Nitrogen and sulfur for phosphorus: Lipidome adaptation of anaerobic sulfate-reducing bacteria in phosphorus-deprived conditions.

Understanding how microbial lipidomes adapt to environmental and nutrient stress is crucial for comprehending microbial survival and functionality. Certain anaerobic bacteria can synthesize glycerolipids with ether/ester bonds, yet the complexities of their lipidome remodeling under varying physicochemical and nutritional conditions remain largely unexplored. In this study, we thoroughly examined the lipidome adaptations of Desulfatibacillum alkenivorans strain PF2803T, a mesophilic anaerobic sulfate-reducing bacterium known for its high proportions of alkylglycerol ether lipids in its membrane, under various cultivation conditions including temperature, pH, salinity, and ammonium and phosphorous concentrations. Employing an extensive analytical and computational lipidomic methodology, we identified an assemblage of nearly 400 distinct lipids, including a range of glycerol ether/ester lipids with various polar head groups. Information theory-based analysis revealed that temperature fluctuations and phosphate scarcity profoundly influenced the lipidome's composition, leading to an enhanced diversity and specificity of novel lipids. Notably, phosphorous limitation led to the biosynthesis of novel glucuronosylglycerols and sulfur-containing aminolipids, termed butyramide cysteine glycerols, featuring various ether/ester bonds. This suggests a novel adaptive strategy for anaerobic heterotrophs to thrive under phosphorus-depleted conditions, characterized by a diverse array of nitrogen- and sulfur-containing polar head groups, moving beyond a reliance on conventional nonphospholipid types.

Network propagation for GWAS analysis: a practical guide to leveraging molecular networks for disease gene discovery.

MOTIVATION: Genome-wide association studies (GWAS) have enabled large-scale analysis of the role of genetic variants in human disease. Despite impressive methodological advances, subsequent clinical interpretation and application remains challenging when GWAS suffer from a lack of statistical power. In recent years, however, the use of information diffusion algorithms with molecular networks has led to fruitful insights on disease genes. RESULTS: We present an overview of the design choices and pitfalls that prove crucial in the application of network propagation methods to GWAS summary statistics. We highlight general trends from the literature, and present benchmark experiments to expand on these insights selecting as case study three diseases and five molecular networks. We verify that the use of gene-level scores based on GWAS P-values offers advantages over the selection of a set of 'seed' disease genes not weighted by the associated P-values if the GWAS summary statistics are of sufficient quality. Beyond that, the size and the density of the networks prove to be important factors for consideration. Finally, we explore several ensemble methods and show that combining multiple networks may improve the network propagation approach.

Sustained Spread of HIV-1 CRF55_01B in Its Place of Initial Origin: Dynamics and Hotspots.

BACKGROUND: Shenzhen, a city with a substantial mobile population, was identified as the first discovered region of HIV-1 CRF55_01B and epicenter of its severe epidemic. During the implementation of venue-based behavioral interventions and the "treat-all" policy, discerning the spread patterns and transmission hotspots of CRF55_01B is imperative. METHODS: In this study, 1,450 partial pol sequences, with demographic information, were collected from all newly diagnosed CRF55_01B infections in Shenzhen from 2008 to 2020. Molecular networks were constructed using the maximum likelihood and time-resolve phylogenies. Transmission rates, effective reproduction numbers (Re) of clusters and viral dispersal were evaluated using Bayesian inference. RESULTS: In total, 526 sequences formed 114 clusters, including seven large clusters. The status and size of clusters were strongly correlated with age, ethnicity, occupation and CD4+ T cell counts. The transmission rates of clusters were significantly higher than the national epidemic estimate. Four large clusters had Re exceeding 1 at the end of sampling period. Immigrants from Guangdong and Hunan, along with local residents, were identified as the transmission hubs, with heterosexual men being the main source and MSM being the main destination. The virus exhibited a high movement frequency from individuals aged 30-49 years toward diverse age groups. CONCLUSIONS: This study demonstrated the hidden CRF55_01B transmissions continued despite current combined interventions in Shenzhen, and special at-risk individuals susceptible to infection or transmission were identified, potentially serving as targets for more effective prevention and control of the local epidemic, thereby mitigating cross-regional spread nationwide due to population migration.

