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Perilla frutescens (PF) is a plant with various biological activities, including antioxidant and antidepressant activities; however, the efficacy of its fermented product has not been evaluated. Herein, we found that the Bacillus subtilis-fermented PF (FPF) increased non-rapid eye movement sleep and slow wave activity. FPF improved the sleep onset latency and sleep duration in a pentobarbital-induced sleep model, and this efficacy was abolished by the adenosine A1 receptor (A1R) antagonist. In addition, FPF increased and decreased the neuronal activity in sleep-activating and inhibiting brain regions, respectively, and these effects were also blocked by A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine. Furthermore, FPF exhibited direct binding and agonistic activities for A1R. HPLC/NMR analysis revealed uracil, protocatechuic acid, apigenin-7-O-diglucuronide, and luteolin-7-O-glucuronide to be the major active components responsible for the sleep-promoting efficacy of FPF. In conclusion, this study suggests that FPF has A1R agonistic activity and this novel activity may be critical for its sleep-promoting effect.

Abstract Background Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS. Results The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ). Conclusion Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture.

Measurement(s) Metabolomics Technology Type(s) high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry Sample Characteristic - Organism Embryophyta

Aquaculture, a crucial sector of the global food industry, faces a myriad of issues due to parasitic invasions. One such parasite, Microcotyle sebastis, which afflicts Korean rockfish in South Korea, has a significant economic impact. The impending danger of resistance to traditional anthelmintics necessitates the exploration of new antiparasitic candidates. Although the efficacy of salinomycin against aquatic parasites such as ciliates and sporozoans is known, its influence on monogeneans has yet to be studied. Therefore, this study investigated the efficacy and safety of salinomycin for the treatment of M. sebastis infections, presenting the first exploration of salinomycin’s therapeutic potential against monogeneans. In vitro examinations revealed a minimum effective concentration of salinomycin of 5 mg/kg, which led to necrosis of the haptor upon dislodging from the gill filaments. The one-time oral administration of the drug at concentrations of 5 mg/kg and 10 mg/kg showed a significant dose-dependent reduction in parasite counts, with no apparent behavioral side effects in Korean rockfish. Biochemical analyses monitored the liver, heart, and kidney enzymes, specifically aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatine kinase–myocardial band (CK-MB). At both 20 °C and 13 °C, no significant differences were observed in the levels of AST and ALT. However, at 20 °C, alterations in BUN levels were evident on Day 14, a deviation not observed at 13 °C. The CK-MB analysis revealed elevated enzyme levels at both temperatures when compared to the control group, reflecting the similar changes observed in terrestrial animals administered salinomycin. The biochemical data suggest that the oral administration of salinomycin is potentially more favorable at 13 °C than at 20 °C. Although our findings warrant further comprehensive studies, including on the long-term and potential effects on nontarget species and water quality, they also suggest that salinomycin could be considered as an alternative or adjunctive treatment if resistance to the currently used praziquantel against M. sebastis is confirmed.

ABSTRACT The genus Streptomyces is one of the richest sources of secondary metabolite biosynthetic gene clusters (BGCs). Sequencing of a large number of genomes has provided evidence that this well-known bacterial genus still harbors a large number of cryptic BGCs, and their metabolites are yet to be discovered. When taking a gene-first approach for new natural product discovery, BGC prioritization would be the most crucial step for the discovery of novel chemotypes. We hypothesized that strains with a greater number of BGCs would also contain a greater number of silent unique BGCs due to the presence of complex regulatory systems. Based on this hypothesis, we employed a comparative genomics approach to identify a specific Streptomyces phylogenetic lineage with the highest and yet-uncharacterized biosynthetic potential. A comparison of BGC abundance and genome size across 158 phylogenetically diverse Streptomyces type strains identified that members of the phylogenetic group characterized by the formation of rugose-ornamented spores possess the greatest number of BGCs (average, 50 BGCs) and also the largest genomes (average, 11.5 Mb). The study of genetic and biosynthetic diversities using comparative genomics of 11 sequenced genomes and a genetic similarity network analysis of BGCs suggested that members of this group carry a large number of unique BGCs, the majority of which are cryptic and not associated with any known natural product. We believe that members of this Streptomyces phylogenetic group possess a remarkable biosynthetic potential and thus would be a good target for a metabolite characterization study that could lead to the discovery of novel chemotypes. IMPORTANCE It is now well recognized that members of the genus Streptomyces still harbor a large number of cryptic BGCs in their genomes, which are mostly silent under laboratory culture conditions. Activation of transcriptionally silent BGCs is technically challenging and thus forms a bottleneck when taking a gene-first approach for the discovery of new natural products. Thus, it is important to focus activation efforts on strains with BGCs that have the potential to produce novel metabolites. The clade-level analysis of biosynthetic diversity could provide insights into the relationship between phylogenetic lineage and biosynthetic diversity. By exploring BGC abundance in relation to Streptomyces phylogeny, we identified a specific monophyletic lineage associated with the highest BGC abundance. Then, using a combined analysis of comparative genomics and a genetic network, we demonstrated that members of this lineage are genetically and biosynthetically diverse, contain a large number of cryptic BGCs with novel genotypes, and thus would be a good target for metabolite characterization studies.

Low-density lipoprotein (LDL) modification such as oxidation and glycation has been known to play a significant role in the initiation and progression of atherosclerosis. The inhibitors on LDL modification have been emerged as a new strategy to be a supplement for prevention of atherosclerosis. In an effort to discover inhibitors on LDL modification from natural products, phytochemical investigation on Cynanchum wilfordii roots were performed, leading to isolation of acetophenones (1–6) including a new one (1) and nine pregnane-type steroids (7–15). The structure of the compound 1 was determined by analysis of 1D and 2D NMR spectral data analysis. All the isolated compounds were evaluated for their inhibitory activities against LDL modification. Among them, compounds 2 and 5 strongly inhibited LDL oxidation and glycation. These results suggest that compounds 2 and 5 from C. wilfordii may provide potential protective effects against atherosclerosis via inhibition of LDL oxidation and glycation.

Cinnamoyl-containing nonribosomal peptides (CCNPs) form a unique family of actinobacterial secondary metabolites and display various biological activities. A new CCNP named epoxinnamide (1) was discovered from intertidal mudflat-derived Streptomyces sp. OID44. The structure of 1 was determined by the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data along with a mass spectrum. The absolute configuration of 1 was assigned by the combination of advanced Marfey’s method, 3JHH and rotating-frame overhauser effect spectroscopy (ROESY) analysis, DP4 calculation, and genomic analysis. The putative biosynthetic pathway of epoxinnamide (1) was identified through the whole-genome sequencing of Streptomyces sp. OID44. In particular, the thioesterase domain in the nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster was proposed as a bifunctional enzyme, which catalyzes both epimerization and macrocyclization. Epoxinnamide (1) induced quinone reductase (QR) activity in murine Hepa-1c1c7 cells by 1.6-fold at 5 μM. It also exhibited effective antiangiogenesis activity in human umbilical vein endothelial cells (IC50 = 13.4 μM).

Six new β-resorcylic acid derivatives (1–5 and 7) were isolated from a halophyte-associated fungus, Colletotrichum gloeosporioides JS0419, together with four previously reported β-resorcylic acid lactones (RALs). The relative and absolute stereochemistry of 1 was completely established by a combination of spectroscopic data and chemical reactions. The structures of the isolated compounds were elucidated by analysis of HRMS and NMR data. Notably, compounds 1–3 had a β-resorcylic acid harboring a long unesterified aliphatic side chain, whereas the long aliphatic chains were esterified to form macrolactones in 4–9. Among the isolated compounds, monocillin I and radicicol showed potent antifungal activities against Cryptococcus neoformans, comparable to clinically available antifungal agents and radicicol showed weak antifungal activity against Candida albicans. These findings provide insight into the chemical diversity of fungal RAL-type compounds and their pharmacological potential.

Estrogen replacement therapy is a treatment to relieve the symptoms of menopause. Many studies suggest that natural bioactive ingredients from plants resemble estrogen in structure and biological functions and can relieve symptoms of menopause. The fruit of V. rotundifolia, called “Man HyungJa” in Korean, is a traditional medicine used to treat headache, migraine, eye pain, neuralgia, and premenstrual syndrome in Korea and China. The aim of the present study was to confirm that V. rotundifolia fruit extract (VFE) exerts biological functions similar to those of estrogen in menopausal syndrome. We investigated its in vitro effects on MCF-7 cells and in vivo estrogen-like effects on weight gain and uterine contraction in ovariectomized rats. Using the polar extract, the active constituents of VFE (artemetin, vitexicarpin, hesperidin, luteolin, vitexin, and vanillic acid) with estrogen-like activity were identified in MCF-7 cells. In animal experiments, the efficacy of VFE in ameliorating body weight gain was similar to that of estrogen, as evidenced from improvements in uterine atrophy. Vitexin and vitexicarpin are suggested as the active constituents of V. rotundifolia fruits.

