Effects of pH alterations on stress- and aging-induced protein phase separation.

Upon stress challenges, proteins/RNAs undergo liquid-liquid phase separation (LLPS) to fine-tune cell physiology and metabolism to help cells adapt to adverse environments. The formation of LLPS has been recently linked with intracellular pH, and maintaining proper intracellular pH homeostasis is known to be essential for the survival of organisms. However, organisms are constantly exposed to diverse stresses, which are accompanied by alterations in the intracellular pH. Aging processes and human diseases are also intimately linked with intracellular pH alterations. In this review, we summarize stress-, aging-, and cancer-associated pH changes together with the mechanisms by which cells regulate cytosolic pH homeostasis. How critical cell components undergo LLPS in response to pH alterations is also discussed, along with the functional roles of intracellular pH fluctuation in the regulation of LLPS. Further studies investigating the interplay of pH with other stressors in LLPS regulation and identifying protein responses to different pH levels will provide an in-depth understanding of the mechanisms underlying pH-driven LLPS in cell adaptation. Moreover, deciphering aging and disease-associated pH changes that influence LLPS condensate formation could lead to a deeper understanding of the functional roles of biomolecular condensates in aging and aging-related diseases.

Genome-wide identification of resistance genes and transcriptome regulation in yeast to accommodate ammonium toxicity.

BACKGROUND: Ammonium is an important raw material for biomolecules and life activities, and the toxicity of ammonium is also an important ecological and agricultural issue. Ammonium toxicity in yeast has only recently been discovered, and information on its mechanism is limited. In recent years, environmental pollution caused by nitrogen-containing wastewater has been increasing. In addition, the use of yeast in bioreactors to produce nitrogen-containing compounds has been developed. Therefore, research on resistance mechanisms that allow yeast to grow under conditions of high concentrations of ammonium has become more and more important. RESULTS: To further understand the resistance mechanism of yeast to grow under high concentration of ammonium, we used NH4Cl to screen a yeast non-essential gene-deletion library. We identified 61 NH4Cl-sensitive deletion mutants from approximately 4200 mutants in the library, then 34 of them were confirmed by drop test analysis. Enrichment analysis of these 34 genes showed that biosynthesis metabolism, mitophagy, MAPK signaling, and other pathways may play important roles in NH4Cl resistance. Transcriptome analysis under NH4Cl stress revealed 451 significantly upregulated genes and 835 significantly downregulated genes. The genes are mainly enriched in: nitrogen compound metabolic process, cell wall, MAPK signaling pathway, mitophagy, and glycine, serine and threonine metabolism. CONCLUSIONS: Our results present a broad view of biological pathways involved in the response to NH4Cl stress, and thereby advance our understanding of the resistance genes and cellular transcriptional regulation under high concentration of ammonium.

ROS1-mediated decrease in DNA methylation and increase in expression of defense genes and stress response genes in Arabidopsis thaliana due to abiotic stresses.

BACKGROUND: Small interfering RNAs (siRNAs) target homologous genomic DNA sequences for cytosine methylation, known as RNA-directed DNA methylation (RdDM), plays an important role in transposon control and regulation of gene expression in plants. Repressor of silencing 1 (ROS1) can negatively regulate the RdDM pathway. RESULTS: In this paper, we investigated the molecular mechanisms by which an upstream regulator ACD6 in the salicylic acid (SA) defense pathway, an ABA pathway-related gene ACO3, and GSTF14, an endogenous gene of the glutathione S-transferase superfamily, were induced by various abiotic stresses. The results demonstrated that abiotic stresses, including water deficit, cold, and salt stresses, induced demethylation of the repeats in the promoters of ACD6, ACO3, and GSTF14 and transcriptionally activated their expression. Furthermore, our results revealed that ROS1-mediated DNA demethylation plays an important role in the process of transcriptional activation of ACD6 and GSTF14 when Arabidopsis plants are subjected to cold stress. CONCLUSIONS: This study revealed that ROS1 plays an important role in the molecular mechanisms associated with genes involved in defense pathways in response to abiotic stresses.

