Biosurfactant Production by Pseudomonas: a Systematic Review.

It is of fundamental interest to research and develop innovative biotechnologies, as well as bioproducts that replace or are alternatives to those of non-renewable origin, such as biosurfactants in relation to traditional surfactants used in various sectors. Consequently, there are a large number of experimental studies addressing different subjects, especially with the use of bacteria of the genus Pseudomonas; however, there is a lack of work that demonstrates the evaluation of this science produced to date. Therefore, this article discusses the production of biosurfactants by Pseudomonas with the aim of surveying and analyzing experimental articles on this topic. To realize this, a systematic search was carried out with well-defined temporal space, databases, and inclusion and exclusion criteria, based on metric studies that guided what information would be collected and the method of evaluation. Therefore, a large number of articles were selected, which demonstrated Pseudomonas aeruginosa as the bioagent mostly used in the tests, which aimed to improve the process in the area. Furthermore, interest in this field has increased over the years, predominantly in emerging market countries, where the most prominent authors on the topic are found. Therefore, it is necessary that there is an expansion of interest in the area to make the production of biosurfactants cheaper in areas that currently have greater development deficiencies, such as means of purifying the bioprocess and reducing foam formation in the bioprocess.

Anti-Müllerian hormone as a promising novel biomarker for litter size in Romanov sheep.

Anti-Müllerian hormone (AMH) is a hormone produced by growing preantral and antral follicles of the ovary. AMH is accepted as an important biomarker for fertility and superovulation parameters in livestock species. This study aimed to evaluate changes in serum AMH level in the oestrous cycle, repeatability of AMH, the effect of age on serum AMH level and the effects of AMH on litter size in Romanov sheep. In the study, a total of 36 Romanov sheep were used as animal material. First blood samples (0th day) were collected from 36 ewes to evaluate AMH and progesterone levels. Second blood samples were collected randomly from 20 ewes 9 days after first sampling to compare AMH levels at two different periods of the oestrous cycle in Romanov ewes. The ewes were categorized into three groups as low, medium and high AMH based on their first AMH levels. Results indicated that serum AMH level did not change during the oestrous and dioestrous phases of the oestrous cycle and two random time points of the oestrous cycle (p > .05). Pearson correlation analysis revealed that there is a high (r = .95) and significant (p < .001) correlation between AMH levels at the 0th (AMH-1) and 9th (AMH-2) days. The effect of AMH level on litter size was found to be significant. Litter size was significantly higher in the high AMH group than in the low AMH group (p < .05). In addition, the age of ewes did not affect serum AMH levels (p > .05). ROC analysis indicates that AMH cut-off value >320 pg/mL with 70% sensitivity and 100% specificity can be used for litter size in Romanov ewes. In conclusion, AMH is highly repeatable and its serum AMH level did not change during the oestrous cycle in Romanov sheep. In addition, AMH affects litter size and can be reliably used as a marker for litter size in Romanov sheep.

Novel metabolite madeirone and neomarinone extracted from Streptomyces aculeoletus as marine antibiofilm and antifouling agents.

Introduction: Biofouling poses a significant economic threat to various marine industries, leading to financial losses that can reach billions of euros annually. This study highlights the urgent need for effective alternatives to traditional antifouling agents, particularly following the global ban on organotin compounds. Material and methods: Streptomyces aculeolatus PTM-346 was isolated from sediment samples on the shores of the Madeira Archipelago, Portugal. The crude extract was fractionated using silica flash chromatography and preparative HPLC, resulting in two isolated marinone compounds: madeirone (1), a novel marinone derivative discovered in this study, and neomarinone (2). The antifouling activities of these compounds were tested against five marine bacterial species and the larvae of the mussel Mytilus galloprovincialis. Additionally, in silico and in vivo environmental toxicity evaluations of madeirone (1) and neomarinone (2) were conducted. Results: Madeirone (1) demonstrated significant antibiofilm efficacy, inhibiting Phaeobacter inhibens by up to 66%, Marinobacter hydrocarbonoclasticus by up to 60%, and Cobetia marina by up to 40%. Neomarinone (2) also exhibited substantial antibiofilm activity, with inhibition rates of up to 41% against P. inhibens, 40% against Pseudo-oceanicola batsensis, 56% against M. hydrocarbonoclasticus, 46% against C. marina, and 40% against Micrococcus luteus. The growth inhibition activity at the same concentrations of these compounds remained below 20% for the respective bacteria, highlighting their effectiveness as potent antibiofilm agents without significantly affecting bacterial viability. Additionally, both compounds showed potent effects against the settlement of Mytilus galloprovincialis larvae, with EC50 values of 1.76 µg/mL and 0.12 µg/mL for compounds (1) and (2), respectively, without impairing the viability of the targeted macrofouling species. In silico toxicity predictions and in vivo toxicity assays both support their potential for further development as antifouling agents. Conclusion: The newly discovered metabolite madeirone (1) and neomarinone (2) effectively inhibit both micro- and macrofouling. This distinct capability sets them apart from existing commercial antifouling agents and positions them as promising candidates for biofouling prevention. Consequently, these compounds represent a viable and environmentally friendly alternative for incorporation into paints, primers, varnishes, and sealants, offering significant advantages over traditional copper-based compounds.

Predicting the Long-Term Stability of Biologics with Short-Term Data.

Understanding the long-term stability of biologics is crucial to ensure safe, effective, and cost-efficient life-saving therapeutics. Current industry and regulatory practices require arduous real-time data collection over three years; thus, reducing this bottleneck while still ensuring product quality would enhance the speed of medicine to patients. We developed a parallel-pathway kinetic model, combined with Monte Carlo simulations for prediction intervals, to predict the long-term (2+ years) stability of biotherapeutic critical quality attributes (aggregates, fragments, charge variants, purity, and potency) with short-term (3-6 months) data from intended, accelerated, and stressed temperatures. We rigorously validated the model with 18 biotherapeutic drug products, composed of IgG1 and IgG4 monoclonal antibodies, antibody-drug conjugates, dual protein coformulations, and a fusion protein, including high concentration (≥100 mg/mL) formulations, in liquid and lyophilized presentations. For each drug product, we accurately predicted the long-term trends of multiple quality attributes using just 6 months of data. Further, we demonstrated superior stability prediction via our methods compared with industry-standard linear regression methods. The robust and repeatable results of this work across an unprecedented suite of 18 biotherapeutic compounds suggest that kinetic models with Monte Carlo simulation can predict the long-term stability of biologics with short-term data.

Nova-ST: Nano-patterned ultra-dense platform for spatial transcriptomics.

Spatial transcriptomics workflows using barcoded capture arrays are commonly used for resolving gene expression in tissues. However, existing techniques are either limited by capture array density or are cost prohibitive for large-scale atlasing. We present Nova-ST, a dense nano-patterned spatial transcriptomics technique derived from randomly barcoded Illumina sequencing flow cells. Nova-ST enables customized, low-cost, flexible, and high-resolution spatial profiling of large tissue sections. Benchmarking on mouse brain sections demonstrates significantly higher sensitivity compared to existing methods at a reduced cost.

Application and research progress of ARTP mutagenesis in actinomycetes breeding.

Atmospheric and room temperature plasma (ARTP) is an emerging artificial mutagenesis breeding technology. In comparison to traditional physical and chemical methods, ARTP technology can induce DNA damage more effectively and obtain mutation strains with stable heredity more easily after screening. It possesses advantages such as simplicity, safety, non-toxicity, and cost-effectiveness, showing high application value in microbial breeding. This article focuses on ARTP mutagenesis breeding of actinomycetes, specifically highlighting the application of ARTP mutagenesis technology in improving the performance of strains and enhancing the biosynthetic capabilities of actinomycetes. We analyzed the advantages and challenges of ARTP technology in actinomycetes breeding and summarized the common features, specific mutation sites and metabolic pathways of ARTP mutagenic strains, which could give guidance for genetic modification. It suggested that the future research work should focus on the establishment of high throughput rapid screening methods and integrate transcriptomics, proteomics, metabonomics and other omics to delve into the genetic regulations and synthetic mechanisms of the bioactive substances in ARTP mutated actinomycetes. This article aims to provide new perspectives for actinomycetes breeding through the establishment and application of ARTP mutagenesis technology, thereby promoting source innovation and the sustainable industrial development of actinomycetes.

