Corrigendum: miR-31-3p functions as a tumor suppressor by directly targeting GABBR2 in prostate cancer.

[This corrects the article DOI: 10.3389/fonc.2022.945057.].

Human Nasal Inferior Turbinate-Derived Neural Stem Cells Improve the Niche of Substantia Nigra Par Compacta in a Parkinson's Disease Model by Modulating Hippo Signaling.

BACKGROUND: Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases, following Alzheimer's disease. The onset of PD is characterized by the loss of dopaminergic neurons in the substantia nigra. Stem cell therapy has great potential for the treatment of neurodegenerative diseases, and human nasal turbinate-derived stem cells (hNTSCs) have been found to share some characteristics with mesenchymal stem cells. Although the Hippo signaling pathway was originally thought to regulate cell size in organs, recent studies have shown that it can also control inflammation in neural cells. METHODS: Dopaminergic neuron-like cells were differentiated from SH-SY5Y cells (DA-Like cells) and treated with 1-Methyl-4-phenylpyridinium iodide to stimulate Reactive oxidative species (ROS) production. A transwell assay was conducted to validate the effect of hNTSCs on the Hippo pathway. We generated an MPTP-induced PD mouse model and transplanted hNTSCs into the substantia nigra of PD mice via stereotaxic surgery. After five weeks of behavioral testing, the brain samples were validated by immunoblotting and immunostaining to confirm the niche control of hNTSCs. RESULTS: In-vitro experiments showed that hNTSCs significantly increased cell survival and exerted anti-inflammatory effects by controlling ROS-mediated ER stress and hippocampal signaling pathway factors. Similarly, the in-vivo experiments demonstrated an increase in anti-inflammatory effects and cell survival rate. After transplantation of hNTSCs, the PD mouse model showed improved mobility and relief from PD symptoms. CONCLUSION: hNTSCs improved the survival rate of dopaminergic neurons by manipulating the hippocampal pathway through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ) by reducing inflammatory cytokines. In this study, we found that controlling the niche of hNTSCs had a therapeutic effect on PD lesions.

Discovery of Orally Bioavailable Phthalazinone Analogues as an ENPP1 Inhibitor for STING-Mediated Cancer Immunotherapy.

A lack of the T cell-inflamed tumor microenvironment limits the efficacy of immune checkpoint inhibitors (ICIs). Activation of stimulator of interferon genes (STING)-mediated innate immunity has emerged as a novel therapeutic approach in cancer therapy. 2',3'-Cyclic GMP-AMP (cGAMP) is a natural STING agonist; however, cGAMP is subjected to endogenous degradation by ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). To improve the ICI response rate, we developed 29f, a novel ENPP1 inhibitor with phthalazin-1(2H)-one as the core scaffold. 29f inhibited the cGAMP hydrolysis by ENPP1 in vitro (IC50 = 68 nM) and enhanced the STING-mediated type I interferon response in both immune and tumor cells. 29f demonstrated excellent metabolic stability and bioavailability (F = 65%). Orally administered 29f promoted tumor growth inhibition in a CT26 syngeneic model and increased the anti-PD-L1 response. Furthermore, 29f-induced immunological memory prevented the tumor relapse against tumor rechallenge, suggesting the promising therapeutic potential of 29f.

Effect of the Duration of NSAID Use on COVID-19.

