A pyridinium-based strategy for lysine-selective protein modification and chemoproteomic profiling in live cells.

Protein active states are dynamically regulated by various modifications; thus, endogenous protein modification is an important tool for understanding protein functions and networks in complicated biological systems. Here we developed a new pyridinium-based approach to label lysine residues under physiological conditions that is low-toxicity, efficient, and lysine-selective. Furthermore, we performed a large-scale analysis of the ∼70% lysine-selective proteome in MCF-7 cells using activity-based protein profiling (ABPP). We quantifically assessed 1216 lysine-labeled peptides in cell lysates and identified 386 modified lysine sites including 43 mitochondrial-localized proteins in live MCF-7 cells. Labeled proteins significantly preferred the mitochondria. This pyridinium-based methodology demonstrates the importance of analyzing endogenous proteins under native conditions and provides a robust chemical strategy utilizing either lysine-selective protein labeling or spatiotemporal profiling in a living system.

ß-Carbonyl sulfonium enables cysteine-specific bioconjugation for activity-based protein profiling in live cells.

Chemical labeling methods for proteins are highly researched. Herein, we introduced ß-carbonyl sulfonium compounds for selective cysteine modification in proteins within biological systems. Structural tuning led to sulfonium-based probes with high reactivity and selectivity. These probes show excellent biocompatibility, cell uptake, and specificity towards cysteine profiling in live cells.

Traceless Peptide and Protein Modification via Rational Tuning of Pyridiniums.

Herein, we report a versatile reaction platform for tracelessly cleavable cysteine-selective peptide/protein modification. This platform offers highly tunable and predictable conjugation and cleavage by rationally estimating the electron effect on the nucleophilic halopyridiniums. Cleavable peptide stapling, antibody conjugation, enzyme masking/de-masking, and proteome labeling were achieved based on this facile pyridinium-thiol-exchange protocol.

Covalent PROTAC design method based on a sulfonyl pyridone probe.

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins. The conventional approach relies on covalent ligands, but our study presents an innovative method employing an N-sulfonyl pyridone warhead to selectively target tyrosine (Tyr) residues. The von Hippel-Lindau (VHL) moiety is transferred from the warhead to the exposed Tyr, allowing us to design a STING degrader (DC50 0.53 µM, Dmax 56.65%). This approach showcases the potential of nucleophilic amino acid labeling probes, particularly for proteins lacking easily accessible cysteine residues, opening new possibilities for covalent PROTAC design and targeted protein degradation therapies.

Pyridinium-Based Strategy for a Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome.

Cell surface proteins (CSPs) are valuable targets for therapeutic agents, but achieving highly selective CSP enrichment in cellular physiology remains a technical challenge. To address this challenge, we propose a newly developed sulfo-pyridinium ester (SPE) cross-linking probe, followed by two-step imaging and enrichment. The SPE probe showed higher efficiency in labeling proteins than similar NHS esters at the level of cell lysates and demonstrated specificity for Lys in competitive experiments. More importantly, this probe could selectively label the cell membranes in cell imaging with only negligible labeling of the intracellular compartment. Moreover, we successfully performed this strategy on MCF-7 live cells to label 425 unique CSPs from 1162 labeled proteins. Finally, we employed our probe to label the CSPs of insulin-cultured MCF-7, revealing several cell surface targets of key functional biomarkers and insulin-associated pathogenesis. The above results demonstrate that the SPE method provides a promising tool for the selective labeling of cell surface proteins and monitoring transient cell surface events.

4-Iodine N-Methylpyridinium-Mediated Peptide Synthesis.

Through systematic optimization of halopyridinium compounds, we established a peptide coupling protocol utilizing 4-iodine N-methylpyridinium (4IMP) for solid-phase peptide synthesis (SPPS). The 4IMP coupling reagent is easily prepared, bench stable, and cost-effective. Employing 4IMP in the SPPS process has showcased remarkable chemoselectivity and efficiency, effectively eliminating racemization and epimerization. This achievement has been substantiated through the successful synthesis of a range of peptides via the direct utilization of commercially available amino acid substrates for SPPS.