Protocol to create phenotypic primary human hepatocyte cultures using the RASTRUM 3D cell model platform.

To improve human hepatotoxicity prediction, in vitro liver cell models replicating hepatocyte function, drug metabolism, and toxicity are required. Here, we present a protocol for creating 3D primary human hepatocyte (PHH) cell models using the RASTRUM Platform. We describe the process for PHH model generation; procedures for characterizing the PHH model, including viability, albumin production, and CYP450 inducibility; and drug treatment using acetaminophen and troglitazone. This protocol has applications in upscaling phenotypic hepatotoxicity applications.

The utility of Streptococcus mutans undecaprenol kinase for the chemoenzymatic synthesis of diverse non-natural isoprenoids.

Isoprene chemoenzymatic cascades (ICCs) overcome the complexity of natural pathways by leveraging a streamlined two-enzyme cascade, facilitating efficient synthesis of C5-isoprene diphosphate precursors from readily available alcohol derivatives. Despite the documented promiscuity of enzymes in ICCs, exploration of their potential for accessing novel compounds remains limited, and existing methods require additional enzymes for generating longer-chain diphosphates. In this study, we present the utility of Streptococcus mutans undecaprenol kinase (SmUdpK) for the chemoenzymatic synthesis of diverse non-natural isoprenoids. Using a library of 50 synthetic alcohols, we demonstrate that SmUdpK's promiscuity extends to allylic chains as small as four carbons and benzylic alcohols with various substituents. Subsequently, SmUdpK is utilized in an ICC with isopentenyl phosphate kinase and aromatic prenyltransferase to generate multiple non-natural isoprenoids. This work provides evidence that, with proper optimization, SmUdpK can act as the first enzyme in these ICCs, enhancing access to both valuable and novel compounds.

The Phenotypic and Molecular Identification of Phyllospheric Bacteria Possessing Antimicrobial Activity from Funtumia elastica (Preuss) Stapf.

Background: Unlike plant phytochemicals, little has been done to explore the metabolites from phyllosphere bacterial flora, some of which enabled them to survive interspecific competition through amensalism. This study evaluated the antimicrobial activity of metabolites from Phyllospheric Bacteria (PB) isolated from Funtumia elastica (FE), against selected bacterial and fungal pathogens. Phenotypic and molecular methods were used to identify the isolated phyllo-microbiota. Methods: The PB were aseptically isolated by sonication. Their metabolites were obtained from the fresh overnight culture of the organisms. The cell-free supernatants containing the metabolites were used for antimicrobial assays against the pathogens. The DNA of the bacterial isolates were isolated using a NIMR-BIOTECH DNA extraction kit, while their 16S rRNA was amplified with the primer: 799F 5'-AACACGGATTA GATACC-3', 1193R 5'- ACGTCATCCCCACCTTCC-3', using SolisFast* Master Mix, (Solis Biodyne-Estonia). The BLAST of the sequence was done from the NCBI Gen-bank. The PB strains identified were submitted to NCBI and accession numbers were assigned to them. Results: The phyllosphere of FE yielded 21 bacterial isolates: 7 Gram-positives and 14 Gram-negatives. The metabolites from these isolates showed varying degrees of bioactivity against Staphylococcus aureus (ATCC29213), Escherichia coli (ATCC 25922) Klebsiella pneumoniae (ATCC 35659); Trychophyton rubrum, Candida albicans and Microsporum canis. Fifteen bioactive isolates sequenced yielded four genera, Enterobacter (E. hormaechei 98.44%), Bacillus (B. cereus 100%), Pontoea (P. dispersa 99.72%), Staphylococcus (S. arlettae 99.72%). Conclusion: Bacteria from FE phyllosphere, produced metabolites antagonistic (cidal) to some human pathogens. This has great potential for possible drug discovery.