UV-guided isolation of enantiomeric polyacetylenes from Bupleurum scorzonerifolium Willd. with inhibitory effects against LPS-induced NO release in BV-2 microglial cells.

UV-guided fractionation led to the isolation of thirteen new polyacetylenes (1-13) from the roots of Bupleurum scorzonerifolium Willd. All polyacetylenes were analyzed as racemates since the lack of optical activity and Cotton effects in the ECD spectra. The sequent chiral-phase HPLC resolution successfully gave twelve pairs of enantiomers 1a/1b and 3a/3b-13a/13b. Their structures were elucidated based on the HRESIMS and NMR data analyses. The absolute configurations were determined by the combination of Snatzke's method, electronic circular dichroism calculations, and single-crystal X-ray diffraction. Using Griess methods and MTT assays, polyacetylenes 1a, 3a, 4a/4b-12a/12b, and 13a displayed inhibitory activities against LPS-induced NO release in BV-2 microglial cells.

Isolation of triterpenoid saponins from Medicago sativa L. with neuroprotective activities.

Three new pentacyclic triterpenoid saponins (1-3), together with medicagenic acid (4) were isolated and purified from 70% EtOH extract of Medicago sativa L. by different column chromatographic and semi-preparative HPLC. Their structures were established by direct interpretation of their spectral data, mainly HR-ESI-MS, 1D-NMR, 2D-NMR, and chemical methods, as well as comparison with literature data. Additionally, all isolates were evaluated for their neuroprotective activities against H2O2-induced damage in human neuroblastoma SHSY5Y cells. As a results, compounds 1 and 2 (67.14% and 73.05%) exhibited potent neuroprotective activities. These findings provide new insights into developing better treatment of neurodegenerative diseases for M. sativa in the future.

Adsorption Properties of Comb-Shaped Polycarboxylate Dispersant onto Different Crystal Pyraclostrobin Particle Surfaces.

The stability of the suspension system of the two crystal forms of pyraclostrobin is evaluated using multiple light technology, and the adsorption performance of polycarboxylate dispersant on the surface of two different crystalline pyraclostrobin particles is compared in combination with XRD, FTIR, XPS, and SEM from the microscopic view. The adsorption kinetics and thermodynamics studies of 2700 on the surfaces of different crystalline forms of pyraclostrobin particles show that the adsorption process of 2700 on the surfaces of pyraclostrobin crystal forms II and IV conform to pseudo-second-order kinetic adsorption model. The Ea values for crystal forms II and IV are 12.93 and 14.39 kJ∙mol-1, respectively, which indicates that both adsorption processes are physical adsorption. The adsorption models of 2700 on the surfaces of pyraclostrobin crystal forms II and IV are in accordance with Langmuir adsorption isotherms. The ∆Gad values of crystal forms II and IV are negative and the ∆Sad values are positive at different temperatures. Therefore, the adsorption processes are spontaneous and accompanied by entropy increase. The results of this study provide an important theoretical basis for the selection of polycarboxylate dispersants in the suspension of pyraclostrobin. This study also provides a reference for the research of polycrystalline pesticide suspension concentrate.

Neuroprotective effects of triterpenoid saponins from Medicago sativa L. against H2O2-induced oxidative stress in SH-SY5Y cells.

Medicago sativa L. is a forage legume plant widely distributed in all continents. Six new triterpenoid saponins, Medicagosides A-F (1-6) and five known ones (7-11) were isolated from M. sativa. Their structures were determined via HRESIMS, 1D and 2D NMR analysis. Biologically, all the isolates displayed neuroprotective activities against H2O2-induced damage in SH-SY5Y cells. Among them, compounds 1, 3-5 and 10 exhibited striking neuroprotective activities at 100 µM, restoring cell viability range from 79.66% to 89.03%, relative to 79.46% (100 µM) of Trolox used as the positive control.

Antioxidant and cytotoxic lignans from the roots of Bupleurum chinense.