Development of Lysine Crotonyl-Mimic Probe to Covalently Identify H3K27Cr Interacting Proteins.

Histone lysine crotonylation (Kcr) is one newly discovered acylation modification and regulates numerous pathophysiological processes. The binding affinity between Kcr and its interacting proteins is generally weak, which makes it difficult to effectively identify Kcr-interacting partners. Changing the amide of crotonyl to an ester increased reactivity with proximal cysteines and retained specificity for Kcr antibody. The probe "H3g27Cr" was designed by incorporating the ester functionality into a H3K27 peptide. Using this probe, multiple Kcr-interacting partners including STAT3 were successfully identified, and this has not been reported previously. Further experiments suggested that STAT3 possibly could form complexes with Histone deacetylase HDACs to downregulate the acetylation and crotonylation of Histone H3K27. Our unique design provided intriguing tools to further explore Kcr-interacting proteins and elucidate their working mechanisms.

Functional Involvement of ADRA1D in Cutaneous Melanoma Progression and Angiogenesis.

Cutaneous melanoma is a highly aggressive and malignant skin cancer, and its high recurrence rate and drug resistance increase the difficulty of treating advanced-stage patients. Studies have revealed that treatment via stimulation of alpha-1 adrenergic receptor (ADRA1) subtypes inhibits melanoma growth in mice. However, the associations between alpha-1D adrenergic receptor (ADRA1D) and cutaneous melanoma are poorly understood. Tissue specimens from 16 pairs of patients with a pigmented nevus and cutaneous melanoma were analyzed for ADRA1D expression using immunohistochemical staining. Western blotting and RT-qPCR were carried out in order to detect ADRA1D expression levels in melanoma cells and human epidermal melanocytes (HEMs), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) levels in HUVECS. A375 cells were transfected with a lentivirus overexpressing ADRA1D. Wound-healing, Transwell, and cell proliferation assays were utilized to identify the ADRA1D effect on the migration, invasion, and proliferation of the two groups of A375 cells in vitro. In order to evaluate the function of ADRA1D in vivo, a melanoma xenograft model was developed in immunodeficient mice. ADRA1D was low expressed in cutaneous melanoma tissues. Overexpression of ADRA1D inhibited the tubulation and migration of HUVECs in vitro. Overexpression of ADRA1D significantly decreased the HIF-1α and VEGF expression. Overexpression of ADRA1D inhibited the invasion and proliferation of A375 melanoma cells in vitro and reduced its angiogenesis in vivo. ADRA1D inhibits cutaneous melanoma growth and angiogenesis. It attenuates melanoma cell proliferation and invasion. Meanwhile, its anti-angiogenic effect is achieved by negatively regulating the HIF-1α/VEGF axis in melanoma tissue, thereby attenuating the growth of cutaneous melanoma and reducing the potential of metastasis.

Ultraviolet B phototherapy does not increase the risk of skin cancer among patients with atopic dermatitis: A population-based retrospective cohort study.

BACKGROUND: UV-B phototherapy is a common treatment modality for patients with atopic dermatitis (AD), but its long-term safety in terms of cutaneous carcinogenic risk has not been studied. OBJECTIVE: To investigate the risk of skin cancer among patients with AD receiving UV-B phototherapy. METHODS: We conducted a nationwide population-based cohort study from 2001 to 2018 to estimate the risk of UV-B phototherapy for skin cancer, nonmelanoma skin cancer, and cutaneous melanoma in patients with AD. RESULTS: Among 6205 patients with AD, the risks of skin cancer (adjusted hazard ratio [HR], 0.91; 95% CI, 0.35-2.35), nonmelanoma skin cancer (adjusted HR, 0.80; 95% CI, 0.29-2.26), and cutaneous melanoma (adjusted HR, 0.80; 95% CI, 0.08-7.64) did not increase among patients with AD treated with UV-B phototherapy, compared with those who did not receive UV-B phototherapy. Additionally, the number of UV-B phototherapy sessions was not associated with an increased risk of skin cancer (adjusted HR, 0.99; 95% CI, 0.96-1.02), nonmelanoma skin cancer (adjusted HR, 0.99; 95% CI, 0.96-1.03), or cutaneous melanoma (adjusted HR, 0.94; 95% CI, 0.77-1.15). LIMITATIONS: Retrospective study. CONCLUSION: Neither UV-B phototherapy nor the number of UV-B phototherapy sessions was associated with an increased risk of skin cancers among patients with AD.