The Role of Alkyl Chains in the Thermoresponse of Supramolecular Network.

Supramolecular materials provide a pathway for achieving precise, highly ordered structures while exhibiting remarkable response to external stimuli, a characteristic not commonly found in covalently bonded materials. The design of self-assembled materials, where properties could be predicted/design from chemical nature of the individual building blocks, hinges upon our ability to relate macroscopic properties to individual building blocks - a feat which has thus far remained elusive. Here, a design approach is demonstrated to chemically engineer the thermal expansion coefficient of 2D supramolecular networks by over an order of magnitude (\boldmath 120 to \boldmath 1000 × 10-6 K-1). This systematic study provides a clear pathway on how to carefully design the thermal expansion coefficient of a 2D molecular assembly. Specifically, a linear relation has been identified between the length of decorating alkyl chains and the thermal expansion coefficient. Counter-intuitively, the shorter the chains the larger is the thermal expansion coefficient. This precise control over thermo-mechanical properties marks a significant leap forward in the de-novo design of advanced 2D materials. The possibility to chemically engineer their thermo-mechanical properties holds promise for innovations in sensors, actuators, and responsive materials across diverse fields.

Integrating single-cell datasets with ambiguous batch information by incorporating molecular network features.

With the rapid development of single-cell sequencing techniques, several large-scale cell atlas projects have been launched across the world. However, it is still challenging to integrate single-cell RNA-seq (scRNA-seq) datasets with diverse tissue sources, developmental stages and/or few overlaps, due to the ambiguity in determining the batch information, which is particularly important for current batch-effect correction methods. Here, we present SCORE, a simple network-based integration methodology, which incorporates curated molecular network features to infer cellular states and generate a unified workflow for integrating scRNA-seq datasets. Validating on real single-cell datasets, we showed that regardless of batch information, SCORE outperforms existing methods in accuracy, robustness, scalability and data integration.

The origin and spread of HIV-1 CRF59_01B epidemic in China: A molecular network and phylogeographic analysis.

Human immunodeficiency virus type 1 CRF59_01B, identified in China in 2013, has been detected nationwide, exhibiting notably high prevalence in Guangzhou and its vicinity. This study aimed to unravel its origin and migration. A data set was established, incorporating all available CRF59_01B pol gene sequences and their metadata from Guangzhou and the public database. Bayesian phylogeographic analysis demonstrated that CRF59_01B originated in Shenzhen, the neighboring city of Guangzhou, around 1998 with posterior probability of 0.937. Molecular network analysis detected 1131 transmission links and showed a remarkably high clustering rate (78.9%). Substantial inter-city transmissions (26.5%, 300/1131) were observed between Shenzhen and Guangzhou while inter-region transmissions linked Guangzhou with South (46) and Southwest (64) China. The centre of Guangzhou was the hub of CRF59_01B transmission, including the inflow from Shenzhen (3.57 events/year) and outflow to the outskirts of Guangzhou (>2 events/year). The large-scale analysis revealed significant migration from Shenzhen to Guangzhou (5.08 events/year) and North China (0.59 events/year), and spread from Guangzhou to Central (0.47 events/year), East (0.42 events/year), South (0.76 events/year), Southwest China (0.76 events/year) and Shenzhen (1.89 events/year). Shenzhen and Guangzhou served as the origin and the hub of CRF59_01B circulation, emphasizing inter-city cooperation and data sharing to confine its nationwide diffusion.

Multilevel Dynamic System as Molecular Morning-After Timer.

Systems chemistry aims to develop molecular systems that display emerging properties arising from their network and absent in their individual constituents. Employing reversible chemistry under thermodynamic control represents a valuable tool for generating dynamic combinatorial libraries of interconverting molecules, which may exhibit intriguing collective behaviour. A simple dynamic combinatorial library was prepared using dithioacetal / thiol / disulfide exchanges. Because of the relative reactivities of these reversible reactions, the library constitutes a two-layer dynamic system with one layer active in an acid medium (thiol/dithioacetal exchange) and one layer active in a basic medium (thiol/disulfide exchange). This property enables the system to respond to momentary changes in acidity of the medium by activating different network regions, channeling some building blocks from one layer to another through shared thiol reagents (nodes). This momentaneous change in wiring affects the final steady state composition of the library, measured the next day, even though the event that caused it vanishes without leaving any residue. Therefore, the final composition of this dynamic system provides information about this transient past perturbation in the environment such as: when it occurred, how long it was, or how intense it was.

