Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time (Wu et al., 2007). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO2 incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A.Figure 1. Overview of seahorse experiment Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.

Microorganisms play essential roles in the health and resilience of cnidarians. Understanding the factors influencing cnidarian microbiomes requires cross study comparisons, yet the plethora of protocols used hampers dataset integration. We unify 16S rRNA gene sequences from cnidarian microbiome studies under a single analysis pipeline. We reprocess 12,010 cnidarian microbiome samples from 186 studies, alongside 3,388 poriferan, 370 seawater samples, and 245 cultured Symbiodiniaceae, unifying ~6.5 billion sequence reads. Samples are partitioned by hypervariable region and sequencing platform to reduce sequencing variability. This systematic review uncovers an incredible diversity of 86 archaeal and bacterial phyla associated with Cnidaria, and highlights key bacteria hosted across host sub-phylum, depth, and microhabitat. Shallow (< 30 m) water Alcyonacea and Actinaria are characterized by highly shared and relatively abundant microbial communities, unlike Scleractinia and most deeper cnidarians. Utilizing the V4 region, we find that cnidarian microbial composition, richness, diversity, and structure are primarily influenced by host phylogeny, sampling depth, and ocean body, followed by microhabitat and sampling date. We identify host and geographical generalist and specific Endozoicomonas clades within Cnidaria and Porifera. This systematic review forms a framework for understanding factors governing cnidarian microbiomes and creates a baseline for assessing stress associated dysbiosis.

Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. Aspergillus species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs).

Fusarium graminearum, the primary cause of Fusarium head blight (FHB) in small-grain cereals, demonstrates remarkably variable levels of aggressiveness in its host, producing different infection dynamics and contrasted symptom severity. While the secreted proteins, including effectors, are thought to be one of the essential components of aggressiveness, our knowledge of the intra-species genomic diversity of F. graminearum is still limited. In this work, we sequenced eight European F. graminearum strains of contrasting aggressiveness to characterize their respective genome structure, their gene content and to delineate their specificities. By combining the available sequences of 12 other F. graminearum strains, we outlined a reference pangenome that expands the repertoire of the known genes in the reference PH-1 genome by 32%, including nearly 21,000 non-redundant sequences and gathering a common base of 9250 conserved core-genes. More than 1000 genes with high non-synonymous mutation rates may be under diverse selection, especially regarding the trichothecene biosynthesis gene cluster. About 900 secreted protein clusters (SPCs) have been described. Mostly localized in the fast sub-genome of F. graminearum supposed to evolve rapidly to promote adaptation and rapid responses to the host’s infection, these SPCs gather a range of putative proteinaceous effectors systematically found in the core secretome, with the chloroplast and the plant nucleus as the main predicted targets in the host cell. This work describes new knowledge on the intra-species diversity in F. graminearum and emphasizes putative determinants of aggressiveness, providing a wealth of new candidate genes potentially involved in the Fusarium head blight disease.

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil‐dwelling Streptomyces sp. (CL12‐4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans ‐fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher's analysis, J ‐based coupling analysis, and computations based on sparse NMR‐derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring‐flipped bicyclic conformations with a rotational barrier of ≈16 kcal mol ⁻¹ in solution. The diterpenes exhibit moderate antibacterial activity against Gram‐positive bacteria including methicillin and multi‐drug resistant Staphylococcus aureus . This is a rare example of an eunicellane‐type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold.