 10.1101/2024.06.11.598473 -
Renal Failure Dec 2024Liraglutide, a glucagon-like peptide-1 receptor agonist, has been shown to regulate blood sugar and control body weight, but its ability to treat obesity-related...
OBJECTIVE
Liraglutide, a glucagon-like peptide-1 receptor agonist, has been shown to regulate blood sugar and control body weight, but its ability to treat obesity-related nephropathy has been poorly studied. Therefore, this study was designed to observe the characteristics and potential mechanism of liraglutide against obesity-related kidney disease.
METHODS
Thirty-six C57BL/6J male mice were randomly divided into six groups ( = 6 per group). Obesity-related nephropathy was induced in mice by continuous feeding of high-fat diet (HFD) for 12 weeks. After 12 weeks, liraglutide (0.6 mg/kg) and AMP-activated protein kinase (AMPK) agonists bortezomib (200 μg/kg) were injected for 12 weeks, respectively. Enzyme-linked immunosorbent assay was employed to detect the levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, blood urea nitrogen, creatinine in serum, as well as urinary protein in urine. Besides, hematoxylin-eosin staining and periodic acid-Schiff staining were used to observe the pathological changes of kidney tissue; immunohistochemistry, western blot, and real-time quantitative PCR to assess the calmodulin-dependent protein kinase kinase beta (CaMKKβ)/AMPK signaling pathway activation.
RESULTS
Liraglutide significantly reduced serum lipid loading, improved kidney function, and relieved kidney histopathological damage and glycogen deposition in the mouse model of obesity-related kidney disease induced by HFD. In addition, liraglutide also significantly inhibited the CaMKKβ/AMPK signaling pathway in kidney tissue of HFD-induced mice. However, bortezomib partially reversed the therapeutic effect of liraglutide on HDF-induced nephropathy in mice.
CONCLUSIONS
Liraglutide has a therapeutic effect on obesity-related kidney disease, and such an effect may be achieved by inhibiting the CaMKKβ/AMPK signaling pathway in kidney tissue.
Topics: Animals; Liraglutide; Male; Diet, High-Fat; Mice; AMP-Activated Protein Kinases; Mice, Inbred C57BL; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Signal Transduction; Obesity; Kidney; Disease Models, Animal; Hypoglycemic Agents
PubMed: 38915241
DOI: 10.1080/0886022X.2024.2351473 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2024Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the...
Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P, P, and P) and three inducible promoters (P, P, and P) on the expression of HLF were compared using G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 μg/L, was achieved when regulated by the P promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SP resulted in increased HLF expression to 1 099.87 μg/L, with 498.68 μg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene was knocked out, leading to an extracellular HLF level of 637.28 μg/L. This study successfully demonstrated the secretory expression of HLF in . through the selection and optimization of expression elements, laying the foundation for the development of efficient . cell factories for lactoprotein synthesis.
Topics: Bacillus subtilis; Lactoferrin; Humans; Promoter Regions, Genetic; Recombinant Proteins
PubMed: 38914499
DOI: 10.13345/j.cjb.230785 -
International Journal of Nanomedicine 2024Combination therapy offers superior therapeutic results compared to monotherapy. However, the outcomes of combination therapy often fall short of expectations, mainly...
BACKGROUND
Combination therapy offers superior therapeutic results compared to monotherapy. However, the outcomes of combination therapy often fall short of expectations, mainly because of increased toxicity from drug interactions and challenges in achieving the desired spatial and temporal distribution of drug delivery. Optimizing synergistic drug combination ratios to ensure uniform targeting and distribution across space and time, particularly in vivo, is a significant challenge. In this study, cRGD-coated liposomes encapsulating optimized synergistic cepharanthine (CEP; a chemotherapy drug) and IR783 (a phototherapy agent) were developed for combined chemotherapy and photothermal therapy in vitro and in vivo.
METHODS
An MTT assay was used to evaluate the combination index of CEP and IR783 in five cell lines. The cRGD-encapsulated liposomes were prepared via thin-film hydration, and unencapsulated liposomes served as controls for the loading of CEP and IR783. Fluorescence and photothermal imaging were used to assess the efficacy of CEP and IR783 encapsulated in liposomes at an optimal synergistic ratio, both in vitro and in vivo.
RESULTS
The combination indices of CEP and IR783 were determined in five cell lines. As a proof-of-concept, the optimal synergistic ratio (1:2) of CEP to IR783 in 4T1 cells was evaluated in vitro and in vivo. The average diameter of the liposomes was approximately 100 nm. The liposomes effectively retained the encapsulated CEP and IR783 in vitro at the optimal synergistic molar ratio for over 7 d. In vivo fluorescence imaging revealed that the fluorescence signal from cRGD-CEP-IR783-Lip was detectable at the tumor site at 4 h post-injection and peaked at 8 h. In vivo photothermal imaging of tumor-bearing mice indicated an increase in tumor temperature by 32°C within 200 s. Concurrently, cRGD-CEP-IR783-Lip demonstrated a significant therapeutic effect and robust biosafety in the in vivo antitumor experiments.
CONCLUSION
The combination indices of CEP and IR783 were successfully determined in vitro in five cell lines. The cRGD-coated liposomes encapsulated CEP and IR783 at an optimal synergistic ratio, exhibiting enhanced antitumor effects and targeting upon application in vitro and in vivo. This study presents a novel concept and establishes a research framework for synergistic chemotherapy and phototherapy treatment.
Topics: Liposomes; Animals; Cell Line, Tumor; Humans; Female; Mice; Indoles; Photothermal Therapy; Benzylisoquinolines; Mice, Inbred BALB C; Peptides, Cyclic; Drug Synergism; Antineoplastic Agents; Combined Modality Therapy; Cell Survival; Drug Delivery Systems; Benzodioxoles
PubMed: 38911506
DOI: 10.2147/IJN.S457008 -
Frontiers in Pharmacology 2024Tirzepatide, a glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor agonist, is indicated for chronic weight...
BACKGROUND
Tirzepatide, a glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor agonist, is indicated for chronic weight management in adults with obesity or overweight as an adjunct to a reduced-calorie diet and increased physical activity. However, the safety profile of Tirzepatide-associated adverse events requires comprehensive evaluation.
METHODS
The AE reports from the first quarter of 2022 to the third quarter of 2023 were selected by exploring the FDA Adverse Event Reporting System (FAERS) database. The new and unexpected potenial AE signals were detected using the disproportionality analysis, including reporting odds ratio(ROR), the proportional reporting ratio (PRR) the Bayesian confidence propagation neural network (BCPNN) and the empirical Bayes geometric mean(EBGM). Then the MedDRA was used to systematically classify the results.
RESULTS
A total of 1,904,481 case reports were obtained from 2022Q2 to 2023Q3. Forty-sixth tirzepatide-induced ADRs at the preferred terms (PTs) level are associated with 8 system organ class In addition, this study uncovered multiple anticipated ADRs, such as gastrooesophageal reflux disease, dyspepsia, and vomiting, in line with the drug labels. Moreover, unexpected and significant ADRs at PTs level, such as incorrect dose administered, injection site haemorrhage, and increased appetite, were discovered and linked to Injury, poisoning, and procedural complications, General disorders and administration site conditions, and Metabolism and nutrition disorders at the System Organ Class level.
CONCLUSION
This study offered new perspectives on the monitoring, surveillance, and management of adverse drug reactions related to tirzepatide. The outcomes of severe adverse events and their respective detection signals, along with unexpected significant adverse event signals, are important to consider in efforts to enhance clinical medication safety when using tirzepatide.
PubMed: 38910884
DOI: 10.3389/fphar.2024.1397029 -
Signal Transduction and Targeted Therapy Jun 2024Pancreatic cancer is one of the deadly malignancies with a significant mortality rate and there are currently few therapeutic options for it. The tumor microenvironment...
Pancreatic cancer is one of the deadly malignancies with a significant mortality rate and there are currently few therapeutic options for it. The tumor microenvironment (TME) in pancreatic cancer, distinguished by fibrosis and the existence of cancer-associated fibroblasts (CAFs), exerts a pivotal influence on both tumor advancement and resistance to therapy. Recent advancements in the field of engineered extracellular vesicles (EVs) offer novel avenues for targeted therapy in pancreatic cancer. This study aimed to develop engineered EVs for the targeted reprogramming of CAFs and modulating the TME in pancreatic cancer. EVs obtained from bone marrow mesenchymal stem cells (BMSCs) were loaded with miR-138-5p and the anti-fibrotic agent pirfenidone (PFD) and subjected to surface modification with integrin α5-targeting peptides (named IEVs-PFD/138) to reprogram CAFs and suppress their pro-tumorigenic effects. Integrin α5-targeting peptide modification enhanced the CAF-targeting ability of EVs. miR-138-5p directly inhibited the formation of the FERMT2-TGFBR1 complex, inhibiting TGF-β signaling pathway activation. In addition, miR-138-5p inhibited proline-mediated collagen synthesis by directly targeting the FERMT2-PYCR1 complex. The combination of miR-138-5p and PFD in EVs synergistically promoted CAF reprogramming and suppressed the pro-cancer effects of CAFs. Preclinical experiments using the orthotopic stroma-rich and patient-derived xenograft mouse models yielded promising results. In particular, IEVs-PFD/138 effectively reprogrammed CAFs and remodeled TME, which resulted in decreased tumor pressure, enhanced gemcitabine perfusion, tumor hypoxia amelioration, and greater sensitivity of cancer cells to chemotherapy. Thus, the strategy developed in this study can improve chemotherapy outcomes. Utilizing IEVs-PFD/138 as a targeted therapeutic agent to modulate CAFs and the TME represents a promising therapeutic approach for pancreatic cancer.
Topics: Pancreatic Neoplasms; Extracellular Vesicles; Humans; Cancer-Associated Fibroblasts; Mice; MicroRNAs; Animals; Tumor Microenvironment; Cellular Reprogramming; Cell Line, Tumor; Mesenchymal Stem Cells; Neoplasm Proteins; Gemcitabine
PubMed: 38910148
DOI: 10.1038/s41392-024-01872-7 -
NPJ Vaccines Jun 2024Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells,...
Vaccines generally require T lymphocytes for B-cell activation and immunoglobulin class switching in response to peptide or protein antigens. In the absence of T cells, limited IgG class switch takes place, germinal centers are short-lived, and the B cells lack memory. Here, immunization of mice with liposomes containing 15mer peptides and monophosphoryl lipid A (MPLA) as adjuvant, induced T-cell independent (TI) IgG class switch within three days, as well as germinal center formation. The antibody responses were long-lived, strictly dependent on Toll-like receptor 4 (TLR4) signaling, partly dependent on Bruton's tyrosine kinase (BTK) signal transmission, and independent of signaling through T-cell receptors, MHC class II and inflammasome. The antibody response showed characteristics of both TI type 1 and TI type 2. All IgG subclasses could be boosted months after primary immunization, and the biological function of the secreted antibodies was demonstrated in murine models of allergic anaphylaxis and of bacterial infection. Moreover, antibody responses after immunization with peptide- and MPLA-loaded liposomes could be triggered in neonatal mice and in mice receiving immune-suppressants. This study demonstrates T-cell independent endogenous B-cell memory and recall responses in vivo using a peptide antigen. The stimulation of these antibody responses required a correct and dense assembly and administration of peptide and adjuvant on the surface of liposomes. In the future, TI vaccines may prove beneficial in pathological conditions in which T-cell immunity is compromised through disease or medicines or when rapid, antibody-mediated immune protection is needed.
PubMed: 38909055
DOI: 10.1038/s41541-024-00902-3