Menopause, caused by decreases in estrogen production, results in symptoms such as facial flushing, vaginal atrophy, and osteoporosis. Although hormone replacement therapy is utilized to treat menopausal symptoms, it is associated with a risk of breast cancer development. We aimed to evaluate the estrogenic activities of Spartina anglica (SA) and its compounds and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic and anti-proliferative effects of extracts of SA and its compounds in MCF-7 breast cancer cells. We performed an uterotrophic assay using an immature female rat model. Among extracts of SA, belowground part (SA-bg-E50) had potent estrogenic activity. In the immature female rat model, the administration of SA-bg-E50 increased uterine weight compared with that in the normal group. Among the compounds isolated from SA, 1,3-di-O-trans-feruloyl-(-)-quinic acid (1) had significant estrogenic activity and induced phosphorylation at serine residues of estrogen receptor (ER)α. All extracts and compounds from SA did not increase MCF-7 cell proliferation. Compound 1 is expected to act as an ERα ligand and have estrogenic effects, without side effects, such as breast cancer development.

Reactive oxygen species (ROS) are a major causative factor of inflammatory responses and extracellular matrix degradation. ROS also cause skin aging and diverse cutaneous lesions. Therefore, antioxidants that inhibit the generation of ROS may be beneficial in the relief of skin aging and diseases. We investigated the anti-skin aging effect of anthraquinones from cultures of Colletotrichum sp., an endophytic fungus isolated from Morus alba L. using human dermal fibroblasts (HDFs). We preferentially evaluated the preventive effects of anti-oxidative anthraquinones (1, 4) against the generation of ROS, nitric oxide (NO), and prostaglandins-E2 (PGE2). Among them, 1,3-dihydroxy-2,8-dimethoxy-6-methylanthraquinone (1) suppressed the generation of ROS, NO, and PGE2 in tumor necrosis factor-alpha (TNF-α)-stimulated HDFs. Compound 1 reversed the TNF-induced increase in matrix metalloproteinase (MMP)-1 and a decrease in procollagen I α1 (COLIA1). It also suppressed inducible NO synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6, and IL-8, which upregulate inflammatory reactions. Mechanistically, compound 1 suppressed nuclear factor-κB, activator protein 1, and mitogen-activated protein kinases in TNF-α-stimulated HDFs. These results suggest that compound 1 may be beneficial for improving skin aging and diverse cutaneous lesions.

Excessive glutamate damages neuronal cells via the accumulation of intracellular reactive oxygen species (ROS), calcium ion (Ca2+) influx, depolarization of mitochondrial membrane potential, and apoptosis, which may result in the development of chronic neurodegenerative diseases. In this study, we evaluated the effects of 4,6′-anhydrooxysporidinone isolated from endophytic fungus Fusarium lateritium SSF2 on glutamate-induced cytotoxicity, accumulation of intracellular ROS, increases in superoxide anion production, Ca2+, depolarization of mitochondrial membrane potential, and apoptotic cell death in hippocampal HT22 cells. 2′,7′-Dichlorofluorescin diacetate (H2DCFDA) staining was used to determine the intracellular reactive oxygen species concentration and dihydroethidine (DHE) staining was used to determine the superoxide radical. Expression of the nuclear factor-erythroid-2–related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was analyzed by Western blot. Fluo-4 staining was used to determine the intracellular Ca2+ levels. In order to explore mitochondrial membrane potential, tetramethylrhodamine methyl ester (TMRM) staining was used. Apoptotic cell death was evaluated using Annexin-V/propidium iodide (PI) staining and TUNEL staining. Expression of the cytochrome c release and cleaved caspase-9, -3 was analyzed by Western blot. Here, we were able to isolate 4,6′-anhydrooxysporidinone from endophytic fungus, Fusarium lateritium SSF2, which was shown to protect HT22 cells from glutamate-induced cytotoxicity, accumulation of intracellular ROS, increases in superoxide anion production, Ca2+, and depolarization of mitochondrial membrane potential. In addition, 4,6′-anhydrooxysporidinone enhanced the expressions of Nrf2 and HO-1. It also inhibited the apoptotic cell death through the inhibition of cytochrome c release and cleaved caspase-9, -3 in glutamate-treated HT22 cells. Therefore, our results provide ample evidence of the neuroprotective properties of 4,6′-anhydrooxysporidinone.

An endophytic fungus, Neofusicoccum parvum JS-0968, was isolated from a plant, Vitex rotundifolia. The chemical investigation of its cultures led to the isolation of a secondary metabolite, (3R)-5-hydroxymellein. It has been reported to have antifungal, antibacterial, and antioxidant activity, but there have been no previous reports on the effects of (3R)-5-hydroxymellein on atherosclerosis. The oxidation of lipoproteins and foam cell formation have been known to be significant in the development of atherosclerosis. Therefore, we investigated the inhibitory effects of (3R)-5-hydroxymellein on atherosclerosis through low-density lipoprotein (LDL) and high-density lipoprotein (HDL) oxidation and macrophage foam cell formation. LDL and HDL oxidation were determined by measuring the production of conjugated dienes and malondialdehyde, the amount of hyperchromicity and carbonyl content, conformational changes, and anti-LDL oxidation. In addition, the inhibition of foam cell formation was measured by Oil red O staining. As a result, (3R)-5-hydroxymellein suppressed the oxidation of LDL and HDL through the inhibition of lipid peroxidation, the decrease of negative charges, the reduction of hyperchromicity and carbonyl contents, and the prevention of apolipoprotein A-I (ApoA-I) aggregation and apoB-100 fragmentation. Furthermore, (3R)-5-hydroxymellein significantly reduced foam cell formation induced by oxidized LDL (oxLDL). Taken together, our data show that (3R)-5-hydroxymellein could be a potential preventive agent for atherosclerosis via obvious anti-LDL and HDL oxidation and the inhibition of foam cell formation.

Glutamate toxicity has been implicated in neuronal cell death in both acute CNS injury and in chronic diseases. In our search for neuroprotective agents obtained from natural sources that inhibit glutamate toxicity, an endophytic fungus, Fusarium solani JS-0169 isolated from the leaves of Morus alba, was found to show potent inhibitory activity. Chemical investigation of the cultures of the fungus JS-0169 afforded isolation of six compounds, including one new γ-pyrone (1), a known γ-pyrone, fusarester D (2), and four known naphthoquinones: karuquinone B (3), javanicin (4), solaniol (5), and fusarubin (6). To identify the protective effects of the isolated compounds (1−6), we assessed their inhibitory effect against glutamate-induced cytotoxicity in HT22 cells. Among the isolates, compound 6 showed significant neuroprotective activity on glutamate-mediated HT22 cell death. In addition, the informatics approach using in silico systems pharmacology identified that compound 6 may exert its neuroprotective effect by controlling the amount of ubiquinone. The results suggest that the metabolites produced by the endophyte Fusarium solani JS-0169 might be related to the neuroprotective activity of its host plant, M. alba.

Seven compounds, including three neolignans 1–3, a norlignan 4, and three diterpenoids 5–7, were isolated from the feces of Trogopterus xanthipes. Structures of these compounds were identified by 1D and 2D NMR as well as MS. The absolute configurations of compounds 1, 2, and 4 were determined by comparing CD spectra and optical rotations. Among the isolated compounds, 1–3 were novel and subsequently named trogopterins A, B, and C, respectively. Likewise, compound 4 was isolated from nature for the first time. Cytotoxic activities of compounds 1–4 were evaluated. Compounds 1–3 exhibited moderate cytotoxic activities against HL-60 cells with IC50 values of 34.77–45.68 μM.