Background and Objectives: Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used to control pain and fever. However, their effect on COVID-19 infected patients has not been fully studied. In this study, we investigated the effect of the duration of NSAIDs use on COVID-19 infection and clinical outcomes. Materials and Methods: In South Korea, 25,739 eligible patients who received COVID-19 testing between 1 January and 31 July 2020, were included in this retrospective observational cohort analysis. Based on the date of the first COVID-19 test for each patient, NSAID prescription dates were used to separate patients into two groups (short-term group: <2 weeks; long-term group: 8−12 weeks). COVID-19 infectivity and clinical outcomes were analyzed. We used the propensity score-matching (PSM) method. Results: Of the 580 patients who had taken NSAIDs before the date of COVID-19 test, 534 and 46 patients were grouped in the short- and long-term NSAID-use groups, respectively. We did not find a statistically significant increased risk of COVID-19 infection (adjustment for age and sex, p = 0.413; adjustment for age, sex, region of residence, comorbidity, Charlson Comorbidity Index, and current use of medication, p = 0.259) or change in clinical outcomes, including conventional oxygen therapy, admission of intensive care unit, artificial ventilation, or death, between the two groups in which the PSM method was applied. Conclusions: The duration of NSAIDs use did not have a statistically significant effect on COVID-19 infectivity or clinical outcomes. However, further studies looking at clinical presentation and laboratory test results in a large number of people should be performed.

The Association of Low Skeletal Muscle Mass with Complex Distal Radius Fracture.

Objectives: Sarcopenia is a skeletal muscle loss disease with adverse outcomes, including falls, mortality, and cardiovascular disease (CVD) in older patients. Distal radius fractures (DRF), common in older people, are strongly related to falls. We aimed to investigate the correlation between DRF and low skeletal muscle mass, which strongly correlated to sarcopenia. Methods: We performed a retrospective review of data from patients diagnosed with or without DRF in our institute between 2015 and 2020. Finally, after propensity score matching, data from 115 patients with and 115 patients without DRF were used for analyses. Multivariate logistic regression analysis was performed for sex, body mass index (BMI), the presence of low skeletal muscle mass, bone quality measured by dual-energy X-ray absorptiometry (DXA), and comorbidities (diabetes mellitus, CVD). Results: We found that female sex (odds ratio = 3.435, p = 0.015), CVD (odds ratio = 5.431, p < 0.001) and low skeletal muscle mass (odds ratio = 8.062, p = 0.001) were significant predictors for DRF. BMI and osteoporosis were not statistically significantly related to DRF. Conclusions: Women with low skeletal muscle mass and CVD may be more responsible for DRF than osteoporosis.

Substance P Inhibitor Promotes Tendon Healing in a Collagenase-Induced Rat Model of Tendinopathy.

BACKGROUND: The substance P-neurokinin 1 receptor pathway has been proposed as a therapeutic target for tendinopathy. However, there is a lack of evidence regarding its practical applications. PURPOSE: To investigate the therapeutic effects of substance P inhibitor (SPI) on inflamed tenocytes in vitro and in a collagenase-induced rat model of tendinopathy in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: We analyzed the mRNA levels of inflammatory (cyclooxygenase [COX]-2 and interleukin [IL]-6) and tenogenic (Mohawk and scleraxis [SCX]) markers using reverse transcription quantitative polymerase chain reaction to demonstrate the effects of SPI on lipopolysaccharide-treated (inflamed) tenocytes. A collagenase-induced rat model of tendinopathy was created by injecting 20 µL of collagenase into the Achilles tendon. A behavior test using an incapacitance apparatus was performed to detect changes in postural equilibrium. The tendon specimens were obtained, and their gross findings were examined. The tensile strength was measured, and histopathological evaluation was performed (hematoxylin and eosin, alcian blue, and immunohistochemical staining). RESULTS: The mRNA levels of COX-2, IL-6, Mohawk, and SCX differed significantly between inflamed tenocytes and those treated with SPI. SPI improved the weight burden in a rat model of tendinopathy in a behavioral test. The specimens of the SPI group showed a normal tendon-like appearance. In the biomechanical test, the tensile strength of the SPI group was significantly greater than that of the tendinopathy group. In the histopathological evaluation, the degree of collagen matrix breakdown was mild in the SPI group. In alcian blue staining, only small focal depositions of proteoglycans and glycosaminoglycans were observed in the SPI group. The SPI group showed decreased expression of IL-6 and neurokinin 1 receptor. CONCLUSION: This study suggests that SPI has therapeutic effects on tendon healing and restoration in a collagenase-induced rat model of tendinopathy. CLINICAL RELEVANCE: SPI is a promising agent for tendinopathy in humans.

