Discovery of Antifungal Norsesquiterpenoids from a Soil-Derived Streptomyces microflavus: Targeting Biofilm Formation and Synergistic Combination with Amphotericin B against Yeast-like Fungi.

Thirty-five norsesquiterpenoids were isolated from the fermentation broth of Streptomyces microflavus from the forest soil of Ailaoshan in China. The structures of new compounds (1-5, 10-26) were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra, as well as Mosher's reagent derivatives method. Norsesquiterpenoids showed different levels of antifungal activity with MIC80 values ranging from 25 to 200 µg/mL against Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. The combining isolated norsesquiterpenoids with amphotericin B resulted in a synergistic interaction against test yeast-like fungi with a fractional inhibitory concentration index < 0.5. Compound 33 significantly inhibited biofilm formation and destroyed the preformed biofilm of fungi. Moreover, 33 downregulated the expression of adhesion-related genes HWP1, ALS1, ALS3, ECE1, EAP1, and BCR1 to inhibit the adhesion of C. albicans. Findings from the current study highlight the potential usage of norsesquiterpenoids from soil-derived Streptomyces for antifungal leads discovery.

Structure-activity relationship and biofilm formation-related gene targets of oleanolic acid-type saponins from Pulsatilla chinensis against Candida albicans.

In the course of our investigations of antifungal natural products, the structure-activity relationship and antifungal activities of oleanolic acid-type saponins (1-28) from Pulsatilla chinensis against human and plant pathogenic fungi were elucidated. The analysis of structure-activity relationship of oleanolic acid-type saponins showed that the free carboxyl at C-28 was essential for their antifungal activities; the free hydroxyl group at the C-23 site of oleanolic acid-type saponins played a crucial role in their antifungal activities; the oligosaccharide chain at C-3 oleanolic acid-type saponins showed significant effects on antifungal efficacy and a disaccharide or trisaccharide moiety at position C-3 displayed optimal antifungal activity. The typical saponin pulchinenoside B3 (16, PB3) displayed satisfactory antifungal activity against human and plant pathogenic fungi, especially, C. albicans with an MIC value of 12.5 µg/mL. Furthermore, PB3 could inhibit the biofilm formation of C. albicans through downregulating the expression of the integrated network of biofilm formation-associated transcription factors (Bcr1 Efg1, Ndt80, Brg1, Rob1 and Tec1) and adhesion-related target genes (HWP1, ALS1, and ALS3). Meanwhile, we found that PB3 could effectively destroy the mature biofilm of C. albicans by the oxidative damage and inducing mitochondria-mediated apoptosis in cells.

Developing a trans-multisynaptic tracer to map the neural circuit of recovered sciatic nerve after treatment with nerve growth factor.

Nerve growth factor (NGF) has been shown to support the survival and differentiation of neurons. In this study, we first developed a retrograde trans-multisynaptic tracer PRV580 expressing the mCherry fluorescent protein based on pseudorabies virus Bartha strain to map the neural circuit of sciatic nerve. Secondly, the newly developed PRV580 was used to map the neural circuit of the recovering sciatic nerve upon treatment with NGF. Our results showed that red signals from PRV580 were observed in various brain regions. Among these regions, many areas of the pyramidal system and the extra-pyramidal system had been mapped, accounting for as much as 56.8 % of the total inputs. Furthermore, we found that NGF could significantly increase the ratio of total input (29.05 %) compared to PBS (3.65 %), indicating that NGF indeed can aid in the repair of injured sciatic nerve. These findings indicated that NGF has therapeutic ability for the treatment of peripheral nerve injuries and virus-based tracers can be used to monitor the recovery.

Senkyunolide B exhibits broad-spectrum antifungal activity against plant and human pathogenic fungi via inhibiting spore germination and destroying the mature biofilm.

BACKGROUND: Aspergillus infection seriously jeopardizes the health and safety of life of immunocompromised patients. The emergences of antifungal resistance highlight a demand to find new effective antifungal drugs. Angelica sinensis is a medicine-food herb and phthalides are its characteristic components. A few of the phthalides have been reported to display satisfactory antifungal activities against plant pathogenic fungi. However, the structure-activity relationships and antifungal action mechanism of phthalides remain to be further explored and elucidated. RESULTS: The antifungal activities of five natural phthalides and four artificial analogs were investigated, and their structure-activity relationships were preliminarily elucidated in the current study. The benzene ring moiety played an essential role in their antifungal activities; the oxygen-containing substituents on the benzene ring obviously impacted their activities, the free hydroxyl was favorable to the activity. Typical phthalide senkyunolide B (SENB) exhibited broad antifungal activities against human and plant pathogenic fungi, especially, Aspergillus fumigatus. SENB affected the spore germination and hyphae growth of Aspergillus fumigatus via down-regulating phosphatidylinositol-PKC-calcineurin axis and the expression of ENG genes. Moreover, SENB disturbed the oxidation-reduction process in Aspergillus fumigatus to destroy the mature biofilms. In vivo experiments indicated SENB significantly prolonged survival and decreased fungal burden in mouse model of invasive pulmonary aspergillosis. CONCLUSIONS: Phthalides could be considered as the valuable leads for the development of antifungal drug to cure plant and human disease. © 2023 Society of Chemical Industry.

Presence of free gallic acid and gallate moieties reduces auto-oxidative browning of epicatechin (EC) and epicatechin gallate (ECg).

Auto-oxidation of flavan-3-ols leads to browning and consequently loss of product quality during storage of ready-to-drink (RTD) green tea. The mechanisms and products of auto-oxidation of galloylated catechins, the major flavan-3-ols in green tea, are still largely unknown. Therefore, we investigated auto-oxidation of epicatechin gallate (ECg) in aqueous model systems. Oxidation products tentatively identified based on MS included δ- or γ-type dehydrodicatechins (DhC2s) as the main contributors to browning. Additionally, various colourless products were detected, including epicatechin (EC) and gallic acid (GA) from degalloylation, ether-linked ε-type DhC2s, and 6 new coupling products of ECg and GA possessing a lactone interflavanic linkage. Supported by density function theory (DFT) calculations, we provide a mechanistic explanation on how presence of gallate moieties (D-ring) and GA affect the reaction pathway. Overall, presence of gallate moieties and GA resulted in a different product profile and less intense auto-oxidative browning of ECg compared to EC.

Outcome analysis of infrapatellar fat pad partial resection or preservation in patients with anterior cruciate ligament reconstruction.

The infrapatellar fat pad (IPFP) is one of the structures surrounding the knee joint that obscures exposure in minimally arthroscopy anterior cruciate ligament reconstruction (ACLR). Most surgeons excise the partial fat pad for better exposure of the knee. However, whether removal of IPFP in ACLR remained inconclusive. The purpose of this study was to investigate clinical outcomes of IPFP preservation or resection in patients with primary hamstring-graft ACLR. A total of 104 patients were assigned to receive either IPFP-R (n = 55) or IPFP-P (n = 49). There were no significant preoperative differences between the two groups. The anterior knee pain (AKP) and the Knee Injury and Osteoarthritis Outcome Score (KOOS) in the two groups both recovered compared with those at baseline, but the IPFP-P group recovered more significantly at 3-, 6-, 12-month, and 3-, 6-month of follow-up, respectively. When assessing the KOOS subclasses using minimum perceptible clinical improvement (MPCI), patients with IPFP-R failed to make significant improvement at 3 months in the symptoms, pain and sports subsets of the KOOS. Knee-related complications were not significantly different between the two groups, while the resection group had a higher incidence. These results suggested that ACLR with primary hamstring grafts can achieve good effects whether performed with IPFP resection or preservation; however, the improvements in anterior knee pain and knee joint functions are better for the patients with IPFP preservation. Therefore, surgeons should avoid the resection of IPFP as much as possible while fully exposing the wild view to ensure the ACLR.

Discovery of antibacterial agents targeting biofilm formation: total synthesis and in vitro investigation of amycolasporins.

Three monoterpene alkaloids amycolasporin A and (±) amycolasporins B and C have been synthesized for the first time from commercially available materials in yields of 31%, 14% and 21%, respectively. Their six analogues (18, 19, 30a and 30d-30f) were synthesized through a similar protocol. Meanwhile, the antibacterial activity of all synthesized molecules was evaluated, showing different levels of bioactivity. Among them, analogue 30d was screened as the most effective antibacterial candidate against E. coli (MIC value, 12.5 µg mL-1) and S. aureus (MIC value, 12.5 µg mL-1). Further investigation showed that 30d obviously inhibited biofilm formation and disrupted the preformed biofilm of E. coli and S. aureus by promoting intracellular ROS release.

Chemical characterization, antiproliferative and antifungal activities of Clinacanthus nutans.

Clinacanthus nutans Lindau (Family: Acanthaceae) is a medicinal herb widely distributed in the tropic and subtropic areas of Asia. C. nutans is traditionally consumed as vegetable or herbal tea, as well as a folk medicine for anticancer and antifungal activities. However, to date, chemical constituent responsible for observed health beneficial effects of this medicinal plant is not clear. In the current study, 32 compounds (1-32), including three new megastigmanes (1-3) were isolated from the aerial parts of C. nutans. Their structures were elucidated on the basis of comprehensive NMR, MS, and CD spectroscopic data analysis, as well as chemical hydrolysis. Among the isolates, cycloartane triterpenoids (9, 10, and 12) displayed moderate anti-proliferative effects against HepG2 cell growth with IC50 values ranging from 9.12 to 19.89 µM. Data obtained from flow cytometry analysis and western blotting assays revealed that compounds 9 and 12 induced apoptosis of HepG2 cells by modulating the expression of proteins associated to mitochondrial-mediated apoptotic pathway. Furthermore, megastigmanes 1, 2, 7, and 8 enhanced the anti-Candida albicans activity of amphotericin B (AmB), supporting the synergistic effects between megastigmanes and AmB. This is the first report of anticancer and antifungal potential of cycloartane triterpenoids and megastigmanes in C. nutans, which shed useful insights on the relationship between C. nutans's chemical constituent and its beneficial effects to health. Findings from this study support further development of this medicinal plant for potential pharmaceutical applications.

Case Report: Disseminated Talaromyces marneffei Infection in a Patient With Chronic Mucocutaneous Candidiasis and a Novel STAT1 Gain-of-Function Mutation.

Chronic mucocutaneous candidiasis (CMC) is a disorder of recurrent or persistent chronic noninvasive symptomatic infections of the skin, nails and mucous membranes. This disorder is primarily caused by Candida albicans. Many factors, including primary immunodeficiencies, can make a host more susceptible to CMC. Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations are the most common genetic etiologies of CMC. We describe a case of CMC with disseminated Talaromyces marneffei infection caused by a new pathogenic Y287N mutation at amino acid 287 in the coiled-coiled domain of STAT1, which was identified using whole-exome sequencing. Position 287 might be a hot spot for missense mutations because several amino acid substitutions were found there. Flow cytometry suggested that the Y287N mutation might reduce the expression of IL-17 of Th17 cells in peripheral blood mononuclear cells stimulated by phorbol myristate acetate and ionomycin. The STAT1 Y287N GOF mutation may be the direct cause of recurrent cutaneous and mucosal candidiasis, including the T. marneffei infection in this patient.

Oxazolomycins produced by Streptomyces glaucus and their cytotoxic activity.

Six oxazolomycins (1-6) were isolated from the fermentation broth of a soil-borne bacterial strain, Streptomyces glaucus. The structures of the new compounds, oxazolomycins D-F (1-3) and glaucumycins A, B (6a/6b), were elucidated by detailed spectroscopic data analysis. Oxazolomycins 1, 2, 4, and 5 demonstrated weak or modest cytotoxic activities against four human cancer cell lines, with IC50 values ranging from 10.6 ± 1.7 to 89.5 ± 6.6 µM (or >100 µM). Further study showed that 4 caused S phase cell cycle arrest in SMMC7721 cells through down-regulating the protein expression of cyclin A2, CDK2. Meanwhile, 4 induced apoptosis in SMMC7721 cells through down-regulating the protein levels of Bcl-2, up-regulating the levels of Bax, and activating the cleavage of caspase-3.

A Hybrid Recommendation System for Marine Science Observation Data Based on Content and Literature Filtering.

With the development of ocean exploration technology and the rapid growth in the amount of marine science observation data, people are faced with a great challenge to identify valuable data from the massive ocean observation data. A recommendation system is an effective method to improve retrieval capabilities to help users obtain valuable data. The two most popular recommendation algorithms are collaborative filtering algorithms and content-based filtering algorithms, which may not work well for marine science observation data given the complexity of data attributes and lack of user information. In this study, an approach was proposed based on data similarity and data correlation. Data similarity was calculated by analyzing the subject, source, spatial, and temporal attributes to obtain the recommendation list. Then, data correlation was calculated based on the literature on marine science data and ranking of the recommendation list to obtain the re-rank recommendation list. The approach was tested by simulated datasets collected from multiple marine data sharing websites, and the result suggested that the proposed method exhibits better effectiveness.

Browning of Epicatechin (EC) and Epigallocatechin (EGC) by Auto-Oxidation.

Green tea catechins are well known for their health benefits. However, these compounds can easily be oxidized, resulting in brown color formation, even in the absence of active oxidative enzymes. Browning of catechin-rich beverages, such as green tea, during their shelf life is undesired. The mechanisms of auto-oxidation of catechins and the brown products formed are still largely unknown. Therefore, we studied auto-oxidative browning of epicatechin (EC) and epigallocatechin (EGC) in model systems. Products of EC and EGC auto-oxidation were analyzed by reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled to mass spectrometry (RP-UHPLC-PDA-MS). In the EC model system, 11 δ-type dehydrodicatechins (DhC2s) and 18 δ-type dehydrotricatechins (DhC3s) that were related to browning could be tentatively identified by their MS2 signature fragments. In the EGC model system, auto-oxidation led to the formation of 13 dihydro-indene-carboxylic acid derivatives and 2 theaflagallins that were related to browning. Based on the products formed, we propose mechanisms for the auto-oxidative browning of EC and EGC. Furthermore, our results indicate that dimers and oligomers that possess a combination of an extended conjugated system, fused rings, and carbonyl groups are responsible for the brown color formation in the absence of oxidative enzymes.

New antimicrobial terpenoids and phloroglucinol glucosides from Syzygium szemaoense.

Six new terpenoids (1-6) and two new phloroglucinol glucosides (7 and 8) were isolated from the extract of Syzygium szemaoense leaves. Among the isolates, compounds 1 and 2 (named syzygiumursanolides A and B) were unusual 28-norursane type triterpenoids with 19(18→17)-abeo spirocyclic skeleton. And syzygiumone B (8) was rare ascorbyl-modified phloroglucinol glucoside. Their structures were elucidated on the basis of comprehensive 1D and 2D NMR, HRESIMS spectroscopic data analysis, as well as experimental and calculated ECD spectra and acid hydrolysis. Antimicrobial bioassay revealed that syzygiumursanolide D (4) displayed the most potent antifungal activities with MIC values ranging from 6.25 to 25 µg/mL against a panel of fungi.

A novel spatial-resolution targeted metabolomics method in a single leaf of the tea plant (Camellia sinensis).

In this study, a novel spatial-resolution targeted metabolomics method was developed for a single leaf on the basis of microscale sample preparation and dansylation derivatization coupled with ultraperformance liquid chromatography-tandem mass spectrometry with a spatial resolution of 1.7 × 1.7 mm2. The practicability of this method was demonstrated by providing absolute quantitative within-leaf distribution information for 56 endogenous metabolites (including 8 catechins, 2 alkaloids, theanine, 4 theaflavins, 14 flavonol/flavone and their glycosides, 21 amino acids, and 6 phenolic acids). An application of this method in a mechanically pierced tea leaf indicated that astringent catechins, quercetin, and quercetin glycosides may be involved in the tea plant response to the wounding. In conclusion, the proposed novel method offered richest information on the within-leaf distribution of metabolites in tea to date and will be greatly helpful in understanding the defensive responses of tea plants against biotic and abiotic stresses.