Discovery of Unusual Ajmaline-Macroline Type Bisindole Alkaloids from Alstonia macrophylla by Building Blocks-Based Molecular Networking.

Three unusual ajmaline-macroline type bisindole alkaloids, alsmaphylines A-C, together with their postulated biogenetic precursors, were isolated from the stem barks and leaves of Alstonia macrophylla via the building blocks-based molecular network (BBMN) strategy. Alsmaphyline A represents a rare ajmaline-macroline type bisindole alkaloid with an S-shape polycyclic ring system. Alsmaphylines B and C are two novel ajmaline-macroline type bisindole alkaloids with N-1-C-21' linkages, and the former possesses an unconventional stacked conformation due to the presence of intramolecular noncovalent interactions. The chemical structures including absolute configurations of alsmaphylines A-C were established by comprehensive spectroscopic analyses, electronic circular dichroism (ECD) calculations, and single-crystal X-ray crystallography. In addition, a plausible biosynthetic pathway of these bisindole alkaloids as well as their ability to promote the protein synthesis on HT22 cells were discussed.

Adsorption of Prochiral Solvent Molecules by Surface-Confined Chiral Supramolecular Assemblies: How Solvent Impacts on-Surface Chirality.

The understanding of supramolecular chirality in self-assembled molecular networks (SAMNs) on surfaces generates a lot of interest because of its relation to the production of chiral sensors, reactors, and catalysts. We herein report the adsorption of a prochiral solvent molecule in porous SAMNs formed by a chiral dehydrobenzo[12]annulene (cDBA) derivative. Through the prochirality recognition of a solvent molecule, the supramolecular chirality of the SAMN is switched: the cDBA exclusively forms a counter-clockwise pore through co-adsorption of the solvent molecule in prochiral 1,2,4-trichlorobenzene, while in 1-phenyloctane it produces the opposite chiral, clockwise pore. The prochirality recognition of the solvent molecule in the chiral SAMN pores is attributed to the adaptable conformational changes of the chiral chains of the cDBA molecule.

Identification of the hub genes in polycystic ovary syndrome based on disease-associated molecule network.

Revealing the key genes involved in polycystic ovary syndrome (PCOS) and elucidating its pathogenic mechanism is of extreme importance for the development of targeted clinical therapy for PCOS. Investigating disease by integrating several associated and interacting molecules in biological systems will make it possible to discover new pathogenic genes. In this study, an integrative disease-associated molecule network, combining protein-protein interactions and protein-metabolites interactions (PPMI) network was constructed based on the PCOS-associated genes and metabolites systematically collected. This new PPMI strategy identified several potential PCOS-associated genes, which have unreported in previous publications. Moreover, the systematic analysis of five benchmarks data sets indicated the DERL1 was identified as downregulated in PCOS granulosa cell and has good classification performance between PCOS patients and healthy controls. CCR2 and DVL3 were upregulated in PCOS adipose tissues and have good classification performance. The expression of novel gene FXR2 identified in this study is significantly increased in ovarian granulosa cells of PCOS patients compared with controls via quantitative analysis. Our study uncovers substantial differences in the PCOS-specific tissue and provides a plethora of information on dysregulated genes and metabolites that are linked to PCOS. This knowledgebase could have the potential to benefit the scientific and clinical community. In sum, the identification of novel gene associated with PCOS provides valuable insights into the underlying molecular mechanisms of PCOS and could potentially lead to the development of new diagnostic and therapeutic strategies.

Combining molecular transmission network analysis and spatial epidemiology to reveal HIV-1 transmission pattern among the older people in Nanjing, China.