Genome-Wide Association Study Identifies Genetic Variants Associated with Rotator Cuff Tear-A Pilot Study.

A rotator cuff is a muscle and tendon surrounding the shoulder joint, and a rotator cuff tear can be caused by overuse or injury, which leads to great pain in affected individuals. However, rotator cuff tear is a multifactorial process whose underlying mechanism is still unclear. Many previous studies have suggested an important role of genetic predisposition, such as single-nucleotide polymorphisms (SNPs), in explaining the genesis of tendinopathy. This study aimed to identify specific genes or genetic variants associated with rotator cuff tears by performing a genome-wide association study (GWAS) using an independent case of rotator cuff tears. GWAS was performed using data from CHA Bundang Medical Center with 20 cases of rotator cuff tears, and 20 cases of healthy controls genotyped on the Illumina HiSeq 2500. Tests of association were performed using the Burrows−Wheeler Aligner (BWA) software at 284,246 SNPs. Data were filtered based on sequence ontology, minor allele frequency, and Hardy−Weinberg equilibrium values, and SNPs were considered significant if the p-value was <0.05. The tests of association revealed more than 20 significantly associated SNPs. SNPs showing the highest significance occurred in candidate genes, including LAIR2 (rs2287828, OR 9.116, p-value 5.49 × 10−4) on chromosome 19 and CRIPAK (rs9328733, OR 6, p-value 1.11 × 10−3) and REST (rs2228991, OR 8.222, p-value 1.20 × 10−3) on chromosome 4. This study attempted to identify genetic variants influencing rotator cuff tears through a genome-wide association study using a dense set of SNPs. More than 20 SNPs were significantly associated with rotator cuff tears. The major limitation of this study is that it was conducted on a small study group and requires further validation. Nevertheless, the identification of potential genetic variants related to rotator cuff injury would aid in the early detection of individuals at risk for the development of tendinopathy and will provide insight into future gene therapies.

Finite element analyses of lateral condyle fracture fixation in paediatrics regarding configuration of Kirschner-wire.

BACKGROUND: This study aimed to discover the most stable outcome among different Kirschner-wire (K-wire) configurations for fixation of a lateral condyle fracture (Milch type II) in different loads of stress by using finite element analyses (FEA). METHODS: The right humerus of a 6-year-old boy with a lateral condyle fracture (Milch type II), was modelled with a computer aided engineering. Using FEA, peak von Mises stress and stiffness were evaluated first for a single K-wire fixation by varying the angle (0, 5, 10, 15, 20, 25, 30 degrees). Then, based on the single K-wire result, assessment of peak von Mises stress and stiffness were evaluated via FEA for two- or three-wire fixation under various configurations (two convergent, two parallel, three divergent). RESULTS: Single K-wire fixation by 5 and 25 degrees had the lowest peak von Mises stress. The fracture site showed higher stiffness at 0, 5 and 15 degrees. Considering the collected results and clinical situation, 5 degree K-wire was selected for the FEA of multiple K-wire fixation. For multiple K-wire fixation, three divergent (5-20-35 degrees) K-wires showed better stability, both in peak von Mises stress and stiffness, than any two-K-wire configurations. Among two K-wire fixations, two divergent (5-50 degrees) K-wires provided the lowest von Mises stress in varus and valgus while two divergent (5-65 degrees) K-wires showed better results in flexion, extension, internal and external rotation, and both configurations showed similar results in stiffness. CONCLUSIONS: We successfully created a paediatric lateral condyle fracture (Milch type II) model which was used to conduct FEA on different K-wire configurations to achieve stability of the fracture. Our results show that an initial K-wire inserted at 5 degrees, followed by the insertion of a second divergent wire at either 45 or 60 degrees provides the most stability in two K-wire fixations in this type of fracture repair.