The Biotechnological Potential of Plant Growth-Promoting Rhizobacteria Isolated from Maize (Zea mays L.) Cultivations in the San Martin Region, Peru.

Maize (Zea mays L.) is an essential commodity for global food security and the agricultural economy, particularly in regions such as San Martin, Peru. This study investigated the plant growth-promoting characteristics of native rhizobacteria isolated from maize crops in the San Martin region of Peru with the aim of identifying microorganisms with biotechnological potential. Soil and root samples were collected from maize plants in four productive zones in the region: Lamas, El Dorado, Picota, and Bellavista. The potential of twelve bacterial isolates was evaluated through traits, such as biological nitrogen fixation, indole acetic acid (IAA) production, phosphate solubilization, and siderophore production, and a completely randomized design was used for these assays. A completely randomized block design was employed to assess the effects of bacterial strains and nitrogen doses on maize seedlings. The B3, B5, and NSM3 strains, as well as maize seeds of the yellow hard 'Advanta 9139' variety, were used in this experiment. Two of these isolates, B5 and NSM3, exhibited outstanding characteristics as plant growth promoters; these strains were capable of nitrogen fixation, IAA production (35.65 and 26.94 µg mL-1, respectively), phosphate solubilization (233.91 and 193.31 µg mL-1, respectively), and siderophore production (34.05 and 89.19%, respectively). Furthermore, molecular sequencing identified the NSM3 isolate as belonging to Sporosarcina sp. NSM3 OP861656, while the B5 isolate was identified as Peribacillus sp. B5 OP861655. These strains show promising potential for future use as biofertilizers, which could promote more sustainable agricultural practices in the region.

Regulated induced proximity targeting chimeras-RIPTACs-A heterobifunctional small molecule strategy for cancer selective therapies.

We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.

Increasing resistance of lawn grasses to zinc and copper pollution.

Coppe and zinc are priority pollutants in city soils. Copper and zinc are the limiting factors for lawn grasses. Cell selection can increase the resistance of lawn grasses to zinc and copper. The effect of zinc on the morphogenic ability of the callus was determined. The results of this study showed that zinc is less toxic to calli than copper. The method of obtaining lawn grass resistant to zinc has been developed. The results were used to develop the cell selection technology for obtaining plants resistant to the complex effect of zinc and copper. Concentrations of Copper (75 mg/l) and zinc (150 mg/L) were selected as selective. The author developed the cell selection scheme for obtaining plants resistant to the complex effect of Cu and Zn. The regenerants showed increased resistance to copper and zinc.

Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes.

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.

Venom Ex Machina? Exploring the Potential of Cell-Free Protein Production for Venom Biodiscovery.

Venoms are a complex cocktail of potent biomolecules and are present in many animal lineages. Owed to their translational potential in biomedicine, agriculture and industrial applications, they have been targeted by several biodiscovery programs in the past. That said, many venomous animals are relatively small and deliver minuscule venom yields. Thus, the most commonly employed activity-guided biodiscovery pipeline cannot be applied effectively. Cell-free protein production may represent an attractive tool to produce selected venom components at high speed and without the creation of genetically modified organisms, promising rapid and highly efficient access to biomolecules for bioactivity studies. However, these methods have only sporadically been used in venom research and their potential remains to be established. Here, we explore the ability of a prokaryote-based cell-free system to produce a range of venom toxins of different types and from various source organisms. We show that only a very limited number of toxins could be expressed in small amounts. Paired with known problems to facilitate correct folding, our preliminary investigation underpins that venom-tailored cell-free systems probably need to be developed before this technology can be employed effectively in venom biodiscovery.

Evolutionary algorithms simulating molecular evolution: a new field proposal.

The genetic blueprint for the essential functions of life is encoded in DNA, which is translated into proteins-the engines driving most of our metabolic processes. Recent advancements in genome sequencing have unveiled a vast diversity of protein families, but compared with the massive search space of all possible amino acid sequences, the set of known functional families is minimal. One could say nature has a limited protein "vocabulary." A major question for computational biologists, therefore, is whether this vocabulary can be expanded to include useful proteins that went extinct long ago or have never evolved (yet). By merging evolutionary algorithms, machine learning, and bioinformatics, we can develop highly customized "designer proteins." We dub the new subfield of computational evolution, which employs evolutionary algorithms with DNA string representations, biologically accurate molecular evolution, and bioinformatics-informed fitness functions, Evolutionary Algorithms Simulating Molecular Evolution.

Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

Developing antibodies with high specificity against post-translationally modified epitopes remains a challenge. Yeast biopanning is well suited in screening for high-specificity binders. Here, we present a protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches. We describe steps for screening a yeast surface display library for antibodies and other binders. We then detail procedures for validating the antibodies found by analyzing their specificity through whole-well image analysis in 96-well plates. For complete details on the use and execution of this protocol, please refer to Arbaciauskaite et al.1.

Exploring industrial lignocellulosic waste: Sources, types, and potential as high-value molecules.

Lignocellulosic biomass has a promising role in a circular bioeconomy and may be used to produce valuable molecules for green chemistry. Lignocellulosic biomass, such as food waste, agricultural waste, wood, paper or cardboard, corresponded to 15.7% of all waste produced in Europe in 2020, and has a high potential as a secondary raw material for industrial processes. This review first presents industrial lignocellulosic waste sources, in terms of their composition, quantities and types of lignocellulosic residues. Secondly, the possible high added-value chemicals obtained from transformation of lignocellulosic waste are detailed, as well as their potential for applications in the food industry, biomedical, energy or chemistry sectors, including as sources of polyphenols, enzymes, bioplastic precursors or biofuels. In a third part, various available transformation treatments, such as physical treatments with ultrasound or heat, chemical treatments with acids or bases, and biological treatments with enzymes or microorganisms, are presented. The last part discusses the perspectives of the use of lignocellulosic waste and the fact that decreasing the cost of transformation is one of the major issues for improving the use of lignocellulosic biomass in a circular economy and green chemistry approach, since it is currently often more expensive than petroleum-based counterparts.

Directional freezing and thawing of biologics in drug substance bottles.

Biological drug substance (DS) is typically stored frozen to increase stability. However, freezing and thawing (F/T) of DS can impact product quality and therefore F/T processes need to be controlled. Because active F/T systems for DS bottles are lacking, freezing is often performed uncontrolled in conventional freezers, and thawing at ambient temperature or using water baths. In this study, we evaluated a novel device for F/T of DS in bottles, which can be operated in conventional freezers, generating a directed air stream around bottles. We characterized the F/T geometry and process performance in comparison to passive F/T using temperature mapping and analysis of concentration gradients. The device was able to better control the F/T process by inducing directional bottom-up F/T. As a result, it reduced cryo-concentration during freezing as well as ice mound formation. However, freezing with the device was dependent on freezer performance, i.e. prolonged process times in a highly loaded freezer were accompanied by increased cryo-concentrations. Thawing was faster compared to without the device, but had no impact on concentration gradients and was slower compared to thawing in a water bath. High-performance freezers might be required to fully exploit the potential of directional freezing with this device and allow F/T process harmonization and scaling across sites.

New solutions in transplantology and graft acquisition.

In view of rapid advancements in the field of transplantology, emerging solutions in tissue procurement for transplantation became a crucial area of research. Tissue transplantation plays a notable role in improving the quality of life for patients afflicted with various ailments, and the increasing number of transplants necessitates the exploration of innovative procurement methods. This study examines a new direction in transplantology, placing focus on innovative approaches to tissue procurement and discussing the commonly used method of "ex mortuo," i.e., retrieving organs from deceased donors. Given the growing demand for organs, the paper discusses the innovative approach slowly emerging as 3D bioprinting. The paper discusses the key challenges associated with the use of this method in transplantology, including issues of biocompatibility, vascularization, and integration with the immune system. The paper also discusses the latest scientific achievements in the field, such as the first transplants of bioprinted organs, demonstrating the practical application of this technology in medicine. It is also the analysis of the ethical, social, and legal aspects related to these new solutions. The article provides a comprehensive overview of the latest trends in transplantology and presents a holistic view of the current state of knowledge and prospects for development in this pivotal area of medicine.