BACKGROUND: In China, the problem of HIV infection among the older people has become increasingly prominent. This study aimed to analyze the pattern and influencing factors of HIV transmission based on a genomic and spatial epidemiological analysis among this population. METHODS: A total of 432 older people who were aged ≥ 50 years, newly diagnosed with HIV-1 between January 2018 and December 2021 and without a history of ART were enrolled. HIV-1 pol gene sequence was obtained by viral RNA extraction and nested PCR. The molecular transmission network was constructed using HIV-TRACE and the spatial distribution analyses were performed in ArcGIS. The multivariate logistic regression analysis was performed to analyze the factors associated with clustering. RESULTS: A total of 382 sequences were successfully sequenced, of which CRF07_BC (52.3%), CRF01_AE (32.5%), and CRF08_BC (6.8%) were the main HIV-1 strains. A total of 176 sequences entered the molecular network, with a clustering rate of 46.1%. Impressively, the clustering rate among older people infected through commercial heterosexual contact was as high as 61.7% and three female sex workers (FSWs) were observed in the network. The individuals who were aged ≥ 60 years and transmitted the virus by commercial heterosexual contact had a higher clustering rate, while those who were retirees or engaged other occupations and with higher education degree were less likely to cluster. There was a positive spatial correlation of clustering rate (Global Moran I = 0.206, P < 0.001) at the town level and the highly aggregated regions were mainly distributed in rural area. We determined three large clusters which mainly spread in the intra-region of certain towns in rural areas. Notably, 54.5% of cases in large clusters were transmitted through commercial heterosexual contact. CONCLUSIONS: Our joint analysis of molecular and spatial epidemiology effectively revealed the spatial aggregation of HIV transmission and highlighted that towns of high aggregation were mainly located in rural area. Also, we found vital role of commercial heterosexual contact in HIV transmission among older people. Therefore, health resources should be directed towards highly aggregated rural areas and prevention strategy should take critical persons as entry points.

Insights into the molecular network characteristics of major HIV-1 subtypes in developed Eastern China: a study based on comprehensive molecular surveillance data.

PURPOSE: This study aimed to conduct a comprehensive molecular epidemiology study of major HIV-1 subtypes in developed Eastern China (Zhejiang Province). METHODS: Plasma samples and epidemiological information were collected from 4180 newly diagnosed HIV-1 positive patients in Zhejiang Province in 2021. Pol sequences were obtained to determine the subtypes via multiple analytical tools. HIV-1 molecular networks were constructed on the basis of genetic distances to analyze transmission patterns among major subtypes. Furthermore, the birth-death skyline (BDSKY) model was utilized to estimate the transmission risks associated with large clusters (LCs). RESULTS: In 4180 patients, 3699 (88.49%) pol sequences were successfully obtained and classified into four subtype groups. In the networks under an optimal genetic distance of 0.01 substitutions/site, the majority of links (74.52%, 1383/1856) involved individuals within the same city, highlighting the predominant role of local transmission in driving the HIV-1 epidemic. In the CRF07_BC, CRF01_AE, and others/URFs networks, men who have sex with men (MSM) were the primary sexual transmission population, with the younger MSM group (< 30 years old) exhibiting higher linkage frequencies. Within the CRF08_BC network, 93.98% of individuals were infected primarily through heterosexual contact and had a significantly greater risk of localized clustering than other subtypes did. Moreover, fifteen identified LCs were predominantly transmitted through commercial heterosexual contact (CHC), exhibiting localized clustering and high potential for sustained diffusion. CONCLUSIONS: Overall, our findings reveal a diverse and heterogeneous distribution of HIV-1 subtypes in Zhejiang Province, with noticeable variations in hotspots across different geographic areas and populations.

Deciphering Microbe-Mediated Dissolved Organic Matter Reactome in Wastewater Treatment Plants Using Directed Paired Mass Distance.

