Using SPAdes De Novo Assembler.

BackgroundCancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities.ResultsSelected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C–C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide.ConclusionsThe ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster—homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)—varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.

Whole-genome analysis and secondary metabolites production of a new strain Brevibacillus halotolerans 7WMA2: A potential biocontrol agent against fungal pathogens

The fungusAspergillus flavus is an opportunistic crop pathogen that produces aflatoxins. Aflatoxins are potent carcinogenic and hepatotoxic secondary metabolites that are highly regulated in most countries.A. flavus also produces many other secondary metabolites and harbours more than 50 putative secondary metabolite biosynthetic gene clusters that have yet to be characterised. Bioactive secondary metabolites that augment the ability of the fungus to infect crops are of particular interest. Biosynthetic gene cluster 11 inA. flavus has been recently shown to encode for the biosynthesis of aspergillic acid, a toxic hydroxamic acid-containing pyrazinone compound that can bind iron, resulting in a red-orange pigment known as ferriaspergillin. A decrease inA. flavus pathogenicity and aflatoxin contamination was observed when aspergillic acid biosynthesis was blocked during maize seed infection. In this study, we probe the available genomes ofAspergillus species for biosynthetic gene cluster 11 homologs. We find that all species possessing gene cluster 11 produce aspergillic acid or a closely related isomer. We demonstrate that theAspergillus sectionFlavi species harbouring biosynthetic gene cluster 11 produce a mixture of aspergillic acid, hydroxyaspergillic acid, and aspergillic acid analogs differing only in the amino acid precursors. Interestingly, manyAspergillus sectionCircumdati species, known mainly for their production of the problematic mycotoxin ochratoxin A, also harbour gene cluster 11 homologs, but do not produce aspergillic acid. Instead, these species produce neoaspergillic acid and its hydroxylated analog neohydroxyaspergillic acid, indicating that cluster 11 is responsible for neoaspergillic acid biosynthesis inAspergillus sectionCircumdati.

Entamoeba histolytica is a parasitic protozoan and the causative agent of amoebiasis in humans. Amoebiasis has a high incidence of disease, resulting in ∼67,900 deaths per year, and it poses a tremendous burden of morbidity and mortality in children. Despite its importance, E. histolytica is an understudied parasite. These protocols describe the in vitro growth, maintenance, cryopreservation, genetic manipulation, and cloning of axenic E. histolytica trophozoites. There has been significant progress in genetic manipulation of this organism over the past decade, and these protocols outline the ways in which these advances can be implemented. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culturing E. histolytica trophozoites Support Protocol 1: Preparation of TYI-S-33 medium Support Protocol 2: Lot testing of Biosate peptone and adult bovine serum for TYI-S-33 medium Basic Protocol 2: Cryopreservation of E. histolytica trophozoites Support Protocol 3: Preparation of cryoprotectant solutions Basic Protocol 3: Transfection of E. histolytica trophozoites with Attractene reagent Basic Protocol 4: Creating clonal lines using limiting dilution Basic Protocol 5: Knockdown of one to two genes with trigger-induced RNA interference Support Protocol 4: Evaluation of RNA interference knockdown with reverse transcriptase PCR Basic Protocol 6: E. histolytica growth curves.

Basophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost-effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Gradient centrifugation-independent basophil isolation Support Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viability Support Protocol 2: Giemsa staining Support Protocol 3: Calcium flux analysis Support Protocol 4: Basophil activation test.

Complete genome sequence of Streptomyces malaysiensis HNM0561, a marine sponge-associated actinomycete producing malaymycin and mccrearamycin E

Immunoheterogeneity is a potential hindrance to the maximum applicability of mesenchymal stromal cells (MSCs) in biotherapeutics and regenerative medicine. The primary causes of this heterogeneityare donor variation, diversity of tissue sources, and the culture conditionsunder which MSCs clonally propagate. Immunoheterogeneity could manifest as functional heterogeneity within these stem cells, affecting their ability to differentiate into adult tissue lineages. The impacted healthy human third molar tooth is a rich source of MSCs, which are isolatable both from the dental pulp and the dental follicle. Dental pulp stem cells (DPSCs), though widely characterized for their multipotential lineage differentiation, demonstrate considerable immunoheterogeneity, which demands the enrichment of these stem cells post-isolation. The aim of this study is to enrich and understand the functional differences in sub-populations of MSCs towards a specific lineage. In this protocol, we have highlighted detailed, stepwise methods for sorting and culturing post-sort and optimized methods for quantifying the differentiation of sorted DPSCs towards the osteogenic lineage. Our workflow is designed with additional considerations for cell sorter availability, maintenance of cell viability during long-distance transport to a state-of-the-art facility, and optimization of post-sorting cell culture and downstream experiments. This protocol can be universally followed to understand functional differences within MSCs isolated from different tissue sources and their implications for multi-lineage differentiation. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Assessing multiparametric expression of MSC surface markers and single cell sorting for enrichment of stem cells Support Protocol 1: Antibody panel design Support Protocol 2: MSC culture, preparation of cell suspension, and sample staining Support Protocol 3: Setting up controls Support Protocol 4: Analysis and data interpretation Basic Protocol 2: Quantitative and qualitative assessment of osteogenic differentiation of post-sorted DPSCs Support Protocol 5: Differentiation of MSCS (pre-sort and post-sort considerations) Support Protocol 6: Cytochemical staining and quantification Support Protocol 7: Immunofluorescence staining Support Protocol 8: RNA isolation and qPCR-based characterization.

