Deep-Sea Sponges and Corals off the Western Coast of Florida-Intracellular Mechanisms of Action of Bioactive Compounds and Technological Advances Supporting the Drug Discovery Pipeline.

The majority of natural products utilized to treat a diverse array of human conditions and diseases are derived from terrestrial sources. In recent years, marine ecosystems have proven to be a valuable resource of diverse natural products that are generated to defend and support their growth. Such marine sources offer a large opportunity for the identification of novel compounds that may guide the future development of new drugs and therapies. Using the National Oceanic and Atmospheric Administration (NOAA) portal, we explore deep-sea coral and sponge species inhabiting a segment of the U.S. Exclusive Economic Zone, specifically off the western coast of Florida. This area spans ~100,000 km2, containing coral and sponge species at sea depths up to 3000 m. Utilizing PubMed, we uncovered current knowledge on and gaps across a subset of these sessile organisms with regards to their natural products and mechanisms of altering cytoskeleton, protein trafficking, and signaling pathways. Since the exploitation of such marine organisms could disrupt the marine ecosystem leading to supply issues that would limit the quantities of bioactive compounds, we surveyed methods and technological advances that are necessary for sustaining the drug discovery pipeline including in vitro aquaculture systems and preserving our natural ecological community in the future. Collectively, our efforts establish the foundation for supporting future research on the identification of marine-based natural products and their mechanism of action to develop novel drugs and therapies for improving treatment regimens of human conditions and diseases.

Evaluating in Vitro Culture Medium of Gut Microbiome with Orthogonal Experimental Design and a Metaproteomics Approach.

In vitro culture based approaches are time- and cost-effective solutions for rapidly evaluating the effects of drugs or natural compounds against microbiomes. The nutritional composition of the culture medium is an important determinant for effectively maintaining the gut microbiome in vitro. This study combines orthogonal experimental design and a metaproteomics approach to obtaining functional insights into the effects of different medium components on the microbiome. Our results show that the metaproteomic profile respond differently to medium components, including inorganic salts, bile salts, mucin, and short-chain fatty acids. Multifactor analysis of variance further revealed significant main and interaction effects of inorganic salts, bile salts, and mucin on the different functional groups of gut microbial proteins. While a broad regulating effect was observed on basic metabolic pathways, different medium components also showed significant modulations on cell wall, membrane, and envelope biogenesis and cell motility related functions. In particular, flagellar assembly related proteins were significantly responsive to the presence of mucin. This study provides information on the functional influences of medium components on the in vitro growth of microbiome communities and gives insight on the key components that must be considered when selecting and optimizing media for culturing ex vivo microbiotas.

Generation of a monoclonal antibody against duck circovirus capsid protein and its potential application for native viral antigen detection.

Introduction: Duck circovirus (DuCV) infection is currently recognized as an important immunosuppressive disease in commercial duck flocks in China. Specific antibodies against DuCV viral proteins are required to improve diagnostic assays and understand the pathogenesis of DuCV infection. Methods and results: To generate DuCV-specific monoclonal antibodies (mAbs), a recombinant DuCV capsid protein without the first 36 N-terminal amino acids was produced in Escherichia coli. Using the recombinant protein as an immunogen, a mAb was developed that reacted specifically with the DuCV capsid protein, expressed in E. coli and baculovirus systems. Using homology modeling and recombinant truncated capsid proteins, the antibody-binding epitope was mapped within the region of 144IDKDGQIV151, which is exposed to solvent in the virion capsid model structure. To assess the applicability of the mAb to probe the native virus antigen, the murine macrophage cell line RAW267.4 was tested for DuCV replicative permissiveness. Immunofluorescence and Western blot analysis revealed that the mAb recognized the virus in infected cells and the viral antigen in tissue samples collected from clinically infected ducks. Discussion: This mAb, combined with the in vitro culturing method, would have widespread applications in diagnosing and investigating DuCV pathogenesis.

ENDOCELL-Seud: a Delphi protocol to harmonise methods in endometrial cell culturing.

In vitro: culturing of endometrial cells obtained from the uterine mucosa or ectopic sites is used to study molecular and cellular signalling relevant to physiologic and pathologic reproductive conditions. However, the lack of consensus on standard operating procedures for deriving, characterising and maintaining primary cells in two- or three-dimensional cultures from eutopic or ectopic endometrium may be hindering progress in this area of research. Guidance for unbiased in vitro research methodologies in the field of reproductive science remains essential to increase confidence in the reliability of in vitro models. We present herein the protocol for a Delphi process to develop a consensus on in vitro methodologies using endometrial cells (ENDOCELL-Seud Project). A steering committee composed of leading scientists will select critical methodologies, topics and items that need to be harmonised and that will be included in a survey. An enlarged panel of experts (ENDOCELL-Seud Working Group) will be invited to participate in the survey and provide their ratings to the items to be harmonised. According to Delphi, an iterative investigation method will be adopted. Recommended measures will be finalised by the steering committee. The study received full ethical approval from the Ethical Committee of the Maastricht University (ref. FHML-REC/2021/103). The study findings will be available in both peer-reviewed articles and will also be disseminated to appropriate audiences at relevant conferences. Lay summary: Patient-derived cells cultured in the lab are simple and cost-effective methods used to study biological and dysfunctional or disease processes. These tools are frequently used in the field of reproductive medicine. However, the lack of clear recommendations and standardised methodology to guide the laboratory work of researchers can produce results that are not always reproducible and sometimes are incorrect. To remedy this situation, we define here a method to ascertain if researchers who routinely culture cells in the lab agree or disagree on the optimal laboratory techniques. This method will be used to make recommendations for future researchers working in the field of reproductive biology to reproducibly culture endometrial cells in the laboratory.