Understanding the reaction mechanism of dissolved organic matter (DOM) during wastewater biotreatment is crucial for optimal DOM control. Here, we develop a directed paired mass distance (dPMD) method that constructs a molecular network displaying the reaction pathways of DOM. It couples direction inference and PMD analysis to extract the substrate-product relationships and delta masses of potentially paired reactants directly from sequential mass spectrometry data without formula assignment. Using this method, we analyze the influent and effluent samples from the bioprocesses of 12 wastewater treatment plants (WWTPs) and build a dPMD network to characterize the core reactome of DOM. The network shows that the first step of the transformation triggers reaction cascades that diversify the DOM, but the highly overlapped subsequent reaction pathways result in similar effluent DOM compositions across WWTPs despite varied influents. Mass changes exhibit consistent gain/loss preferences (e.g., +3.995 and -16.031) but different occurrences across WWTPs. Combined with genome-centric metatranscriptomics, we reveal the associations among dPMDs, enzymes, and microbes. Most enzymes are involved in oxygenation, (de)hydrogenation, demethylation, and hydration-related reactions but with different target substrates and expressed by various taxa, as exemplified by Proteobacteria, Actinobacteria, and Nitrospirae. Therefore, a functionally diverse community is pivotal for advanced DOM degradation.

The origin, dissemination, and molecular networks of HIV-1 CRF65_cpx strain in Hainan Island, China.

BACKGROUND: HIV-1 CRF65_cpx strain carries drug-resistant mutations, which raises concerns about its potential for causing virologic failure. The CRF65_cpx ranks as the fourth most prevalent on Hainan Island, China. However, the origin and molecular epidemiology of CRF65_cpx strains in this area remain unclear. This study aims to estimate the spatial origins and dissemination patterns of HIV-1 CRF65_cpx in this specific region. METHODS: Between 2018 and 2021, a total of 58 pol sequences of the CRF65_cpx were collected from HIV-positive patients on Hainan Island. The available CRF65_cpx pol sequences from public databases were compiled. The HIV-TRACE tool was used to construct transmission networks. The evolutionary history of the introduction and dissemination of HIV-1 CRF65_cpx on Hainan Island were analyzed using phylogenetic analysis and the Bayesian coalescent-based approach. RESULTS: Among the 58 participants, 89.66% were men who have sex with men (MSM). The median age was 25 years, and 43.10% of the individuals had a college degree or above. The results indicated that 39 (67.24%) sequences were interconnected within a single transmission network. A consistent expansion was evident from 2019 to 2021, with an incremental annual addition of four sequences into the networks. Phylodynamic analyses showed that the CRF65_cpx on Hainan Island originated from Beijing (Bayes factor, BF = 17.4), with transmission among MSM on Hainan Island in 2013.2 (95%HPD: 2012.4, 2019.5), subsequently leading to an outbreak. Haikou was the local center of the CRF65_cpx epidemic. This strain propagated from Haikou to other locations, including Sanya (BF > 1000), Danzhou (BF = 299.3), Chengmai (BF = 27.0) and Tunchang (BF = 16.3). The analyses of the viral migration patterns between age subgroups and risk subgroups revealed that the viral migration directions were from "25-40 years old" to "17-24 years old" (BF = 14.6) and to "over 40 years old" (BF = 17.6), and from MSM to heterosexuals (BF > 1000) on Hainan Island. CONCLUSION: Our analyses elucidate the transmission dynamics of CRF65_cpx strain on Hainan Island. Haikou is identified as the potential hotspot for CRF65_cpx transmission, with middle-aged MSM identified as the key population. These findings suggest that targeted interventions in hotspots and key populations may be more effective in controlling the HIV epidemic.

How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent.

Pharmaceuticals and their human metabolites are contaminants of emerging concern in the aquatic environment. Most monitoring studies focus on a limited set of parent compounds and even fewer metabolites. However, more than 50% of the most consumed pharmaceuticals are excreted in higher amounts as metabolites than as parents, as confirmed by a literature analysis within this study. Hence, we applied a wide-scope suspect screening approach to identify human pharmaceutical metabolites in wastewater influent from three Swiss treatment plants. Based on consumption amounts and human metabolism data, a suspect list comprising 268 parent compounds and over 1500 metabolites was compiled. Online solid phase extraction combined with liquid chromatography coupled to high-resolution tandem mass spectrometry was used to analyze the samples. Data processing, annotation, and structure elucidation were achieved with various tools, including molecular networking as well as SIRIUS/CSI:FingerID and MetFrag for MS2 spectra rationalization. We confirmed 37 metabolites with reference standards and 16 by human liver S9 incubation experiments. More than 25 metabolites were detected for the first time in influent wastewater. Semiquantification with MS2Quant showed that metabolite to parent concentration ratios were generally lower compared to literature expectations, probably due to further metabolite transformation in the sewer system or limitations in the metabolite detection. Nonetheless, metabolites pose a large fraction to the total pharmaceutical contribution in wastewater, highlighting the need for metabolite inclusion in chemical risk assessment.