Targeted Biomolecule Regulation Platform: A Split-and-Mix PROTAC Approach.

The development of bifunction al molecules, which can enable targeted RNA degradation, targeted protein acetylation, or targeted protein degradation, remains a time-consuming process that requires tedious optimization. We propose a split-and-mix nanoplatform that serves as a self-adjustable platform capable of facile screening, programmable ligand ratios, self-optimized biomolecule spatial recognition, and multifunctional applications. Herein, we demonstrate the potential of our proposed nanoplatform by showcasing proteolysis-targeting chimeras (PROTACs), namely, split-and-mix PROTAC (SM-PROTAC). We highlight the scope of our platform through the targeted disruption of intracellular therapeutic targets involving ERα, CDK4/6, AR, MEK1/2, BRD2/4, BCR-ABL, etc. These studies confirm the effectiveness and universality of the SM-PROTAC platform for proximity-induced applications. This platform is programmable, with significant potential applications to biomolecule regulation, including the fields of epigenetics, gene editing, and biomolecule modification regulation.

The thiol-sulfoxonium ylide photo-click reaction for bioconjugation.

Visible-light-mediated methods were heavily studied as a useful tool for cysteine-selective bio-conjugation; however, many current methods suffer from bio-incompatible reaction conditions and slow kinetics. To address these challenges, herein, we report a transition metal-free thiol-sulfoxonium ylide photo-click reaction that enables bioconjugation under bio-compatible conditions. The reaction is highly cysteine-selective and generally finished within minutes with naturally occurring riboflavin derivatives as organic photocatalysts. The catalysts and substrates are readily accessible and bench stable and have satisfactory water solubility. As a proof-of-concept study, the reaction was smoothly applied in chemo-proteomic analysis, which provides efficient tools to explore the druggable content of the human proteome.

Extremely Low Dose of Erythropoiesis-Stimulating Agent May Be Associated with Increased Mortality in Hemodialysis Patients.

INTRODUCTION: Although high-dose erythropoiesis-stimulating agent (ESA) has been shown to increase mortality risk and adverse cardiovascular events in hemodialysis patients, the safety of extremely low-dose ESA is unclear. METHODS: We retrospectively analyzed the association between ESA dose and mortality in the monthly dosing range of 0-43,000 U of equivalent epoetin alfa in 304 Taiwan hemodialysis patients by using Cox proportional hazard model and cubic spline model. RESULTS: Compared with mean monthly ESA dose of 15,000-25,000 U (mean ± standard deviation 20,609 ± 2,662 U), monthly ESA dose of less than 15,000 U (mean ± standard deviation 7,413 ± 4,510 U) is associated with increased mortality. Monthly ESA dose of 25,001-43,000 U (mean ± standard deviation 31,160 ± 4,304 U) is not associated with higher mortality risk than monthly ESA dose of 15,000-25,000 U. The results were consistent in Cox proportional hazard models and cubic spline models. Subgroup analyses showed no significant heterogeneities among prespecified subgroups. CONCLUSIONS: Extremely low dose of ESA in hemodialysis patients may be associated with increased mortality risk. Future studies are warranted to prove this association.

Three novel Actinoplanes species isolated by using polyaspartic acid as a water-retaining agent for the enrichment in situ.

