Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

Postbiotic potential of Bacillus velezensis KMU01 cell-free supernatant for the alleviation of obesity in mice.

Attention toward the preventive effects of postbiotics on metabolic diseases has increased because of greater stability and safety over probiotics. However, studies regarding the bioactive effects of postbiotics, especially from probiotic Bacillus strains, are relatively limited. The anti-obesity effects of the cell-free culture supernatant of Bacillus velezensis KMU01 (CFS-B.vele) were evaluated using high-fat-diet (HFD)-induced mice. HFD-induced mice (n = 8 per group) received equal volumes of (1) CFS-B.vele (114 mg/kg) in PBS, (2) Xenical in PBS, or (3) PBS alone by oral gavage daily for 13 weeks. The results demonstrated that CFS-B.vele changed the gut microbiota and showed anti-obesity effects in HFD-induced obese mice. The elevated Firmicutes/Bacteroidota ratio induced by HFD was decreased in the CFS-B.vele group compared to the other groups (p < 0.05). The CFS-B.vele intervention led to the enrichment of SCFA-producers, such as Roseburia and Eubacterium, in the cecum, suggesting their potential involvement in the amelioration of obesity. Due to these changes, the various obesity-related biomarkers (body weight, fat in tissue, white adipose tissue weight and size, serum LDL-cholesterol level, hepatic lipid accumulation, and adipogenesis/lipogenesis-related gene/protein expression) were improved. Our findings suggest that CFS-B.vele has potential as a novel anti-obesity agent through modulation of the gut microbiota.

Anti-Inflammatory Effect and Signaling Mechanism of Glycine max Hydrolyzed with Enzymes from Bacillus velezensis KMU01 in a Dextran-Sulfate-Sodium-Induced Colitis Mouse Model.

The purpose of this study was to investigate the effect that Glycine max hydrolyzed with enzymes from Bacillus velezensis KMU01 has on dextran-sulfate-sodium (DSS)-induced colitis in mice. Hydrolysis improves functional health through the bioconversion of raw materials and increase in intestinal absorption rate and antioxidants. Therefore, G. max was hydrolyzed in this study using a food-derived microorganism, and its anti-inflammatory effect was observed. Enzymatically hydrolyzed G. max (EHG) was orally administered once daily for four weeks before DSS treatment. Colitis was induced in mice through the consumption of 5% (w/v) DSS in drinking water for eight days. The results showed that EHG treatment significantly alleviated DSS-induced body weight loss and decreased the disease activity index and colon length. In addition, EHG markedly reduced tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 production, and increased that of IL-10. EHG improved DSS-induced histological changes and intestinal epithelial barrier integrity in mice. Moreover, we found that the abundance of 15 microorganisms changed significantly; that of Proteobacteria and Escherichia coli, which are upregulated in patients with Crohn's disease and ulcerative colitis, decreased after EHG treatment. These results suggest that EHG has a protective effect against DSS-induced colitis and is a potential candidate for colitis treatment.

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile.

Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.

Postbiotics Enhance NK Cell Activation in Stress-Induced Mice through Gut Microbiome Regulation.

Recent studies have revealed that probiotics and their metabolites are present under various conditions; however, the role of probiotic metabolites (i.e., postbiotics in pathological states) is controversial. Natural killer (NK) cells play a key role in innate and adaptive immunity. In this study, we examined NK cell activation influenced by a postbiotics mixture in response to gut microbiome modulation in stress-induced mice. In vivo activation of NK cells increased in the postbiotics mixture treatment group in accordance with Th1/Th2 expression level. Meanwhile, the Red Ginseng treatment group, a reference group, showed very little expression of NK cell activation. Moreover, the postbiotics mixture treatment group in particular changed the gut microbiome composition. Although the exact role of the postbiotics mixture in regulating the immune system of stress-induced mice remains unclear, the postbiotics mixture-induced NK cell activation might have affected gut microbiome modulation.

Glucose as a Protein-Condensing Cellular Solute.

The present study reports a surprising protein-condensing effect of glucose, prompted by our accidental observation during chemical library screening under a high-glucose condition. We noticed "glucosing-out" of certain compounds, in which physiological concentrations of glucose induced compound aggregation. Adapting the "glucosing-out" concept to proteins, our proteomic analysis identified three cellular proteins (calmodulin, rho guanine nucleotide exchange factor 40, and polyubiquitin-C) that displayed robust glucose-dependent precipitation. One of these proteins, calmodulin, formed glucose-dependent condensates that control cellular glycogenolysis in hepatic cells. Our findings suggest that glucose is a heretofore underappreciated driver of protein phase separation that may have profound effects on cellular homeostasis.

Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe.

Covalent inhibitors of enzymes are increasingly appreciated as pharmaceutical seeds, yet discovering non-cysteine-targeting inhibitors remains challenging. Herein, we report an intriguing experience during our activity-based proteomic screening of 1601 reactive small molecules, in which we monitored the ability of library molecules to compete with a cysteine-reactive iodoacetamide probe. One epoxide molecule, F8, exhibited unexpected enhancement of the probe reactivity for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-limiting glycolysis enzyme. In-depth mechanistic analysis suggests that F8 forms a covalent adduct with an aspartic acid in the active site to displace NAD+, a cofactor of the enzyme, with concomitant enhancement of the probe reaction with the catalytic cysteine. The mechanistic underpinning permitted the identification of an optimized aspartate-reactive GAPDH inhibitor. Our findings exemplify that activity-based proteomic screening with a cysteine-reactive probe can be used for discovering covalent inhibitors that react with non-cysteine residues.

The effect of smartphone uses on gait and obstacle collision during walking.

OBJECTIVE: This study aims to investigate the impact of obstacle collision according to the presence or absence of light and the change of gait when using smartphones while walking. STUDY DESIGN/SETTING: A cross-sectional study. SUBJECTS: 24 healthy young people. METHODS: Obstacle collision was measured in participants as they passed through four obstacles at different heights (eyes, waist, knees, ankle level), with the lights turned on. The same was measured with the lights turned off. To measure spatiotemporal variables, GAITRite was used. Obstacle collision was used in the frequency analysis, and the spatiotemporal variables were assessed using one-way ANOVA test. The post-hoc Tukey's test was used to detect spatiotemporal variables differences (p < 0.05). RESULTS: Obstacle collision was the highest at eye level when playing games and messaging while walking. It was also higher when lights were turned off rather than when lights were on. Gait function decreased while walking when messaging or playing games on a smartphone as compared to when walking without a smartphone. CONCLUSION: Understanding the collision rate of various obstacles encountered when using smartphones while walking could contribute to the prevention of accidents.

Homologous U-box E3 Ubiquitin Ligases OsPUB2 and OsPUB3 Are Involved in the Positive Regulation of Low Temperature Stress Response in Rice (Oryza sativa L.).

Rice U-box E3 Ub ligases (OsPUBs) are implicated in biotic stress responses. However, their cellular roles in response to abiotic stress are poorly understood. In this study, we performed functional analyses of two homologous OsPUB2 and OsPUB3 in response to cold stress (4°C). OsPUB2 was up-regulated by high salinity, drought, and cold, whereas OsPUB3 was constitutively expressed. A subcellular localization assay revealed that OsPUB2 and OsPUB3 were localized to the exocyst positive organelle (EXPO)-like punctate structures. OsPUB2 was also localized to the nuclei. OsPUB2 and OsPUB3 formed a hetero-dimeric complex as well as homo-dimers in yeast cells and in vitro. OsPUB2/OsPUB3 exhibited self-ubiquitination activities in vitro and were rapidly degraded in the cell-free extracts with apparent half-lives of 150-160 min. This rapid degradation of OsPUB2/OsPUB3 was delayed in the presence of the crude extracts of cold-treated seedlings (apparent half-lives of 200-280 min). Moreover, a hetero-dimeric form of OsPUB2/OsPUB3 was more stable than the homo-dimers. These results suggested that OsPUB2 and OsPUB3 function coordinately in response to cold stress. OsPUB2- and OsPUB3-overexpressing transgenic rice plants showed markedly better tolerance to cold stress than did the wild-type plants in terms of survival rates, chlorophyll content, ion leakage, and expression levels of cold stress-inducible marker genes. Taken together, these results suggested that the two homologous rice U-box E3 Ub ligases OsPUB2 and OsPUB3 are positive regulators of the response to cold stress.

Germacrane sesquiterpenes isolated from the rhizome of Curcuma xanthorrhiza Roxb. inhibit UVB-induced upregulation of MMP-1, -2, and -3 expression in human keratinocytes.

Four sesquiterpenes were isolated from the rhizome of Curcuma xanthorrhiza Roxb.: furanodiene (1), germacrone (2), furanodienone (3), and 13-hydroxygermacrone (4). Importantly, this was the first time compounds 1 and 4 were isolated from this plant. The chemical structures of these compounds were determined using 1D- and 2D-nuclear magnetic resonance, infrared spectroscopy, and electron ionization mass spectrometry analyses. Among the isolated compounds, compounds 2 and 4 inhibited UVB-induced upregulation of the mRNA and protein expression levels of MMP-1, MMP-2, and MMP-3 in human keratinocytes (HaCaT). Moreover, this upregulation occurred in a dose-dependent manner over the range of 1-10 µM for each compound.

Two new isoarylbenzofuran diglucosides from the root bark of Morus alba.

Two new arylbenzofuran diglucopyranosides, (2″R)-(-)-moracin-O-5',3″-ß-D-diglucopyranoside (1) and (2″R)-(-)-moracin-P-5',2″-ß-d-diglucopyranoside (2), along with known arylbenzofurans, moracin M 6-ß-D-glucopyranoside (3), and an isomeric mixture of R-(-)-moracin O (4) and R-(-)-moracin P (5), were isolated from the root bark of Morus alba L. The structure of the compounds was elucidated based on mass spectrometry, infrared, 1D and 2D nuclear magnetic resonance spectroscopic data.

Lignan and flavonoids from the stems of Zea mays and their anti-inflammatory and neuroprotective activities.

The stems of Zea mays L., otherwise known as cornstalks, were extracted with 80 % aqueous MeOH, and the concentrated extract was successively partitioned with ethyl acetate (EtOAc), normal butanol, and water. From the EtOAc fraction, a new lignan along with three known flavonoids, tricin (1), salcolin A (2), and salcolin B (3), were isolated. The chemical structure of the lignan was determined to be tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1-one (4) through spectroscopic data analyses including NMR, MS, and IR. All compounds were isolated for the first time from this plant. The isolated compounds were evaluated for their inhibitory activity against NO production in Lipopolysaccharide-induced RAW 264.7 cells and their protective activity in glutamate-induced cell death in HT22 cells. The compounds 1, 2 and 4 showed anti-inflammatory effects with IC50 values of 2.63, 14.65, and 18.91 µM, respectively, as well as neuroprotective effects with EC50 values of 25.14, 47.44, and >80 µM, respectively.

Long-term utility and durability of the therapeutic effects of bimatoprost 0.03% for eyelash augmentation in healthy Asian subjects.

BACKGROUND: Eyelashes of Asians differ from those of Caucasians in morphology and growth characteristics. Ethnic differences also exist for the tolerability profile of prostaglandin analogues. OBJECTIVE: To evaluate the long-term utility and durability of bimatoprost 0.03% in eyelash augmentation in Asian females. METHODS: One cohort received bimatoprost 0.03% for 36 weeks and another for 20 weeks, with the latter cohort followed for 16 weeks after treatment cessation. The primary endpoint was the percent change in eyelash length at week 20. Secondary measures included percent change in eyelash thickness and darkness, physician's Global Eyelash Assessment and patient satisfaction. RESULTS: At week 20, eyelash length was enhanced in a time-dependent manner, with maximum improvement achieved (19.3%; p < 0.0001). Significant improvements in thickness and darkness were also achieved (22.9%, 6.0%; p < 0.0001). 77.8% of subjects improved by ≥1 grade on Global Eyelash Assessment, with 83.1% satisfied/very satisfied. Improvements were maintained with ongoing treatment to 36 weeks, while these effects were progressively lost with discontinuation. CONCLUSION: Bimatoprost 0.03% safely enhanced eyelashes in Asian females, maintained with ongoing treatment. Cessation of treatment was associated with progressive loss of effects.

A new flavonoid glycoside from the root bark of Morus alba L.

A new guibourtinidol glycoside, (2R,3S)-guibourtinidol-3-O-α-d-apiofuranosyl-(1 → 6)-O-ß-D-glucopyranoside (1), and three known compounds, quercetin 7-O-ß-D-glucopyranoside (2), syringaresinol-4-O-ß-D-glucopyranoside (3) and dehydrodiconiferyl alcohol 4,9'-di-O-ß-D-glucopyranoside (4), were isolated from the root bark of Morus alba L. through repeated silica gel, octadecyl silica gel and Sephadex LH-20 column chromatography for the n-BuOH fraction. The chemical structure of the compounds was elucidated based on MS, infrared, 1D and 2D NMR spectroscopic data. Compounds 2-4 were also isolated for the first time from the root bark of M. alba L. in this study.

Re-evaluation of physicochemical and NMR data of triol ginsenosides Re, Rf, Rg2, and 20-gluco-Rf from Panax ginseng roots.