Cultivable epiphytic bacteria of the Chlorophyta Ulva sp.: diversity, antibacterial, and biofilm-modulating activities.

AIMS: Macroalgae harbor a rich epiphytic microbiota that plays a crucial role in algal morphogenesis and defense mechanisms. This study aims to isolate epiphytic cultivable microbiota from Ulva sp. surfaces. Various culture media were employed to evaluate a wide range of cultivable microbiota. Our objective was to assess the antibacterial and biofilm-modulating activities of supernatants from isolated bacteria. METHODS AND RESULTS: Sixty-nine bacterial isolates from Ulva sp. were identified based on 16S rRNA gene sequencing. Their antibacterial activity and biofilm modulation potential were screened against three target marine bacteria: 45%, mostly affiliated with Gammaproteobacteria and mainly grown on diluted R2A medium (R2Ad), showed strong antibacterial activity, while 18% had a significant impact on biofilm modulation. Molecular network analysis was carried out on four bioactive bacterial supernatants, revealing new molecules potentially responsible for their activities. CONCLUSION: R2Ad offered the greatest diversity and proportion of active isolates. The molecular network approach holds promise for both identifying bacterial isolates based on their molecular production and characterizing antibacterial and biofilm-modulating activities.

A ternary correlation multi-symptom network strategy based on in vivo chemical profile identification and metabolomics to explore the molecular basis of Ephedra herb against viral pneumonia.

Ephedra herb (EH), an important medicine prescribed in herbal formulas by Traditional Chinese Medicine practitioners, has been widely used in the treatment of viral pneumonia in China. However, the molecular basis of EH in viral pneumonia remains unclear. In this study, a ternary correlation multi-symptom network strategy was established based on in vivo chemical profile identification and metabolomics to explore the molecular basis of EH against viral pneumonia. Results showed that 143 compounds of EH and 70 prototype components were identified in vivo. EH could reduce alveolar-capillary barrier disruption in rats with viral pneumonia and significantly downregulate the expression of inflammatory factors and bronchoalveolar lavage fluid. Plasma metabolomics revealed that EH may be involved in the regulation of arachidonic acid, tryptophan, tyrosine, nicotinate, and nicotinamide metabolism. The multi-symptom network showed that 12 compounds have an integral function in the treatment of viral pneumonia by intervening in many pathways related to viruses, immunity and inflammation, and lung injury. Further verification demonstrated that sinapic acid and frambinone can regulate the expression of related genes. It has been shown to be a promising representative of the pharmacological constituents of ephedra.

Streptomyces-Fungus Co-Culture Enhances the Production of Borrelidin and Analogs: A Genomic and Metabolomic Approach.

The marine Streptomyces harbor numerous biosynthetic gene clusters (BGCs) with exploitable potential. However, many secondary metabolites cannot be produced under laboratory conditions. Co-culture strategies of marine microorganisms have yielded novel natural products with diverse biological activities. In this study, we explored the metabolic profiles of co-cultures involving Streptomyces sp. 2-85 and Cladosporium sp. 3-22-derived from marine sponges. Combining Global Natural Products Social (GNPS) Molecular Networking analysis with natural product database mining, 35 potential antimicrobial metabolites annotated were detected, 19 of which were exclusive to the co-culture, with a significant increase in production. Notably, the Streptomyces-Fungus interaction led to the increased production of borrelidin and the discovery of several analogs via molecular networking. In this study, borrelidin was first applied to combat Saprolegnia parasitica, which caused saprolegniosis in aquaculture. We noted its superior inhibitory effects on mycelial growth with an EC50 of 0.004 mg/mL and on spore germination with an EC50 of 0.005 mg/mL compared to the commercial fungicide, preliminarily identifying threonyl-tRNA synthetase as its target. Further analysis of the associated gene clusters revealed an incomplete synthesis pathway with missing malonyl-CoA units for condensation within this strain, hinting at the presence of potential compensatory pathways. In conclusion, our findings shed light on the metabolic changes of marine Streptomyces and fungi in co-culture, propose the potential of borrelidin in the control of aquatic diseases, and present new prospects for antifungal applications.