Nontargeted metabolomics predicts the storage duration of white teas with 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols as marker compounds.

Storage has a dramatic influence on the chemical composition, sensory qualities, biological activity, and therefore the commercial value of white tea. In this study, the metabolites in white teas stored for 1, 3, 7, and ≥ 10 years were comprehensively compared by a nontargeted metabolomics investigation. Most metabolites, including catechins, flavonol/flavone glycosides, amino acids, nucleosides, organic acids, aroma precursors, lipids, and carbohydrates, decreased with increasing storage duration, while 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) and pyroglutamic acid increased. The absolute quantifications of 24 storage-related compounds combined with linear regression analysis showed that a panel of 5 indexes based on EPSFs has a good predictive ability for the storage duration of white teas (correlation coefficients were 0.9294 and 0.8812 in the model and test sets, respectively). The errors between the predicted and the actual storage durations ranged from -1.75 to 1.84 years for the white teas stored for <10 years.

Identification and quantification of key odorants in the world's four most famous black teas.

Keemun, Assam, Darjeeling and Ceylon black teas are honored as the world's four most famous black teas, and their excellent aroma qualities are well received by people around the world. In this study, aroma components in these four types of teas were analyzed by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) and gas chromatography-olfactometry (GC-O) technologies. A total of 42 aroma-active compounds were ultimately identified, especially benzeneacetaldehyde, geraniol, (Z)-3-hexen-1-yl hexanoate, trans-ß-ionone, cis-linalool oxide (pyranoid), hotrienol, and methyl salicylate presented the strongest aroma strengths with pleasant scents in all tested teas. The quantification results indicated that 19 compounds including (Z)-3-hexenol, 1-octen-3-ol, linalool, phenylethyl alcohol, hexanal, benzeneacetaldehyde, limonene, heptanoic acid, (Z)-3-hexen-1-ol, acetate, benzyl alcohol, trans-linalool oxide (furanoid), hotrienol, 1-octen-3-one, 2-nonanone, (E)-2-octenal, nonanal, ß-myrcene, 2-pentylfuran, and methylpyrazine were identified as the key compounds with odor activity values (OAVs) higher than 1.0 in the world's four most famous black teas. Notably, the comparison of GC-O and OAV calculation results showed that methyl salicylate (Ceylon), (E)-2-octenal (Assam), benzeneacetaldehyde (Keemun) and linalool and trans-linalool oxide (furanoid) (Darjeeling) might be the most definitive odorants in the corresponding tea categories.

Variation patterns in the content of glycosides during green tea manufacturing by a modification-specific metabolomics approach: Enzymatic reaction promoting an increase in the glycosidically bound volatiles at the pan firing stage.

The glycosides are presumed to influence the quality of green tea but the molecular mechanism behind remains unclear. To elucidate the contribution of glycosides to the flavor formation of green tea, changes of both glycosidically bound non-volatiles (GBNVs) and glycosidically bound volatiles (GBVs) during the manufacturing of green tea were investigated using a modification-specific metabolomics method. A total of 64 glycosides (47 GBNVs and 17 GBVs) were identified and their contents mainly changed during the pan firing and drying stages of green tea manufacturing. Notably, the contents of GBVs significantly increased by 1.12-4.46-fold during pan firing. Correlation analysis showed that the GBVs contents were negatively related to the contents of volatiles and glucose. Model experiments revealed that enzymatic synthesis contributed to the increase in the content of GBVs during the pan firing. This comprehensive study on the glycosides changes revealed the molecular bases for GBVs increments during the pan firing.

Aroma formation and dynamic changes during white tea processing.

The formation of and dynamic changes in aroma during white tea processing have not previously been systematically investigated. In this study, advanced comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry was employed to investigate the mechanism of white tea aroma formation. A total of 172 volatiles were identified and mainly comprising endogenous volatiles, which displayed diverse change trends during the withering period. In this process, free aroma precursor amino acids and glycosidically bound volatiles (GBVs) were found to contribute to the formation of white tea aroma, with the differential expression of aroma-related key genes accounting for various accumulation of endogenous volatiles and GBVs. In addition, the drying was also shown to play an important role in the formation of white tea aroma. Our study provides the first characterization of white tea aroma formation and establishes a theoretical basis for quality control during white tea processing operations.

[Prognostic Value of Morphology and Hans Classification in Diffuse Large B Cell Lymphoma].

OBJECTIVE: To investigate the prognostic value of morphology and Hans classification in diffuse large B cell lymphoma（DLBCL）. METHODS: Clinical data of 249 patients diagnosed with DLBCL in our hospital and Hangzhou Xixi hospital during Jan 2006 to Dec 2016 were analyzed retrospectively. These patients were classified into 3 groups: immunoblastic variant（IB） group, centroblastic variant（CB） group and others group according to the cell morphology. And DLBCL was also divided into GCB（germinal center B-cell-like）or non-GCB（non-germinal center B-cell-like） group by analyzing the expression of CD10, BCL6 and MUM1 (GCB: CD10 +,BCL6+-,MUM1+-/CD10-,BCL6+,MUM1-；non-GCB：CD10-,BCL6-,MUM1+-/CD10-,BCL6+,MUM1+). RESULTS: The univariate analysis displayed that the age，LDH level，IPI，IB，non-GCB，B-symptoms and rituximab all could influence the OS and EFS, the CR rate of CB subtype patients was significantly higher than that of the patients with IB subtype （68.3% vs 38.9%)(P=0.02）. IB subtype was the in dependent prognostic factor for both EFS and OS in the whole study. In multivariate analysis, IPI and IB were the independent prognostic factors for OS and EFS. IB subtype was also an independent prognostic factor in EFS and OS with or without rituximab. The expression of BCL2 and BCL6 was related with prognosis in R-CHOP, but not in CHOP treated patients. Other markers (CD5, CD10, IRF4/MUM1, HLA-DR and Ki-67 proliferation index) were not of the significant prognostic value for DLBCL. When accepted rituximab, the GCB and non-GCB were not different significantly for prognosis. However, the non-GCB group showed a poor prognosis without using rituximab （EFS P=0.020；OS P=0.020）. Multivariate Cox models showed that OS and EFS were not significantly different between GCB and non-GCB group, however, the IB subtype had a very significantly poor prognosis in OS and EFS (P=0.001, P=0.002). When the analysis was restricted to DLBCL with CB morphology only, no prognostic value was observed in Hans classification. CONCLUSION: The subtype of immunoblast is a major risk factor in patients treated with CHOP or R-CHOP. There is a significant association between the Hans classification and the morphologic subclassification. Results of this study have supplemented the data for the prognostic factor of DLBCL and demonstrated that the cytomorphologic diagnosis can be reproducible.

Metabolomics Investigation Reveals That 8-C N-Ethyl-2-pyrrolidinone-Substituted Flavan-3-ols Are Potential Marker Compounds of Stored White Teas.

White teas of different stored ages have varied flavor, bioactivity, and commercial value. In this study, a liquid chromatography-mass spectrometry-based metabolomics investigation revealed that there are distinct differences among the compound patterns of Baihaoyinzhen (BHYZ) and Baimudan (BMD) white teas with various storage durations. The levels of flavan-3-ols, procyanidins, theasinensins, theaflavins, flavonol- O-glycosides, flavone- C-glycosides, and most of the amino acids were reduced after long-term (>4 years) storage. More importantly, 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs), including seven novel compounds discovered in white teas for the first time, were formed from theanine and flavan-3-ols during storage, and their contents were positively correlated with the storage duration. These findings were further confirmed by the linearly increasing formation of EPSFs in reaction solution and BMD white teas stored in an environment-controlled cabinet. In conclusion, EPSFs were detected in white teas for the first time and were discovered as marker compounds and potential indicators for long-term storage of white tea.