RapidAIM: a culture- and metaproteomics-based Rapid Assay of Individual Microbiome responses to drugs.

BACKGROUND: Human-targeted drugs may exert off-target effects or can be repurposed to modulate the gut microbiota. However, our understanding of such effects is limited due to a lack of rapid and scalable assay to comprehensively assess microbiome responses to drugs. Drugs and other compounds can drastically change the overall abundance, taxonomic composition, and functions of a gut microbiome. RESULTS: Here, we developed an approach to screen compounds against individual microbiomes in vitro, using metaproteomics to both measure absolute bacterial abundances and to functionally profile the microbiome. Our approach was evaluated by testing 43 compounds (including 4 antibiotics) against 5 individual microbiomes. The method generated technically highly reproducible readouts, including changes of overall microbiome abundance, microbiome composition, and functional pathways. Results show that besides the antibiotics, the compounds berberine and ibuprofen inhibited the accumulation of biomass during in vitro growth of the microbiota. By comparing genus and species level-biomass contributions, selective antibacterial-like activities were found with 35 of the 39 non-antibiotic compounds. Seven of the compounds led to a global alteration of the metaproteome, with apparent compound-specific patterns of functional responses. The taxonomic distributions of altered proteins varied among drugs, i.e., different drugs affect functions of different members of the microbiome. We also showed that bacterial function can shift in response to drugs without a change in the abundance of the bacteria. CONCLUSIONS: Current drug-microbiome interaction studies largely focus on relative microbiome composition and microbial drug metabolism. In contrast, our workflow enables multiple insights into microbiome absolute abundance and functional responses to drugs. The workflow is robust, reproducible, and quantitative and is scalable for personalized high-throughput drug screening applications.

Metaproteomics Reveals Growth Phase-Dependent Responses of an In Vitro Gut Microbiota to Metformin.

Metaproteomics has been used in combination with in vitro gut microbiota models to study drug-microbiome interactions. However, it remains unexplored whether the metaproteomics profile of in vitro gut microbiota responds differently to a same stimulus added at different growth phases. In this study, we cultured a human gut microbiota in 96-deep well plates using a previously validated model. Metformin was added during the lag, log, and stationary phases. Microbiome samples, collected at different time points, were analyzed by optical density and function by metaproteomic. The in vitro gut microbiota growth curves, taxonomy, and functional responses were different depending whether metformin was added during the lag, log, or stationary phases. The addition of drugs at the log phase may lead to the greatest decline of bacterial growth. Metaproteomic analysis suggests that the strength of the metformin effect on the gut microbiome functional profile may be ranked as lag phase > log phase > stationary phase. Metformin added at the lag phase may result in a significantly reduced level of the Clostridiales order and an increased level of the Bacteroides genus, which is different from stimulations during the rest of the growth phases. Metformin may also result in alterations of several pathways, including energy production and conversion, lipid transport and metabolism, translation, ribosomal structure, and biogenesis. Our results indicate that the timing for drug stimulation should be considered when studying drug-microbiome interactions in vitro.

The Development of Whole Sporozoite Vaccines for Plasmodium falciparum Malaria.

Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is in vitro culturing methodology, which has been described for several Plasmodium species, including the major disease-causing human malaria parasite, Plasmodium falciparum. Here, we review whole sporozoite vaccine immunogenicity and in vitro culturing platforms for sporozoite production.

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro.

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications.

Evaluating the Biostability of Yellow and Clear Intraocular Lenses with a System Simulating Natural Intraocular Environment.

PURPOSE: Blue light-filtering intraocular lenses (IOLs) are thought to protect the retina from blue light damage after cataract surgery, and the implantation of yellow-tinted IOLs has been commonly used in cataract surgery. To our knowledge, this is the first investigation measuring the long-term biostability of yellow-tinted IOLs using an in vitro system simulating natural intraocular environment. METHODS: Six hydrophobic acrylic IOLs, three clear IOLs, and three yellow-tinted IOLs were included in the study. Each yellow-tinted IOL was a matching counterpart of a clear IOL, with the only difference being the lens color. The IOLs were kept in conditions replicating the intraocular environment using a perfusion culture system for 7 months. Resolution, light transmittance rate, and the modulation transfer function (MTF) were measured before and after culturing. Surface roughness of the anterior and posterior surfaces was also measured. RESULTS: After culturing for 7 months, there were no changes in the resolution, the light transmittance rate, and MTF. The surface roughness of the anterior and posterior surfaces increased after culturing; however, this increase was clinically insignificant. There were no differences in surface roughness between the clear and yellow-tinted IOLs, either before or after culturing. CONCLUSIONS: A novel in vitro system replicating intraocular environment was used to investigate the biostability of yellow-tinted IOLs. The surface roughness showed no clinically significant increase after culturing for 7 months. TRANSLATIONAL RELEVANCE: This system is useful for evaluating the biostability of IOLs.