Three novel actinomycete strains, designated TRM66264-DLMT, TRM88002T and TRM88003T, were isolated by using polyaspartic acid as a water-retaining agent for the enrichment in situ. The 16S rRNA gene sequence and phylogenetic analyses of three strains indicated that they belonged to the genus Actinoplanes. The phylogenetically closest strains of TRM66264-DLMT, TRM88002T and TRM88003T were Actinoplanes bogorensis LIPI11-2-Ac043T (98.4 %), Actinoplanes abujensis A4029T (98.0 %) and Actinoplanes ferrugineus IFO15555T (98.1 %), respectively. The major polar lipids of strains TRM66264-DLMT and TRM88002T were phosphatidylethanolamine and disphosphatidylglycerol, while strain TRM88003T only had phosphatidylethanolamine. The predominant menaquinones of strain TRM66264-DLMT were identified as MK-9(H4) and MK-9 (H6). Strains TRM88002T and TRM88003T had MK-9(H4). The cell-wall peptidoglycan of three strains contained meso-diaminopimelic acid. The whole-cell sugars of strain TRM66264-DLMT were identified as arabinose, glucose, galactose and xylose. Strains TRM88002T and TRM88003T mainly had arabinose and glucose. The DNA G+C content of strains TRM66264-DLMT, TRM88002T and TRM88003T were 70.48, 70.46 and 70.64 mol%, respectively. Genotypic and phenotypic analysis confirmed that all three strains sre new members of the genus Acinoplanes. Therefore, it is proposed that strains TRM66264-DLMT, TRM88002T and TRM88003T represent three novel species of the genus Actinoplanes, for which the names Actinoplanes polyasparticus sp. nov. (type strain TRM66264-DLMT=CCTCC AA 2021015T=LMG 32389T), Actinoplanes hotanensis sp. nov. (type strain TRM88002T=CCTCC AA 2021036T=LMG 32621T) and Actinoplanes aksuensis sp. nov. (type strain TRM88003T=CCTCC AA 2021037 T=LMG 32622T) are proposed.

Classic Eosinophilic Pustular Folliculitis in an Immunocompetent Patient with Syphilis: Are They Related?

Eosinophilic pustular folliculitis (EPF) is a rare, chronic, non-infectious inflammatory skin disease. Although the pathogenesis of EPF is unknown, eosinophilic pustular folliculitis may be associated with human immunodeficiency virus (HIV) infection, malignancies or syphilis. Here, we report the first case of EPF associated with syphilis, indicating that syphilis and EPF are correlated with T-helper type 2 immune responses. A 48-year-old man gradually developed erythema and pustules on the face, neck. Physical examination revealed multiple infiltrative red patches and plaques on the face, neck with tiny pustules. Skin biopsy results revealed that the dermal follicular sebaceous gland unit was infiltrated by a large number of neutrophils and eosinophils, forming eosinophilic microabscesses. Therefore, the patient was diagnosed with EPF associated with syphilis and received drug treatment. After the treatment, the pustules markedly decreased, leaving behind pigmentation. Furthermore, the patient is still being followed up.

Clinical value of the prognostic nutritional index and red blood cell distribution width-to-albumin ratio for the prediction of severity of and mortality associated with Stevens-Johnson syndrome/toxic epidermal necrolysis.