Endophytes have been recognized as a source for structurally novel and biologically active secondary metabolites. Among the host plants for endophytes, some medicinal plants that produce pharmaceuticals have been reported to carry endophytes, which could also produce bioactive secondary metabolites. In this study, the medicinal plant Aconitum carmichaeli was selected as a potential source for endophytes. An endophytic microorganism, Aureobasidium pullulans AJF1, harbored in the flower of Aconitum carmichaeli, was cultured on a large scale and extracted with an organic solvent. Extensive chemical investigation of the extracts resulted in isolation of three lipid type compounds (1−3), which were identified to be (3R,5R)-3,5-dihydroxydecanoic acid (1), (3R,5R)-3-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-5-hydroxydecanoic acid (2), and (3R,5R)-3-(((3R,5R)-5-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-3-hydroxydecanoyl)oxy)-5-hydroxydecanoic acid (3) by chemical methods in combination with spectral analysis. Compounds 2 and 3 had new structures. Absolute configurations of the isolated compounds (1−3) were established using modified Mosher’s method together with analysis of NMR data for their acetonide derivatives. All the isolates (1−3) were evaluated for antibiotic activities against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and their cytotoxicities against MCF-7 cancer cells. Unfortunately, they showed low antibiotic activities and cytotoxic activities.

Atherosclerosis is the key factor responsible for cardiovascular events, which is a major cause of morbidities and mortalities worldwide. It is well known that high-density lipoprotein (HDL) oxidation and glycation increases the risk for atherosclerosis. Epimedium koreanum has been used as a traditional oriental medicine for treating erectile dysfunction, kidney diseases, osteoporosis, and breast cancer. However, no reports on the effects of E. koreanum on HDL modification exist. In this study, we investigated the inhibitory effects of E. koreanum extract and its eight flavonoids, which are: (1) anhydroicaritin 3-O-rhamnoside, (2) β-anhydroicaritin, (3−5) epimedins A-C, (6) epimedoside A, (7) icariin, and (8) des-O-methyl-β-anhydroicaritin, against HDL modification. HDLs obtained from pooled human plasma samples were incubated in vitro with E. koreanum extract or each compound in the presence of copper sulfate or fructose. The HDL modifications were evaluated by measuring generation of conjugated dienes, production of thiobarbituric acid reactive substances, change in electrophoretic mobility of apoA-I, advanced glycation end products formation, and apoA-I aggregation. Consequently, E. koreanum extract and compound 8 suppressed HDL modification through inhibition of lipid peroxidation, apoA-I aggregation, negative charge increase, and AGEs formation. In particular, compound 8 showed more potent inhibitory effect on HDL modification than the extracts, suggesting its protective role against atherosclerosis via inhibition of HDL oxidation and glycation.

Fungal endophytes have attracted attention due to their functional diversity. Secondary metabolites produced by Pestalotiopsis sydowiana from a halophyte, Phragmites communis Trinus, were investigated. Eleven compounds, including four penicillide derivatives (1–4) and seven α-pyrone analogues (5–10) were isolated from cultures of P. sydowiana. The compounds were identified based on spectroscopic data. The inhibitory activities against the 20S proteasome were evaluated. Compounds 1–3, 5, and 9–10 showed modest proteasome inhibition activities, while compound 8 showed strong activity with an IC50 of 1.2 ± 0.3 μM. This is the first study on the secondary metabolites produced by P. sydowiana and their proteasome inhibitory activities. The endophytic fungus P. sydowiana might be a good resource for proteasome inhibitors.

Decalin-containing tetramic acid derivatives, especially 3-decalinoyltetramic acids (3-DTAs), are commonly found as fungal secondary metabolites. Numerous biological activities of this class of compounds, such as antibiotic, antiviral, antifungal, antiplasmodial, and antiprotozoal properties, have been the subject of ongoing research. For this reason, these molecules have attracted a lot of interest from the scientific community and various efforts including semi-synthesis, co-culturing with bacteria and biosynthetic gene sequencing have been made to obtain more derivatives. In this review, 3-DTAs are classified into four major groups based on the absolute configuration of the bicyclic decalin ring. Their biosynthetic pathways, various biological activities, and structure-activity relationship are then introduced. [ABSTRACT FROM AUTHOR]

A number of characteristics identified in two-dimensional-infrared spectra that could be used to track different secondary structural elements during amyloid formation without error-prone deconvolution are described. The computed results obtained from 2D-IR spectra could be used to test simulations and aggregation mechanisms.

The article discusses a study by Wei Li and colleagues which aimed to evaluate the inhibitory effects of each compound of the soluble epoxide hydrolase (sEH) of perennial herbaceous plant Gentiana scabra (G. scabra). Topics discussed include the role of sEH on eicosanoid epoxides metabolism and epoxyeicosatrienoic acids (EETs) hydrolisis, and the isolation of nine phenolic compounds including siringin, berchemol, and koaburaside from G. Scabra's rhizomes and roots.

Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we analysed the recombinant protein produced from rock bream thioredoxin 1 cDNA (rRbTRx1). To verify the immune-stimulatory effect of this recombinant protein, changes in the expression level of several genes were investigated using the cDNA microarray chips in rock bream peripheral blood leukocytes stimulated with rRbTRx1. Furthermore, the immune responses of rock bream to Streptococcus iniae FKC (formalin-killed cell) vaccination alone or in combination with recombinant proteins were analysed after an experimental challenge with living S. iniae. Microarray analysis showed that 237 unique genes were upregulated more than two-fold after rRbTRx1 stimulation. Serum agglutination titres were relatively low; however, the FKC vaccine still conferred protection against S. iniae. Moreover, the adverse effects showed no statistically significant difference between fish injected with a concentration and non-injected fish. After experimental challenge to the rock bream by injection with living bacteria (S. iniae), the relative percent survival in the vaccinated groups with FKC + rRbTRx1 was significantly higher than that of the vaccinated groups with FKC alone, which were 85.9% and 68.2%, respectively. This indicated that the recombinant protein as an adjuvant showed synergism with the injected vaccine in rock bream. [ABSTRACT FROM AUTHOR]

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution ¡mages of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes. [ABSTRACT FROM AUTHOR]

Three new neo-clerodane diterpenoids, barbatellarines C-E (-), were isolated from the CHCl-soluble fraction of the aerial part of Scutellaria barbata. Their chemical structures were elucidated by detailed analysis of NMR and MS data. Compounds and were C(13) epimers, which was confirmed by an NOE difference experiment and the NOESY spectrum. The relative configuration was determined on the basis of the H-NMR J-value and NOE data, while the absolute configuration of the previously isolated analogue, barbatellarine B (), as a representative member of the group, was assigned by CD analysis. [ABSTRACT FROM AUTHOR]

There is considerable interest in uncovering the pathway of amybid formation because the toxic properties of amyboid likely stems from prefibril intermediates and not the fully formed fibrils. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyboid polypeptide (hIAPP). By monitoring the kinetics at 6 different labeled sites, we find that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils, followed by formation of the 2 parallel β-sheets with the N-terminal β-sheet likely forming before the C-terminal sheet. This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation as well as a general methodology for studying other amyboid forming proteins without the use of structure-perturbing labels. [ABSTRACT FROM AUTHOR]

We have recently developed a new and simple way of collecting 2D infrared and visible spectra that utilizes a pulse shaper and a partly collinear beam geometry. 2D IR and Vis spectroscopies are powerful tools for studying molecular structures and their dynamics. They can be used to correlate vibrational or electronic eigenstates, measure energy transfer rates, and quantify the dynamics of lineshapes, for instance, all with femtosecond time-resolution. As a result, they are finding use in systems that exhibit fast dynamics, such as sub-millisecond chemical and biological dynamics, and in hard-to-study environments, such as in membranes. While powerful, these techniques have been difficult to implement because they require a series of femtosecond pulses to be spatially and temporally overlapped with precise time-resolution and interferometric phase stability. However, many of the difficulties associated with implementing 2D spectroscopies are eliminated by using a pulse shaper and a simple beam geometry, which substantially lowers the technical barriers required for researchers to enter this exciting field while simultaneously providing many new capabilities. The aim of this paper is to provide an overview of the methods for collecting 2D spectra so that an outsider considering using 2D spectroscopy in their own research can judge which approach would be most suitable for their research aims. This paper focuses primarily on 2D IR spectroscopy, but also includes our recent work on adapting this technology to collecting 2D Vis spectra. We review work that has already been published as well as cover several topics that we have not reported previously, including phase cycling methods to remove background signals, eliminate unwanted scatter, and shift data collection into the rotating frame. [ABSTRACT FROM AUTHOR]

The capability of 2D IR spectroscopy to elucidate time-evolving structures is enhanced by a programmable mid-IR pulse shaper that greatly improves the ease, speed, and accuracy of data collection. Traditional ways of collecting 2D IR spectra are difficult to implement, cause distorted peak shapes, and result in poor time resolution and/or phase problems. We report on several methods for collecting 2D IR spectra by using a computer-controlled germanium acoustooptic modulator that overcomes the above problems. The accuracy and resolution of each method is evaluated by using model metal carbonyl compounds that have well defined line-shapes. Furthermore, phase cycling can now be employed to largely alleviate background scatter from heterogeneous samples. With these methods in hand, we apply 2D IR spectroscopy to study the structural diversity in amyloid fibers of aggregated human islet amyloid polypeptide (hIAPP), which is involved with type 2 diabetes. The 2D IR spectra reveal that the β-sheet fibers have a large structural distribution, as evidenced by an inhomogeneously broadened β-sheet peak and strong coupling to random coil conformations. Structural diversity is an important characteristic of hIAPP because it may be that partly folded peptides cause the disease. This experiment on hIAPP is an example of how computer generation of 2D IR pulse sequences is a key step toward automating 2D IR spectroscopy, so that new pulse sequences can be implemented quickly and a diverse range of systems can be studied more easily. [ABSTRACT FROM AUTHOR]

Abstract Specimen-induced aberration has been a major factor limiting the imaging depth of single-molecule localization microscopy (SMLM). Here, we report the application of label-free wavefront sensing adaptive optics to SMLM for deep-tissue super-resolution imaging. The proposed system measures complex tissue aberrations from intrinsic reflectance rather than fluorescence emission and physically corrects the wavefront distortion more than three-fold stronger than the previous limit. This enables us to resolve sub-diffraction morphologies of cilia and oligodendrocytes in whole zebrafish as well as dendritic spines in thick mouse brain tissues at the depth of up to 102 μm with localization number enhancement by up to 37 times and localization precision comparable to aberration-free samples. The proposed approach can expand the application range of SMLM to whole zebrafish that cause the loss of localization number owing to severe tissue aberrations.

The article presents a study which explores the formation of cellular close-ended tunneling nanotubes through mechanical deformation. It mentions that membrane nanotubes or tunneling nanotubes (TNTs) that connect cells have been recognized as a previously unidentified pathway for intercellular transport between distant cells.

Abstract Photobleaching is the permanent loss of fluorescence after extended exposure to light and is a major limiting factor in super‐resolution microscopy (SRM) that restricts spatiotemporal resolution and observation time. Strategies for preventing or overcoming photobleaching in SRM are reviewed developing new probes and chemical environments. Photostabilization strategies are introduced first, which are borrowed from conventional fluorescence microscopy, that are employed in SRM. SRM‐specific strategies are then highlighted that exploit the on–off transitions of fluorescence, which is the key mechanism for achieving super‐resolution, which are becoming new routes to address photobleaching in SRM. Off states can serve as a shelter from excitation by light or an exit to release a damaged probe and replace it with a fresh one. Such efforts in overcoming the photobleaching limits are anticipated to enhance resolution to molecular scales and to extend the observation time to physiological lifespans.

Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin.

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

We report that the Gm7068 (CatSpere) and Tex40 (CatSperz) genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of CatSperz reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.8 μm intervals along the flagellum. This disruption renders the proximal flagellum inflexible and alters the 3D flagellar envelope, thus preventing sperm from reorienting against fluid flow in vitro and efficiently migrating in vivo. Ejaculated CatSperz-null sperm cells retrieved from the mated female uterus partially rescue in vitro fertilization (IVF) that failed with epididymal spermatozoa alone. Human CatSperε is quadrilaterally arranged along the flagella, similar to the CatSper complex in mouse sperm. We speculate that the newly identified CatSperζ subunit is a late evolutionary adaptation to maximize fertilization inside the mammalian female reproductive tract.

A novel ergosterol conjunction-type alkaloid, hericirine (1), was isolated from the dried fruiting bodies of Hericium erinaceum. The overall framework of 1 was isolated, and combined with hericerin and ergosterol. Its structure was elucidated based on spectroscopic analysis using 1D and 2D NMR, IR and HRESIMS techniques. We found that hericirine significantly inhibited protein expression of iNOS and COX-2 and reduced NO, PGE2, TNF-α, IL-6 and IL-1β production in RAW264.7 cells exposed to LPS. [ABSTRACT FROM AUTHOR]