Colchicine as a novel drug for the treatment of osteosarcoma through drug repositioning based on an FDA drug library.

Background: Colchicine is a traditional medication that is currently approved to treat gout and familial Mediterranean fever (FMF). However, colchicine has a wide range of anti-inflammatory activities, and several studies have indicated that it may be useful in a variety of other conditions, such as rheumatic disease, cardiac disease, and cancer. Osteosarcoma, the most common type of bone sarcoma, is derived from primitive bone-forming mesenchymal cells. In this study, we investigated whether colchicine could be used to treat osteosarcoma through the regulation of cell cycle signaling. Methods: Two human osteosarcoma cell lines, U2OS and Saos-2, were used. A clonogenic assay was used to determine the antiproliferative effects of colchicine on osteosarcoma cells. Reactive oxygen species (ROS) production and apoptosis were measured by flow cytometry. Migration and invasion assays were performed to investigate the inhibitory effects of colchicine. The signaling pathways related to colchicine treatment were verified by GO biological process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results: Colchicine was selected as the lead compound based on the results of initial screening and cell viability assays conducted in Saos-2 and U2Os cells. Colchicine reduced the viability of Saos-2 and U2OS cells in a concentration-dependent manner. It also significantly inhibited colony-forming ability and induced ROS production and apoptosis. It also inhibited the migration and invasion of both Saos-2 and U2OS cells. GOBP and KEGG enrichment analyses indicated the involvement of microtubule-based processes and cancer-related pathways. Conclusions: These findings suggest that colchicine has therapeutic potential in osteosarcoma.

miR-31-3p functions as a tumor suppressor by directly targeting GABBR2 in prostate cancer.

MicroRNAs are key regulators of gene expression in tumorigenesis. In this study, we investigated the tumor-suppressive function of miR-31-3p. Analysis of the Gene Expression Omnibus database revealed that the expression of miR-31-3p in prostate cancer tissues is lower than that in adjacent normal tissues from patients with prostate cancer. Moreover, miR-31-3p induces apoptosis in DU145, PC-3, and LNCap prostate cancer cells, while those transfected with miR-31-3p exhibit significantly decreased cell proliferation, migration, invasiveness, and tumor sphere-forming ability, as determined using the cell counting kit-8, transwell, and sphere-forming assays. Further analysis revealed that GABBR2 is a direct target of miR-31-3p. Within a DU145 xenograft murine model, intratumoral injection of a miR-31-3p mimic suppresses tumor growth. Taken together, the findings of this study suggest that miR-31-3p performs a novel tumor-suppressive function in prostate cancer and may represent a novel target for anti-prostate cancer miRNA therapeutics.

Hair methylmercury levels are inversely correlated with arterial stiffness.

BACKGROUND AND AIMS: Cardiovascular diseases (CVD), including coronary heart disease, are the leading cause of death worldwide. Several studies investigating the relationship between fish intake, methylmercury exposure, and CVDs in adults have reported inconsistent results. This study aimed to determine the association between hair methylmercury levels and arterial stiffness using brachial-ankle pulse wave velocity (baPWV). METHODS: This cross-sectional study included 891 seemingly healthy Korean adults (418 men and 473 women). The anthropometric and biochemical profiles, including methylmercury levels in the hair, were measured. Arterial stiffness was measured using baPWV, wherein high baPWV was defined as >1375 cm/s (>75th percentile). The odds ratios for high baPWVs were examined using multivariable logistic regression analysis after adjusting for potential confounders across the quintiles of hair methylmercury levels (Q1 = ≤0.6, Q2 = 0.6-0.8, Q3 = 0.8-1.1, Q4 = 1.1-1.5, and Q5=>1.5 µg/g). RESULTS: After adjusting for multiple confounders-age, sex, height, body weight, smoking status, weekly alcohol consumption, total metabolic equivalent of task, mean arterial blood pressure, resting heart rate, triglycerides, low density lipoprotein cholesterol, fasting plasma glucose, uric acid and white blood cell count-the odds ratios (95% confidence intervals) for high baPWVs in each quintile of hair methylmercury levels were 1.00, 0.36 (0.17-0.76), 0.38 (0.20-0.76), 0.28 (0.13-0.61), and 0.49 (0.24-0.99), respectively. CONCLUSIONS: Within non-toxic low levels, higher hair methylmercury levels are independently associated with lower arterial stiffness in seemingly healthy Korean adults regardless of classical cardiovascular risk factors.