Ginseng roots were extracted with aqueous methanol, and extracts were suspended in water and extracted successively with ethyl acetate and n-butanol. Column chromatography using the n-butanol fraction yielded four purified triol ginseng saponins: the ginsenosides Re, Rf, Rg2, and 20-gluco-Rf. The physicochemical, spectroscopic, and chromatographic characteristics of the ginsenosides were measured and compared with reports from the literature. For spectroscopic analysis, two-dimensional nuclear magnetic resonance (NMR) methods such as (1)H-(1)H correlation spectroscopy, nuclear Overhauser effect spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond connectivity were employed to identify exact peak assignments. Some peak assignments for previously published (1)H- and (13)C-NMR spectra were found to be inaccurate. This study reports the complete NMR assignment of 20-gluco-Rf for the first time.

Neolignans from the fruits of Magnolia obovata and their inhibition effect on NO production in LPS-induced RAW 264.7 cells.

Three new neolignans, named 9-methoxyobovatol (6), magnobovatol (7), and 2-hydroxyobovaaldehyde (9), along with six known ones, magnolol (1), honokiol (2), isomagnolol (3), obovatol (4), obovatal (5), and obovaaldehyde (8), were isolated from the fruits of Magnolia obovata using silica gel and ODS column chromatography. From the results of spectroscopic data including EIMS, IR, 1H- and 13C-NMR, DEPT, and 2D-NMR (gCOSY, gHSQC, gHMBC), the chemical structures were determined. All isolated compounds were evaluated for inhibition activity on nitric oxide production in LPS-induced RAW 264.7 cells, and compounds 1-4, 6, 7, and 9 showed significant activity with IC50 values of 15.8 ± 0.3, 3.3 ± 1.2, 14.1 ± 0.9, 6.2 ± 1.2, 14.8 ± 2.3, 14.2 ± 1.2, and 14.8 ± 3.2 µM, respectively, without any visible toxic effect.

Late-onset eccrine angiomatous hamartoma associated with a ganglion cyst on the sole of the foot.

Eccrine angiomatous hamartoma (EAH) is a benign, uncommon, combined vascular and eccrine malformation. Most cases of this disorder have been single or multiple nodules or plaques that appear red, yellow, blue, violaceous, or skin colored. EAH may be congenital or appear later in childhood; it rarely arises during puberty or adulthood. A 52-year-old female patient visited our department for tender subcutaneous cystic tumor on the right sole with a one month history. Histopathologic examination confirmed EAH. During excisional biopsy procedure, mucinous discharges were observed which were histopathologically diagnosed as ganglion.

A case of acrodermatitis enteropathica localized on the hands and feet with a normal serum zinc level.

Acrodermatitis enteropathica is classified as a congenital autosomal recessive type and an acquired transient type. This disease manifests as acral and periorificial dermatitis, alopecia, intractable diarrhea, and failure to thrive. Whereas the autosomal hereditary type is caused by malabsorption of zinc in the intestine, the acquired type is caused by low nutritional support or decreased peripheral release of zinc from blood. We experienced a case of a 5-month old, breast feeding, full-term female presenting with only acral bullous dermatitis without diarrhea, periorificial dermatitis and an abnormal serum zinc level.

A Case of Lichen Planus Pigmentosus-inversus in a Korean Patient.

Lichen planus pigmentosus-inversus is a rare variant of lichen planus pigmentosus. The eruption of lichen planus pigmentosus-inversus occurs mainly in the flexural regions and presents with brownish macules and patches. Here we describe the case of a 31-year-old Korean woman who presented with hyperpigmented lesions over her antecubital and popliteal area and groin. Physical examination revealed multiple brownish macules and patches on the intertriginous area without pruritis, and histologic findings showed a regressive pattern of lichen planus. These clinical and histological findings were consistent with a diagnosis of lichen planus pigmentosus-inversus.

A herpes simplex virus infection secondary to acupuncture and cupping.

Acupuncture and cupping have a public reputation as being safe even though these practices can lead to complications such as trauma or infection. We report here on a case of herpes simplex virus (HSV) infection secondary to acupuncture and cupping in a 56-year-old woman. The patient, who had a history of acupuncture and cupping on her left forearm for treating her myalgia, developed painful papules. Histologically, the biopsy specimen showed characteristic ballooning degeneration and inclusion bodies in the epidermis and mid-dermis. These clinical and histological findings were consistent with the diagnosis of HSV infection.