The prognostic nutritional index (PNI) and red blood cell distribution width-to-albumin ratio (RAR) are considered to be related to the prognosis of disease severity. However, the role of these biomarkers in predicting Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) severity and mortality is unclear. The aim of the current study was to investigate the association of PNI and RAR with severity and mortality in individuals with SJS/TEN. Clinical data were retrospectively collected from 74 individuals with SJS/TEN and 74 healthy individuals, who were matched for age and sex during the same period. PNI, RAR, and other indicators were compared between individuals with SJS/TEN and healthy controls. The association of PNI and RAR with SJS/TEN severity was assessed using Spearman or Pearson correlation analyses. Individuals with SJS/TEN were categorized into two groups, either survivors or nonsurvivors. The correlation between PNI, RAR, and SJS/TEN mortality was analyzed using univariate and multivariate logistic regression. The predictive value of the previously mentioned indicators on the mortality of patients with SJS/TEN was assessed using receiver operating characteristic curve analysis. The RAR level of patients with SJS/TEN was greater than that of the control group (p < 0.05), whereas PNI was lower. In compliance with correlation analysis, RAR was positively correlated with SCORTEN (Score of Toxic Epidermal Necrolysis) and ABCD-10 (age, bicarbonate, cancer, dialysis, 10% body surface area) (p < 0.05), and PNI was negatively correlated (p < 0.05). RAR is a risk factor for death in patients with SJS/TEN, but an elevated PNI level is a protective factor for mortality. The best cutoff values of PNI and RAR for predicting death in patients with SJS/TEN were 31.375 (sensitivity, 84.7%; specificity, 80%) and 0.486 (sensitivity, 73.3%; specificity, 84.7%). These results underscore the potential clinical value of PNI and RAR as appropriate and meaningful biomarkers to assess the severity of SJS/TEN and the mortality associated with it.

Synthesis and characterization of a macromolecular magnetic resonance imaging and delivery system with hyaluronic acid as a carrier.

Using hyaluronic acid (HA) as macromolecular drug carriers, a glutathione-responsive imaging drug delivery system HA-SS-a-Gd-DOTA was formed by conjugating gadolinium chelates and cytarabine. This system exhibited T1-reflexivity (21.9 mmol-1 L s-1, 0.5 T) that was higher than that of gadoterate meglumine. In an acidic environment, in vitro drug release reached 63.4% in 24 h. Low cytotoxicity indicated that this system has good biocompatibility. In vivo mouse imaging studies showed that tumor signaling was significantly enhanced. About 58% of the signal enhancement was obtained 50 min after injection of the drug. The degradation of the hyaluronic acid macromolecular chains in vivo makes it an ideal tumor imaging diagnostic agent because it did not cause damage to important organs of the mice.

Prediction of Diagnostic Gene Biomarkers Associated with Immune Infiltration for Basal Cell Carcinoma.

Purpose: Basal cell carcinoma (BCC) is a frequent tumor of the surface layer of skin or its accessories, and ranks first among the prevalence of skin cancer cases. However, its pathogenesis remains unclear. The purpose of this analysis was to scientifically evaluate the role of mRNAs in the occurrence and progression of BCC and further elucidate their underlying potential molecular mechanisms of action. Methods: Differentially expression genes (DEGs) between nineteen BCC cases and five controls which initiate from the GSE103439 and GSE7553 datasets were identified and the transcriptome sequencing information was obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and Gene Ontology (GO) annotation were performed. Logistic regression (LASSO) and support vector machine (SVM) analyses were performed to identify candidate biomarkers obtained from protein-protein interaction (PPI) analysis. The tumor microenvironment comprising hub genes in BCC was investigated by immune infiltration analysis. The expression of two representative hub genes (KIF23 and NCAPG) was measured by qRT-PCR. Finally, the potential miRNAs and lncRNAs related to the hub genes were analyzed on relevant websites to obtain a ceRNA interaction network. Results: Twenty-seven DEGs were identified. Fifteen hub genes were screened in the protein-protein interaction network. These showed marked enrichment in the cell cycle and p53 signaling pathway. FGF20, KIF23, and NCAPG were identified as the diagnostic markers of BCC. Immune cell infiltration analysis suggested their significant association with T cells CD4 memory activated, macrophages M1, and natural killer (NK) resting cells. Two miRNAs and twelve lncRNAs were used to construct the lncRNA-miRNA-mRNA ceRNA network. Conclusion: FGF20, KIF23, and NCAPG are potential diagnostic markers of BCC. Our findings may shed new light on the molecular mechanisms underlying BCC occurrence.