Synthesis of Benzoxaphosphole 1-Oxide Heterocycles via a Three-Component Coupling Reaction Involving Arynes, Phosphites, and Ketones.

An efficient and transition-metal-free three-component reaction with benzynes formed in situ from 2-(trimethylsilyl)aryl triflate, phosphites, and ketones was developed for the synthesis of benzoxaphosphole 1-oxides. An array of benzoxaphosphole 1-oxides were prepared from both activated and non-activated ketones in moderate to good yields with a broad functional group tolerance. This reaction is useful for preparing organophosphorus compounds encountered in natural products and materials.

Deep Learning-based Diagnosis of Glaucoma Using Wide-field Optical Coherence Tomography Images.

PURPOSE: (1) To evaluate the performance of deep learning (DL) classifier in detecting glaucoma, based on wide-field swept-source optical coherence tomography (SS-OCT) images. (2) To assess the performance of DL-based fusion methods in diagnosing glaucoma using a variety of wide-field SS-OCT images and compare their diagnostic abilities with that of conventional parameter-based methods. METHODS: Overall, 675 eyes, including 258 healthy eyes and 417 eyes with glaucoma were enrolled in this retrospective observational study. Each single-page wide-field report (12×9 mm) of wide-field SS-OCT imaging provides different types of images that reflect the state of the eyes. A DL-based automated diagnosis system was proposed to detect glaucoma and identify its stage based on such images. We applied the convolutional neural network to each type of image to detect glaucoma. In addition, 2 fusion strategies, fusion by convolution network (FCN) and fusion by fully connected network (FFC) were developed; they differ in terms of the level of fusion of features derived from convolutional neural networks. The diagnostic models were trained using 382 and 293 images in the training and test data sets, respectively. The diagnostic ability of this method was compared with conventional parameters of the thickness of the retinal nerve fiber layer and ganglion cell complex. RESULTS: FCN achieved an area under the receiver operating characteristic curve (AUC) of 0.987 (95% confidence interval, CI: 0.968-0.996) and an accuracy of 95.22%. In contrast, FFC achieved an AUC of 0.987 (95% CI, 0.971-0.998) and an accuracy of 95.90%. Both FCN and FFC outperformed the conventional method (P<0.001). In detecting early glaucoma, both FCN and FFC achieved significantly higher AUC and accuracy than the conventional approach (P<0.001). In addition, the classification performance of the DL-based fusion methods in identifying the 5 stages of glaucoma is presented via a confusion matrix. CONCLUSION: DL protocol based on wide-field OCT images outperformed the conventional method in terms of both AUC and accuracy. Therefore, DL-based diagnostic methods using wide-field OCT images are promising in diagnosing glaucoma in clinical practice.

Attenuating Effects of Dieckol on Hypertensive Nephropathy in Spontaneously Hypertensive Rats.