Mucopolysaccharidoses (MPSs) are complex lysosomal diseases that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic/iduronic acid-N-acetylhexosamine or uronic/iduronic acid-N-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2025 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable-isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of methanolysis time Support Protocol 5: Measurement of methanolic HCl concentration Support Protocol 6: Preparation of working methanolic HCl solution (1.1 M) Support Protocol 7: Dilution of prepared urine sample.

Cell‐cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell‐cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less‐fit cells from mosaic cultures with fitter counterparts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Electroporation of hPSCs to establish a fluorescent reference cell line Support Protocol 1: Single‐cell dissociation of hPSCs Support Protocol 2: Single‐cell cloning of fluorescently labeled hPSCs Basic Protocol 2: Separate culture and co‐culture proliferation assays Basic Protocol 3: Assessing levels of apoptosis in hPSC cultures using flow cytometry Basic Protocol 4: Transwell assay Support Protocol 3: Immunohistochemistry and image quantification of cleaved caspase‐3 Basic Protocol 5: Cell confrontation assay Basic Protocol 6: Cell compression assay Basic Protocol 7: Time‐lapse imaging to assess mechanical extrusion

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions.

Natural product discovery campaigns aim to identify compounds with the desired bioactivity, for example, metabolites with antibiotic activity. The major driver of many projects is still the finding of bioactive extracts, which will be followed up to isolate the activity-causing agent as pure compound. However, nowadays also additional strategies can be used to increase the probability of success. Metabolomic approaches indicate chemical novelty, and genomics allow identification of putative biosynthetic gene clusters (BGCs) of interest, even though the corresponding metabolite is unknown. Whatever the entry to the campaign is, at one point the scientists need to have the desired compound in hand to analyze it in detail. Hence, expression must be achieved to yield the compound of interest, either to link it to the corresponding putative BGC or to overcome the bottleneck of sparse compound supply. Therefore, homologous and heterologous expression approaches are feasible ways forward to increase production yield, shorten fermentation time, or to get BGCs expressed at all for which no suitable fermentation condition was identified.In this chapter, expression approaches in bacteria are described to biosynthesize compounds of interest. Homologous expression, by genetic manipulation of the original Streptomyces producer strain, and heterologous expression in the microbial workhorse Escherichia coli are exemplified.

Specialized metabolites encoded by biosynthetic gene clusters (BGCs) in the oral microbiome remain largely unexplored in the context of oral health and disease. Previous genome-centric surveys have cataloged hundreds of uncharacterized BGCs in the oral cavity associated with health and disease, but these studies relied on reference genomes and did not capture strain-level variation or the native distribution of BGCs. Here, we assembled three independently sourced metagenomic datasets from healthy and dental caries samples, extracted BGCs, and quantified their abundance alongside expression in a metatranscriptomic dataset. We first identified that aryl polyene, ribosomally synthesized and post-translationally modified peptide (RiPP), and nonribosomal peptide (NRP) encoding BGCs were the most abundant BGC classes across all three metagenomic datasets. We then grouped these BGCs into homology-based families and found that homologous clusters were usually consistently associated with either health or dental caries, suggesting conserved community-level roles for BGCs. An elastic-net regression model further selected 45 BGCs out of >5000 that could distinguish healthy and dental caries samples in the metatranscriptomic dataset, which demonstrated that BGCs could be predictive markers of disease. This analysis emphasizes the importance of high-quality metagenomic and metatranscriptomic datasets to resolve BGC expression patterns and to guide discovery of metabolites relevant to oral health and disease.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

Polycyclic tetramate macrolactams (PTMs) biosynthetic gene cluster are widely distributed in different bacterial types, especially in Streptomyces species. The mining of the genomic data of marine-derived Streptomyces sp. SCSIO 40010 reveals the presence of a putative PTM-encoding biosynthetic gene cluster (ptm′ BGC) that features a genetic organization for potentially producing 5/5/6 type of carbocyclic ring-containing PTMs. A fermentation of Streptomyces sp. SCSIO 40010 led to the isolation and characterization of six new PTMs 1–6. Comprehensive spectroscopic analysis assigned their planar structures and relative configurations, and their absolute configurations were deduced by comparing the experimental electronic circular dichroism (ECD) spectra with the reported spectra of the known PTMs. Intriguingly, compounds 1–6 were determined to have a trans-orientation of H-10/H-11 at the first 5-membered ring, being distinct from the cis-orientation in their known PTM congeners. PTMs 1–5 displayed cytotoxicity against several cancer cell lines, with IC50 values that ranged from 2.47 to 17.68 µM.