In the search for biologically active compounds from the roots of Bupleurum chinense D C., phytochemical investigation of its ethanol extract led to the isolation and identification of a new 8-O-4' neolignan glucoside, saikolignanoside A (1), along with eight known lignans (2-9). Their structures were determined on the basis of IR, UV, HRESIMS, and NMR spectroscopic analyses. The antioxidant and cytotoxic effects of isolated compounds were evaluated in vitro. The isolated compounds (IC50 > 200 µM) did not display 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Whereas compounds 1-2, 5, 7, and 9 exhibited potent 2, 2'-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging properties with IC50 values ranging from 8.34 to 15.24 µM, while compounds 3-4, 6, 8 showed moderate properties. In addition, all compounds were evaluated for cytotoxicities against A549, HepG2, U251, Bcap-37, and MCF-7 cell lines. Compounds 5 and 9 (IC50 < 51.62 µM) possessed stronger cytotoxic activities against all the tested tumor cell lines, compared with the positive control 5-Fluorouracil.

Neuroprotective oleanane triterpenes from the roots of Bupleurum chinense.

The discovery of new natural compounds with pharmacological properties is an increasingly important field, and a continuous phytochemical investigation of the roots of Bupleurum chinense D.C. has led to the isolation of 17 triterpenoids, including three new oleanane triterpenes (1-3) together with 14 known ones. Their structures were determined on the basis of 1D and 2D NMR spectra as well as HR-ESI-MS data. The cytotoxicities of all compounds against five selected human cancer cell lines were assayed. Only compounds 9 and 14 exhibited moderate activities. Recently, a number of investigations have focused on the neuroprotective properties of triterpenoids in B. chinense. In order to expand our knowledge about this herb, the neuroprotective effects of compounds 1-17 against hydrogen peroxide (H2O2)-induced neuronal cell damage in human neuroblastoma SH-SY5Y cells were evaluated. Compounds 1-3, 6-7 showed significant neuroprotective effects against H2O2-induced SH-SY5Y cell death. Preliminary structure-activity relationships (SARs) between neuroprotective effects and the isolates were also discussed.

Cytotoxic triterpenoid glycosides (saikosaponins) from the roots of Bupleurum chinense.

As a part of our ongoing studies on cytotoxic triterpenoid saponins from herbal medicines, phytochemical investigation of the roots of Bupleurum chinense DC. afforded four new saikosaponins (1-4), along with 16 known ones (5-20). Their structures were established by direct interpretation of their spectral data, mainly HR-ESI-MS, 1D NMR and 2D NMR, and by comparison with literature data. Among them, compound 20 was isolated from the natural product for the first time. The cytotoxicities of all compounds against five selected human cancer cell lines (A549, HepG2, Hep3B, Bcap-37 and MCF-7) were assayed. In general, a number of the isolated compounds exhibited potent cytotoxic activities against the five selected human cancer cell lines. In particular, compounds 3, 8-9, 11-13, 16 and 20 showed more potent cytotoxic activities against the HepG2 and A549 cell lines than the positive control 5-fluorouracil. Based on the primary screening results, the preliminary structure-activity relationship (SAR) studies were also discussed. The SAR results suggest that the 13,28-epoxy bridge, the orientation of the hydroxyl group and the type of the sugar units are important requirements for cytotoxicity and selectivity.

Biosynthesis of Gold Nanoparticles Using Novel Bamboo (Bambusa chungii) Leaf Extracts.

Gold nanoparticles (Au NPs) have drawn significant interest because of their antisotropic physical properties in biomedical applications. In this paper, we reported the application of bamboo (Bambusa chung) leaf extracts, previously not exploited, in the synthesis of Au NPs at ambient temperature. The average dimension of quasi-spherical Au NPs was 28.8±4.5 nm by transmission electron microscopy (TEM). The UV-vis spectroscopy gave an optimal reaction time of 180 min in the process of bioreduction. The organic shell of Au NPs was characterized by Fourier transform infrared (FTIR) spectra and thermogravimetric analysis (TGA), suggesting that the main compositions of the organic shell were hydroxyflavones. The X-ray diffraction (XRD) studies indicated the Au NPs were (111) oriented. This eco-friendly method for the synthesis of Au NPs was simple, amenable for large scale commercial production and biological applications to future in vivo imaging and cancer therapy.

Separation and identification of multiple constituents in Xiao Chai Hu Decoction (Sho-saiko-to) by bioactivity-guided fractionation combined with LC-ESI-QTOFMS/MS.

Xiao Chai Hu Decoction (XCHD), named Sho-saiko-to in Japanese, is a well-known traditional Chinese medicine formula used in Asia. However, the characterization methods used in the past have lacked sensitivity and the nature of the active constituents of XCHD remains unclear. This study was carried out to establish the hyphenated method of bioactivity-guided fractionation and liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOFMS/MS) in order to identify the major bioactive constituents of XCHD. D101 macroporous resin was used to separate and enrich the material base into four fractions, XCHD-1, XCHD-2, XCHD-3 and XCHD-4. Each fraction was then evaluated for its antidepressant effect using depression-related parameters. An LC-ESI-QTOFMS/MS method in both positive and negative ion mode was also applied for separation and identification of the biological active fractions of XCHD. As a result, 79 compounds including polysaccharides, flavonoids, saikosaponins, ginsenosides, licoricesaponins and gingerols were detected, 69 of them were identified or tentatively characterized. Based on our preliminary characterization investigations, polysaccharides, gingerols and flavonoids in XCHD may contribute to the antidepressant effect of XCHD. In conclusion, the hyphenated method of bioactivity-guided fractionation and LC-ESI-QTOFMS/MS was meaningful for the isolation and preliminary identification of the biological active components in complex matrices of traditional Chinese medicine.

A new cyclolignan glycoside from the tubers of Pinellia ternata.

A new 2,7'-type cyclolignan glycoside, cyclolignanyingoside A (1), together with six known compounds (2-7) were isolated from the tubers of Pinellia ternata (Thunb.) Breit. The structure of 1 was elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses, HR-ESI-MS, and CD spectrometry. The cytotoxic, antioxidant and tyrosinase-inhibiting activities of all the isolates were determined. However, all the isolates exhibited no activity on the selected cell lines (Hep-3B, Bcap-37, and MCF-7). In addition, compounds 1-3 and 7 exhibited strong 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) free radical scavenging activity, and compounds 2 and 4 showed a moderate mushroom tyrsinase inhibitory activity.

A rapid extraction and analysis method for the simultaneous determination of 26 bioflavonoids in Portulaca oleracea L.

INTRODUCTION: Portulaca oleracea L. (P. oleracea, purslane) is an edible plant that is widely distributed around the world, and flavonoids are its main bioactive constituents. Therefore, the detection of flavonoids is very important for a better understanding of its pharmacological actions and to monitor the product quality control of P. oleracea. OBJECTIVE: To develop a rapid method to extract and determine 26 bioflavonoids in P. oleracea, based on microwave extraction (MWE) and triple quadrupole-linear ion trap mass spectrometry. METHODS: The optimal conditions of MWE for the extraction of flavonoids from P. oleracea involved the use of methanol as the extraction solvent, a microwave power of 300 W, an extraction time of 450 s, and a solvent-to-solid ratio of 30 mL/g. The samples were analysed using an ultra-performance liquid chromatograph coupled with a triple quadrupole-linear ion trap mass spectrometer (UPLC-MS/MS) system. RESULTS: The calibration curves of all 26 analytes showed good linearity (r ≥ 0.999) and the intra- and interday precisions and repeatability were all within required limits. The mean recoveries measured at three concentrations were higher than 94.2%, with RSDs lower than 2.94% for the targets. CONCLUSION: The established MWE/UPLC-MS/MS method is a rapid and effective method for quality evaluation of P. oleracea from different production regions and different harvest periods.

Two new alkaloids from Portulaca oleracea and their cytotoxic activities.