Hypertension induces renal fibrosis or tubular interstitial fibrosis, which eventually results in end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is one of the underlying mechanisms of renal fibrosis. Though previous studies showed that Ecklonia cava extracts (ECE) and dieckol (DK) had inhibitory action on angiotensin (Ang) I-converting enzyme, which converts Ang I to Ang II. It is known that Ang II is involved in renal fibrosis; however, it was not evaluated whether ECE or DK attenuated hypertensive nephropathy by decreasing EMT. In this study, the effect of ECE and DK on decreasing Ang II and its down signal pathway of angiotensin type 1 receptor (AT1R)/TGFß/SMAD, which is related with the EMT and restoring renal function in spontaneously hypertensive rats (SHRs), was investigated. Either ECE or DK significantly decreased the serum level of Ang II in the SHRs. Moreover, the renal expression of AT1R/TGFß/SMAD was decreased by the administration of either ECE or DK. The mesenchymal cell markers in the kidney of SHRs was significantly decreased by ECE or DK. The fibrotic tissue of the kidney of SHRs was also significantly decreased by ECE or DK. The ratio of urine albumin/creatinine of SHRs was significantly decreased by ECE or DK. Overall, the results of this study indicate that ECE and DK decreased the serum levels of Ang II and expression of AT1R/TGFß/SMAD, and then decreased the EMT and renal fibrosis in SHRs. Furthermore, the decrease in EMT and renal fibrosis could lead to the restoration of renal function. It seems that ECE or DK could be beneficial for decreasing hypertensive nephropathy by decreasing EMT and renal fibrosis.

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses.

Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogues that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal reporter reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

Gamma-aminobutyric acid-salt attenuated high cholesterol/high salt diet induced hypertension in mice.

Excessive salt intake induces hypertension, but several gamma-aminobutyric acid (GABA) supplements have been shown to reduce blood pressure. GABAsalt, a fermented salt by L. brevis BJ20 containing GABA was prepared through the post-fermentation with refined salt and the fermented GABA extract. We evaluated the effect of GABA-salt on hypertension in a high salt, high cholesterol diet induced mouse model. We analyzed type 1 macrophage (M1) polarization, the expression of M1 related cytokines, GABA receptor expression, endothelial cell (EC) dysfunction, vascular smooth muscle cell (VSMC) proliferation, and medial thicknesses in mice model. GABA-salt attenuated diet-induced blood pressure increases, M1 polarization, and TNF-α and inducible nitric oxide synthase (NOS) levels in mouse aortas, and in salt treated macrophages in vitro. Furthermore, GABA-salt induced higher GABAB receptor and endothelial NOS (eNOS) and eNOS phosphorylation levels than those observed in salt treated ECs. In addition, GABA-salt attenuated EC dysfunction by decreasing the levels of adhesion molecules (E-selectin, Intercellular Adhesion Molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) and of von Willebrand Factor and reduced EC death. GABA-salt also reduced diet-induced reductions in the levels of eNOS, phosphorylated eNOS, VSMC proliferation and medial thickening in mouse aortic tissues, and attenuated Endothelin-1 levels in salt treated VSMCs. In summary, GABA-salt reduced high salt, high cholesterol diet induced hypertension in our mouse model by reducing M1 polarization, EC dysfunction, and VSMC proliferation.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Radiofrequency irradiation attenuates angiogenesis and inflammation in UVB-induced rosacea in mouse skin.

Rosacea is a skin inflammatory condition accompanied by cutaneous signs such as oedema, flushing, erythema, telangiectasia and pustules. Generally, rosacea is triggered by ultraviolet B (UVB) exposure. When exposed to UVB, skin epidermis thickens and produces elevated levels of pro-inflammatory cytokines, especially keratinocyte-related VEGF, a potent angiogenic factor. The upregulations of VEGF expression and its secretion promote the formation of new blood vessels and exacerbates rosacea. In this study, radiofrequency (RF) irradiation reduced keratinocyte proliferation in the epidermal layer, the expressions of pro-inflammatory cytokines, angiogenesis-related inflammatory factors and VEGF in our UVB-induced model of rosacea in vitro and in vivo. RF irradiation attenuated VEGF-induced angiogenesis-associated processes such as tube formation, cell migration and endothelial cell proliferation. Notably, blood vessel densities in the skins of UVB-treated mice and rosacea patients were significantly decreased by RF irradiation. These results provide experimental and molecular evidence regarding the effectiveness of RF irradiation for the treatment of rosacea.

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells.

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.