Metabolomics-Guided Discovery of New Dimeric Xanthones from Co-Cultures of Mangrove Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11.

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool for the activation of biosynthetic gene clusters (BGCs) to generate new metabolites, as well as to increase the yield of respective target metabolites. As part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, we selected the co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophyted in mangrove Rhizophora mangle considering the impart of the taxonomic criteria and ecological data. The competition interaction of the two strains was investigated through morphology observation and scanning electron microscopy (SEM), and it was found that the mycelia of the DHS-48 and DHS-11 compacted and tangled with each other with an interwoven pattern in the co-culture system. A new approach that integrates HPLC chromatogram, 1HNMR spectroscopy, UPLC-MS-PCA, and molecular networking enabled the targeted isolation of the induced metabolites, including three new dimeric xanthones phomoxanthones L-N (1-3), along with six known analogs (4-9). Their planar structures were elucidated by an analysis of their HRMS, MS/MS, and NMR spectroscopic data and the absolute configurations based on ECD calculations. These metabolites showed broad cytotoxic activity against the cancer cells assessed, of which compounds 7-9 displayed significant cytotoxicity towards human liver cells HepG-2 with IC50 values ranging from 4.83 µM to 12.06 µM. Compounds 1-6 exhibited weak immunosuppressive activity against the proliferation of ConA-induced (T-cell) and LPS-induced (B-cell) murine splenic lymphocytes. Therefore, combining co-cultivation with a metabolomics-guided strategy as a discovery tool will be implemented as a systematic strategy for the quick discovery of target bioactive compounds.

The Impact of the Culture Regime on the Metabolome and Anti-Phytopathogenic Activity of Marine Fungal Co-Cultures.

Co-cultivation, coupled with the OSMAC approach, is considered an efficient method for expanding microbial chemical diversity through the activation of cryptic biosynthetic gene clusters (BGCs). As part of our project aiming to discover new fungal metabolites for crop protection, we previously reported five polyketides, the macrolides dendrodolides E (1) and N (2), the azaphilones spiciferinone (3) and 8α-hydroxy-spiciferinone (4), and the bis-naphtho-γ-pyrone cephalochromin (5) from the solid Potato Dextrose Agar (PDA) co-culture of two marine sediment-derived fungi, Plenodomus influorescens and Pyrenochaeta nobilis. However, some of the purified metabolites could not be tested due to their minute quantities. Here we cultivated these fungi (both axenic and co-cultures) in liquid regime using three different media, Potato Dextrose Broth (PDB), Sabouraud Dextrose Broth (SDB), and Czapek-Dox Broth (CDB), with or without shaking. The aim was to determine the most ideal co-cultivation conditions to enhance the titers of the previously isolated compounds and to produce extracts with stronger anti-phytopathogenic activity as a basis for future upscaled fermentation. Comparative metabolomics by UPLC-MS/MS-based molecular networking and manual dereplication was employed for chemical profiling and compound annotations. Liquid co-cultivation in PDB under shaking led to the strongest activity against the phytopathogen Phytophthora infestans. Except for compound 1, all target compounds were detected in the co-culture in PDB. Compounds 2 and 5 were produced in lower titers, whereas the azaphilones (3 and 4) were overexpressed in PDB compared to PDA. Notably, liquid PDB co-cultures contained meroterpenoids and depside clusters that were absent in the solid PDA co-cultures. This study demonstrates the importance of culture regime in BGC regulation and chemical diversity of fungal strains in co-culture studies.