Two new alkaloids named (3R)-3,5-bis(3-methoxy-4-hydroxyphenyl)-2,3-dihydro-2(1H)-pyridinone (1) and 1,5-dimethyl-6-phenyl-1,2-dihydro-1,2,4-triazin-3(2H)-one (2), together with two known compounds (7'R)-N-feruloyl normetanephrine (3) and N-trans-feruloyl tyramine (4) were isolated from the air-dried aerial parts of Portulaca oleracea L. Their structures and configurations were elucidated by spectroscopic methods including 1D NMR, 2D NMR, and HR-MS techniques. In addition, compounds 1-4 were tested for in vitro cytotoxic activities against human lung (K562 and A549) and breast (MCF-7 and MDA-MB-435) cancer cell lines.

Study on characterization of Bupleurum chinense polysaccharides with antioxidant mechanisms focus on ROS relative signaling pathways and anti-aging evaluation in vivo model.

This study explored the structures of three polysaccharides from Bupleurum chinense DC. (BCPRs), and evaluated their antioxidant and anti-aging properties. The HPGPC and ion chromatography analyses revealed that the molecular weights of the BCPRs ranged from 12.05 to 21.20 kDa, and were primarily composed of rhamnose, arabinose, xylose, galactose, glucose and galacturonic acid. Methylation and NMR studies identified 10 PMAAs, establishing the various backbones of BCPRs 1-3. BCPR-3 demonstrated potent antioxidant activities, including DPPH, ABTS, hydroxy, and superoxide radicals scavenging in vitro. At concentrations between 125 and 500 µg/mL, BCPR-3 increased T-AOC, SOD and GSH-Px activities, while decreasing MDA levels in H2O2-induced SH-SY5Y cells. In addition, RNA-seq results indicated that BCPR-3 considerably downregulated the expression of 49 genes and upregulated five genes compared with the control group. KEGG analysis suggested that these differentially expressed genes (DEGs) were predominantly involved in the TNF and PI3K/Akt signaling pathways. Furthermore, in vivo experiment with Drosophila melanogaster showed that BCPR-3 could extend the average lifespan of flies. In conclusion, polysaccharides from B. chinense exhibited potential antioxidant and anti-aging activities, which could be developed as new ingredients to combat oxidative stress damage and slow the aging process.

GRP78 Downregulation in Keratinocytes Promotes Skin Inflammation through the Recruitment and Activation of CCR6+ IL-17A-Producing γδ T Cells.

The migration of γδ T lymphocytes toward skin lesions and their concomitant pathogenic IL-17A production play a crucial role in the pathogenesis of psoriasis. However, the regulatory mechanisms of IL-17A production by γδ T cells and their migration remain to be fully explored. Intracellular GRP78 is a molecular chaperone that regulates endoplasmic reticulum stress, whereas secretory GRP78, as a member of the resolution-associated molecular patterns, exerts immunoregulatory effects. In this study, we reported that both the intracellular GRP78 in skin lesions and secretory GRP78 in the serum were significantly decreased in patients with psoriasis. A GRP78 knockdown exacerbated imiquimod-induced skin inflammation, whereas the application of recombinant GRP78 protein or BIP inducer X (a GRP78 inducer) attenuated the dermatitis. Mechanistically, the GRP78 knockdown in keratinocytes enhanced the production of chemokines, specifically CCL20, which regulates γδ T-cell migration. Moreover, recombinant GRP78 was found to directly bind to γδ T cells to suppress its migration ability and proinflammatory capacities by downregulating the CCR6 and IL-17A expression. Collectively, our results uncovered a pivotal role of GRP78 in the pathogenesis of psoriasis, which was mainly exerted by regulating the interaction between keratinocytes and γδ T cells, and might provide a promising target for psoriasis therapy.

Dupilumab therapy improves gut microbiome dysbiosis and tryptophan metabolism in Chinese patients with atopic dermatitis.

BACKGROUND: Dupilumab has demonstrate its potential to orchestrate inflammatory skin microenvironment, enhance skin barrier and shift skin microbiome dysbiosis, collectively contributing to clinical improvement in patients with atopic dermatitis (AD). As the second genome of human body, growing evidence suggests that the gut microbiome might relate to the host response to treatments. Little is known about the association between dupilumab treatment and gut microbiome in AD patients. OBJECTIVE: We aimed to characterize the gut microbiome among Chinese subjects with or without AD and determine the potential effect of dupilumab on the gut microbiome. RESULTS: The 16 s rRNA gene sequencing was conducted on 48 healthy controls (HC), 44 AD patients and 27 AD patients who received dupilumab for 16 weeks. Prior to treatment, we identified the changed beta-diversity, increased Firmicutes/Bacteroidetes ratio, decreased Bifidobacterium and expanded Faecalibacterium among the AD patients compared to HC. After 16 weeks of dupilumab treatment, gut microbiome dysbiosis of the AD patients improved with reversed beta-diversity, closer bacterial connections, increased colonization of Bifidobacterium, Ruminococcus gnavus, and Coprococcus, which were negatively correlated with disease severity indicators. This shift was largely independent of the degree of clinical improvement. Bacterial function analysis revealed further metabolic alterations following dupilumab treatment, including up-regulated expression of genes involved in the indole pathway of tryptophan metabolism, corroborated by quantitative UHPLC-MS/MS analysis. CONCLUSION: Dupilumab treatment tends to help shift the gut microbial dysbiosis in AD patients to a healthier state, along with improved intestinal tryptophan metabolism, suggesting the gut flora and its metabolites may mediate part of the synergistic therapeutic effects on the host.

NIR regulated upconversion nanoparticles@metal-organic framework composite hydrogel dressing with catalase-like performance and enhanced antibacterial efficacy for accelerating wound healing.

Developing a hydrogel dressing with excellent antibacterial efficacy for accelerating wound healing is high desirable in clinical applications. In this work, NIR regulated metal-organic framework composite hydrogel dressing was constructed for enhanced antibacterial efficacy and accelerated wound healing via the compounding between hydrogel and UCNPs@ZrMOF-Pt nanoparticles. The visible light emitted from upconvertion nanoparticles (UCNPs) activated porphyrin based metal-organic framework (MOF) in composite hydrogel to generate 1O2 for photodynamic antibacterial therapy under NIR laser irradiation. Moreover, the UCNPs@ZrMOF-Pt in composite hydrogel with catalase-like performance could effectively convert the high concentration H2O2 in wound to abundant O2, which relieved the hypoxic in infected wound. Thus, the photodynamic antibacterial efficacy was remarkably enhanced, leading to accelerate the wound healing. This work presented a novel strategy for high efficient antibacterial therapy and accelerated wound healing.

Differentiation of Morphological Traits and Genome-Wide Expression Patterns between Rice Subspecies Indica and Japonica.

Changes in gene expression patterns can lead to the variation of morphological traits. This phenomenon is particularly evident in recent evolution events such as crop domestication and responses to environmental stress, where alterations in expression levels can efficiently give rise to domesticated syndromes and adaptive phenotypes. Rice (Oryza sativa L.), one of the world's most crucial cereal crops, comprises two morphologically distinct subspecies, Indica and Japonica. To investigate the morphological divergence between these two rice subspecies, this study planted a total of 315 landrace individuals of both Indica and Japonica under identical cultivation conditions. Out of the 16 quantitative traits measured in this study, 12 exhibited significant differences between the subspecies. To determine the genetic divergence between Indica and Japonica at the whole-genome sequence level, we constructed a phylogenetic tree using a resequencing dataset encompassing 95 rice landrace accessions. The samples formed two major groups that neatly corresponded to the two subspecies, Indica and Japonica. Furthermore, neighbor-joining (NJ) trees based on the expression quantity of effectively expressed genes (EEGs) across five different tissues categorized 12 representative samples into two major clades aligning with the two subspecies. These results imply that divergence in genome-wide expression levels undergoes stabilizing selection under non-stressful conditions, with evolutionary trends in expression levels mirroring sequence variation levels. This study further supports the pivotal role of changes in genome-wide expression regulation in the divergence of the two rice subspecies, Indica and Japonica.