Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.

Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible.

Ampullaviruses: From Extreme Environments to Biotechnological Innovation.

Ampullaviruses are unique among viruses. They live in extreme environments and have special bottle-shaped architecture. These features make them useful tools for biotechnology. These viruses have compact genomes. They encode a range of enzymes and proteins. Their natural environment highlights their suitability for industrial applications. Ongoing research explores ways in which these viruses can improve enzyme stability. They are also employed in the creation of new biosensors and the development of new bioremediation techniques. High coinfection rates and the ecology of ampullaviruses at larger scales can also reveal new viral vectors. They can also help improve phage therapy. Here, we have explored the structure and function of ampullaviruses. We have focused on their use in biotechnology. We have also identified their characteristics that could prove to be useful. We have also pointed out key knowledge gaps and bridging them could further extend the biotechnological uses.

Unlocking the potential of cultivated meat through cell line engineering.

Cultivated meat has the potential to revolutionize food production, but its progress is hindered by fundamental shortcomings of mammalian cells with respect to industrial-scale bioprocesses. Here, we discuss the essential role of cell line engineering in overcoming these limitations, highlighting the balance between the benefits of enhanced cellular traits and the associated regulatory and consumer acceptance challenges. We believe that careful selection of cell engineering strategies, including both genetic and non-genetic modifications, can address this trade-off and is essential to advancing the field.

Mimicking and analyzing the tumor microenvironment.

The tumor microenvironment (TME) is increasingly appreciated to play a decisive role in cancer development and response to therapy in all solid tumors. Hypoxia, acidosis, high interstitial pressure, nutrient-poor conditions, and high cellular heterogeneity of the TME arise from interactions between cancer cells and their environment. These properties, in turn, play key roles in the aggressiveness and therapy resistance of the disease, through complex reciprocal interactions between the cancer cell genotype and phenotype, and the physicochemical and cellular environment. Understanding this complexity requires the combination of sophisticated cancer models and high-resolution analysis tools. Models must allow both control and analysis of cellular and acellular TME properties, and analyses must be able to capture the complexity at high depth and spatial resolution. Here, we review the advantages and limitations of key models and methods in order to guide further TME research and outline future challenges.

Transitioning ecosystems: how will permafrost cryophiles respond to a changing climate?

Permafrost harbours a diversity of cryophilic microorganisms that can be metabolically active at sub-zero temperatures and likely play a role in global carbon cycling. This forum article explores possible impacts of permafrost warming on cold-adapted microbiota, highlights underexplored areas of research, and suggests future short and long-term research foci.

Capturing carbon to mitigate climate change: storage or use?

Reducing atmospheric CO2 is vital to combat climate change. Alongside reducing emissions, it is essential to capture atmospheric CO2 and either use it or store it, depending on which option yields the best outcomes. Government policies should coordinate actions in areas such as the bioeconomy and avoid creating perverse incentives.

Global regulatory policies for animal biotechnology: overview, opportunities and challenges.

Genome editing (GnEd) has the potential to provide many benefits to animal agriculture, offering a means for achieving rapid growth, disease resistance, and novel phenotypes. The technology has the potential to be useful for rapidly incorporating traits into existing selectively bred animals without the need for crossbreeding and backcrossing. Yet only four products from animals created via biotechnology, all growth-enhanced fishes, have reached commercialization and only on a limited scale. The past failure of genetically engineered (or GM) products to reach conventional producers can largely be attributed to the high cost of meeting GMO regulatory requirements. We review the history of GMO regulations internationally, noting the influence of Codex Alimentarius on the development of many existing regulatory frameworks. We highlight new regulatory approaches for GnEd organisms, first developed by Argentina, and the adoption of similar approaches by other countries. Such new regulatory approaches allow GnEd organisms that could have been developed by conventional means to be regulated under the same rules as conventional organisms and in the future is likely to enhance the opportunity for biotech animals to enter production. Treating certain GnEd products as conventional has had a large impact on the variety of biotechnological innovations successfully navigating regulatory processes. We suggest that for the full potential of GnEd technologies to be realized, enabling public policies are needed to facilitate use of GnEd as a breeding tool to incorporate new traits within existing animal breeding programs, rather than only a tool to create distinct new products.

Thinking globally, acting locally in the 21st century: Bamboo to bioproducts and cleaned mine sites.

Current solutions to global challenges place tension between global benefits and local impacts. The result is increasing opposition to implementation of beneficial climate policies. Prioritizing investment in projects with tangible local benefits that also contribute to global climate change can resolve this tension and make local communities' partners instead of antagonists to change; the approach advocated is a new take on "thinking globally, acting locally". This approach is a departure from the usual strategy of focusing resources on solutions perceived to have the largest potential global impact, without regards to local concerns. Reclamation of polluted mine sites by using fast growing bamboo to remove heavy metals provides a case study to show what is possible. Effective implementation of thinking globally while acting locally will require increased coordination between different types of researchers, new educational models, and greater stakeholder participation in problem identification and solution development.

Is cardiovascular risk profiling from UK Biobank retinal images using explicit deep learning estimates of traditional risk factors equivalent to actual risk measurements? A prospective cohort study design.

OBJECTIVE: Despite extensive exploration of potential biomarkers of cardiovascular diseases (CVDs) derived from retinal images, it remains unclear how retinal images contribute to CVD risk profiling and how the results can inform lifestyle modifications. Therefore, we aimed to determine the performance of cardiovascular risk prediction model from retinal images via explicitly estimating 10 traditional CVD risk factors and compared with the model based on actual risk measurements. DESIGN: A prospective cohort study design. SETTING: The UK Biobank (UKBB), a prospective cohort study, following the health conditions including CVD outcomes of adults recruited between 2006 and 2010. PARTICIPANTS: A subset of data from the UKBB which contains 52 297 entries with retinal images and 5-year cumulative incidence of major adverse cardiovascular events (MACE) was used. Our dataset is split into 3:1:1 as training set (n=31 403), validation set (n=10 420) and testing set (n=10 474). We developed a deep learning (DL) model to predict 5-year MACE using a two-stage DL neural network. PRIMARY AND SECONDARY OUTCOME MEASURES: We computed accuracy, area under the receiver operating characteristic curve (AUC) and compared variations in the risk prediction models combining CVD risk factors and retinal images. RESULTS: The first-stage DL model demonstrated that the 10 CVD risk factors can be estimated from a given retinal image with an accuracy ranging between 65.2% and 89.8% (overall AUC of 0.738 with 95% CI: 0.710 to 0.766). In MACE prediction, our model outperformed the traditional score-based models, with 8.2% higher AUC than Systematic COronary Risk Evaluation (SCORE), 3.5% for SCORE 2 and 7.1% for the Framingham Risk Score (with p value<0.05 for all three comparisons). CONCLUSIONS: Our algorithm estimates the 5-year risk of MACE based on retinal images, while explicitly presenting which risk factors should be checked and intervened. This two-stage approach provides human interpretable information between stages, which helps clinicians gain insights into the screening process copiloting with the DL model.

Electrochemical Glucose Sensors: Classification, Catalyst Innovation, and Sampling Mode Evolution.

Glucose sensors are essential tools for monitoring blood glucose concentration in diabetic patients. In recent years, with the increasing number of individuals suffering from diabetes, blood glucose monitoring has become extremely necessary, which expedites the iteration and upgrade of glucose sensors greatly. Currently, two main types of glucose sensors are available for blood glucose testing: enzyme-based glucose sensor (EBGS) and enzyme-free glucose sensor (EFGS). For EBGS, several progresses have been made to comprehensively improve detection performance, ranging from enhancing enzyme activity, thermostability, and electron transfer properties, to introducing new materials with superior properties. For EFGS, more and more new metallic materials and their oxides are being applied to further optimize its blood glucose monitoring. Here the latest progress of electrochemical glucose sensors, their manufacturing methods, electrode materials, electrochemical parameters, and applications were summarized, the development glucose sensors with various noninvasive sampling modes were also compared.

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (Gs, Gi/o, Gq/11, G12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.

Rapid and specific detection of nanoparticles and viruses one at a time using microfluidic laminar flow and confocal fluorescence microscopy.

Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we introduce an approach for rapid and specific detection of single nanoparticles using a confocal-based flow virometer. The combination of laminar flow in a microfluidic channel and correlated fluorescence signals emerging from both free dyes and fluorescently labeled primary antibodies provide insights into nanoparticle volumes and specificities. We evaluate and validate the assay using fluorescent beads and viruses, including SARS-CoV-2 with fluorescently labeled primary antibodies. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting viruses at relevant loads. Based on our results, we envision the future use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup.

Pyriofenone Interacts with the Major Facilitator Superfamily Transporter of Phytopathogenic Fungi to Potentially Control Tea Leaf Spot Caused by Lasiodiplodia theobromae.

Tea leaf spot caused by Lasiodiplodia theobromae is a newly discovered fungal disease in southwest China. Due to a lack of knowledge of its epidemiology and control strategies, the disease has a marked impact on tea yield and quality. Pyriofenone is a new fungicide belonging to the aryl phenyl ketone fungicide group, which has shown marked efficacy in controlling various fungal diseases. However, its mechanism of action is not yet understood. This study found that pyriofenone exhibits strong in vitro inhibitory activity against various phytopathogenic fungi. Specifically, it showed strong inhibitory activity against L. theobromae, with a half-maximal effective concentration (EC50) value of 0.428 µg/ml determined by measuring mycelial growth rate. Morphological observations, using optical, scanning electron, and transmission electron microscopy, revealed that pyriofenone induces morphological abnormalities in L. theobromae hyphae. At lower doses, the hyphae became swollen, the distance between septa decreased, and the hyphal growth rate slowed. At higher doses and longer exposures, the hyphae collapsed. Transcriptomic and bioinformatic analyses indicated that pyriofenone can affect the expression of genes related to membrane transporters. Homology modeling suggested that pyriofenone may bind to a candidate target protein of the major facilitator superfamily (MFS) transporter, with a free binding energy of -7.1 kcal/mol. This study suggests that pyriofenone may potentially regulate the transport of metabolites in L. theobromae, thus affecting hyphal metabolism and interfering with hyphal growth. Pyriofenone exhibits in vitro inhibitory activity against various tea foliar pathogens and holds promise for future applications to the control of tea foliar diseases.

A screen for cell envelope stress uncovers an inhibitor of prolipoprotein diacylglyceryl transferase, Lgt, in Escherichia coli.

The increasing prevalence of antibiotic resistance demands the discovery of antibacterial chemical scaffolds with unique mechanisms of action. Phenotypic screening approaches, such as the use of reporters for bacterial cell stress, offer promise to identify compounds while providing strong hypotheses for follow-on mechanism of action studies. From a collection of ∼1,800 Escherichia coli GFP transcriptional reporter strains, we identified a reporter that is highly induced by cell envelope stress-pProm rcsA -GFP. After characterizing pProm rcsA -GFP induction, we assessed a collection of bioactive small molecules for reporter induction, identifying 24 compounds of interest. Spontaneous suppressors to one compound in particular, MAC-0452936, mapped to the gene encoding the essential prolipoprotein diacylglyceryl transferase, lgt. Lgt inhibition by MAC-0452936 inhibition was confirmed through genetic, phenotypic, and biochemical approaches. The oxime ester, MAC-0452936, represents a useful small molecule inhibitor of Lgt and highlights the potential of using pProm rcsA -GFP as a phenotypic screening tool.

Tailoring hydrophobicity and strength in spider silk-inspired coatings via thermal treatments.

The advent of advanced coatings has transformed material functionalities, extending their roles from basic coverage and visual appeal to include unique properties such as self-healing, superior hydrophobicity, and antimicrobial action. However, the traditional dependency on petrochemical-derived materials for these coatings raises environmental concerns. This study proposes the use of renewable and alternative materials for coating development. We present the use of bioengineered spider silk-inspired protein (SSIP), produced through recombinant technology, as a viable, eco-friendly alternative due to their ease of processing under ambient pressure and the utilization of water as a solvent, alongside their exceptional physicochemical properties. Our research investigates the effects of different thermal treatments and protein concentrations on the mechanical strength and surface water repellency of coatings on silica bases. Our findings reveal a direct correlation between the temperature of heat treatment and the enhancements in surface hydrophobicity and mechanical strength, where elevated temperatures facilitate increased resistance to water and improved mechanical integrity. Consequently, we advocate SSIPs present a promising, sustainable choice for advanced coatings, providing a pathway to fine-tune coating recipes for better mechanical and hydrophobic properties with a reduced ecological footprint, finding potential uses in various fields such as electronics.

Novel Administration Routes, Delivery Vectors, and Application of Vaccines Based on Biotechnologies: A Review.

Traditional vaccines can be classified into inactivated vaccines, live attenuated vaccines, and subunit vaccines given orally or via intramuscular (IM) injection or subcutaneous (SC) injection for the prevention of infectious diseases. Recently, recombinant protein vaccines, DNA vaccines, mRNA vaccines, and multiple/alternative administering route vaccines (e.g., microneedle or inhalation) have been developed to make vaccines more secure, effective, tolerable, and universal for the public. In addition to preventing infectious diseases, novel vaccines have currently been developed or are being developed to prevent or cure noninfectious diseases, including cancer. These vaccine platforms have been developed using various biotechnologies such as viral vectors, nanoparticles, mRNA, recombination DNA, subunit, novel adjuvants, and other vaccine delivery systems. In this review, we will explore the development of novel vaccines applying biotechnologies, such as vaccines based on novel administration routes, vaccines based on novel vectors, including viruses and nanoparticles, vaccines applied for cancer prevention, and therapeutic vaccines.

Stability Modeling Methodologies to Enable Earlier Patient Access.

Over recent years, confidence has been gained that predictive stability modeling approaches using statistical tools, prior knowledge and industry experience enable, in many instances, a robust and reliable shelf-life/expiry or retest period prediction for medicinal products. These science and risk-based approaches can compensate for not having a complete real-time stability data set to be included in regulatory applications at the time of initial submission and, thereby, accelerate the availability of new medicines. Examples of predictive stability modeling include accelerated stability assessment procedure (ASAP), advanced kinetic modeling (AKM), and novel modeling approaches that involve the use of Bayesian statistics and Artificial Intelligence (AI) applications such as Machine Learning (ML), with applicability to both synthetic and biological molecules. For biologics, product-specific and platform prior knowledge could be used to overcome model limitations known for non-quantitative stability indicating attributes. A successful ongoing verification approach by comparing the predicted data with real-time stability data would be an appropriate risk management approach which is intended to address regulatory concerns, and further build confidence in the robustness of these predictive modelling approaches with regulatory agencies. Global regulatory acceptance of stability modeling could allow patients to receive potential life-saving medications faster without compromising quality, safety or efficacy.

Whole-genome sequences of vaginal isolates Lactobacillus crispatus CECT30647 and Lactobacillus gasseri CECT30648.

We present the whole-genome sequences of Lactobacillus crispatus CECT30647 and Lactobacillus gasseri CECT30648, two vaginal isolates with probiotic potential.

Aquatic Biomaterial Repositories: Comprehensive Guidelines, Recommendations, and Best Practices for Their Development, Establishment, and Sustainable Operation.

The alarming pace of species extinction severely threatens terrestrial and aquatic ecosystems, undermining the crucial ecological services vital for environmental sustainability and human well-being. Anthropogenic activities, such as urbanization, agriculture, industrialization, and those inducing climate change, intensify these risks, further imperiling biodiversity. Of particular importance are aquatic organisms, pivotal in biodiscovery and biotechnology. They contribute significantly to natural product chemistry, drug development, and various biotechnological applications. To safeguard these invaluable resources, establishing and maintaining aquatic biomaterial repositories (ABRs) is imperative. This review explores the complex landscape of ABRs, emphasizing the need for standardized procedures from collection to distribution. It identifies key legislative and regulatory frameworks, such as the Nagoya Protocol and EU directives, essential for ensuring responsible and equitable biorepository operations. Drawing on extensive literature and database searches, this study compiles existing recommendations and practices into a cohesive framework with which to guide the establishment and sustainable management of ABRs. Through collaborative efforts and adherence to best practices, ABRs can play a transformative role in the future of marine biotechnology and environmental conservation.