Nanoparticle-loaded microbubbles for treatment of lung cancer.

Lung cancer is one of the most common cancers and a leading cause of death, with poor prognosis and high unmet clinical need. Chemotherapy is a common part of the treatment, either alone or in combination with other treatment modalities, but with limited efficacy and severe side effects. Encapsulation of drugs into nanoparticles can enable a more targeted delivery with reduced off-target toxicity. Delivery to the lungs is however often insufficient due to various biological barriers in the body and in the tumor microenvironment. Here we demonstrate that by incorporating drug-loaded nanoparticles into air-filled microbubbles, a more effective targeting to the lungs can be achieved. Fluorescence imaging and mass spectrometry revealed that the microbubbles could significantly improve accumulation of drug in the lungs of mice, compared to injecting either the free drug by itself or only the drug-loaded nanoparticles. Therapeutic efficacy was verified in a preclinical mouse model with non-small cell lung cancer, monitoring tumor growth by luminescence.

Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics.

Magnetic-Responsive Carbon Nanotubes Composite Scaffolds for Chondrogenic Tissue Engineering.

The demand for engineered scaffolds capable of delivering multiple cues to cells continues to grow as the interplay between cell fate with microenvironmental and external cues is revealed. Emphasis has been given to develop stimuli-responsive scaffolds. These scaffolds are designed to sense an external stimulus triggering a specific response (e.g., change in the microenvironment, release therapeutics, etc.) and then initiate/modulate a desired biofunction. Here, magnetic-responsive carboxylated multi-walled carbon nanotubes (cMWCNTs) are integrated into 3D collagen/polylactic acid (PLA) scaffold via a reproducible filtration-based method. The integrity and biomechanical performance of the collagen/PLA scaffolds are preserved after cMWCNT integration. In vitro safety assessment of cMWCNT/collagen/PLA scaffolds shows neither cytotoxicity effects nor macrophage pro-inflammatory response, supporting further in vitro studies. The cMWCNT/collagen/PLA scaffolds enhance chondrocytes metabolic activity while maintaining high cell viability and extracellular matrix (i.e., type II collagen and aggrecan) production. Comprehensive in vitro study applying static and pulsed magnetic field on seeded scaffolds shows no specific cell response in dependence with the applied field. This result is independent of the presence or absence of cMWCNT into the collagen/PLA scaffolds. Taken together, these findings provide additional evidence of the benefits to exploit the CNTs outstanding properties in the design of stimuli-responsive scaffolds.

Sperm quality parameters, fertilizing potential, metabolites, and DNA methylation in cold-stored and cryopreserved milt from Atlantic salmon (Salmo salar L.).

Cold storage and freezing/thawing of milt may affect sperm functionality and the subsequent fertilization ability of milt. This study aimed to investigate sperm quality parameters and fertilization potential of Atlantic salmon milt, stored cold and subsequently cryopreserved, using different storage conditions. The objective was also to assess if analysis of milt metabolites and sperm DNA methylation signatures could be applicable to further elucidate sperm quality and fertilization following preservation. Milt samples were collected from eight mature Atlantic salmon males and stored for 4 days at 2°C and 8°C. Samples were taken on day one of storage at 2°C and on day four of storage at 2°C and 8°C. Storage for 4 days at 8°C is expected to be detrimental to sperm quality, and was included to create contrasts. Correspondingly, aliquots of cold-stored milt were prepared for cryopreservation, resulting in a total of six experimental conditions. Samples from all six experimental conditions were used in fertilization trials and analyzed for sperm viability, motility, ATP content, DNA fragmentation index, and High DNA stainability. In addition, milt samples from four of the males were analyzed for targeted metabolites and DNA methylation signatures by reduced representation bisulfite sequencing. The fertilization trials were performed using sperm:egg ratios of 75 × 103 and 500 × 103, respectively. Storage duration, temperature, and cryopreservation of cold-stored milt influenced several sperm quality parameters, metabolites, and DNA methylation signatures. The total motility, progressive motility, ATP, and velocity parameters were the sperm parameters with the strongest correlation to fertilization rates (p < 0.01). Several metabolites were correlated with fertility rates in both cold-stored and cryopreserved samples (p < 0.05). The fertilizing capacity of cold-stored milt was significantly reduced after 4 days of storage at 8°C, while corresponding cryopreserved milt showed reduced fertilization at both storage temperatures (2°C and 8°C) (p < 0.05). The results indicate that cryopreservation of milt stored for 1 day does not compromise either fertilization ability or DNA methylation signatures.

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors.

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20-25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reduction.

Effect of Radical Polymerization Method on Pharmaceutical Properties of Π Electron-Stabilized HPMA-Based Polymeric Micelles.

Polymeric micelles are among the most extensively used drug delivery systems. Key properties of micelles, such as size, size distribution, drug loading, and drug release kinetics, are crucial for proper therapeutic performance. Whether polymers from more controlled polymerization methods produce micelles with more favorable properties remains elusive. To address this question, we synthesized methoxy poly(ethylene glycol)-b-(N-(2-benzoyloxypropyl)methacrylamide) (mPEG-b-p(HPMAm-Bz)) block copolymers of three different comparable molecular weights (∼9, 13, and 20 kDa), via both conventional free radical (FR) and reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers were subsequently employed to prepare empty and paclitaxel-loaded micelles. While FR polymers had relatively high dispersities (D ∼ 1.5-1.7) compared to their RAFT counterparts (D ∼ 1.1-1.3), they formed micelles with similar pharmaceutical properties (e.g., size, size distribution, critical micelle concentration, cytotoxicity, and drug loading and retention). Our findings suggest that pharmaceutical properties of mPEG-b-p(HPMAm-Bz) micelles do not depend on the synthesis route of their constituent polymers.

High-throughput assay for effect screening of amphotericin B and bioactive components on filamentous Candida albicans.

AIMS: The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB). METHODS AND RESULTS: Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 µg ml-1 , and this was eightfold reduced (0.0038 µg ml-1 ) in the presence of the nutritional drinks. CONCLUSIONS: The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development.

Identification of novel cyanoacrylate monomers for use in nanoparticle drug delivery systems prepared by miniemulsion polymerisation - A multistep screening approach.

Poly (alkyl cyanoacrylate) (PACA) polymeric nanoparticles (NPs) are promising drug carriers in drug delivery. However, the selection of commercially available alkyl cyanoacrylate (ACA) monomers is limited, because most monomers were designed for use in medical and industrial glues and later repurposed for drug encapsulation. This study therefore aimed to seek out novel ACA materials for use in NP systems using a toxicity led screening approach. A multistep strategy, including cytotoxicity screening of alcohols as degradation products of PACA (44 alcohols), NPs (14 polymers), and a final in vivo study (2 polymers) gave poly (2-ethylhexyl cyanoacrylate) PEHCA as a promising novel PACA candidate. For the first time, this work presents cytotoxicity data on several novel ACAs, PEHCA in vivo toxicity data, and miniemulsion polymerisation-based encapsulation of the cabazitaxel and NR688 in novel PACA candidates. Furthermore, several of the ACA candidates were compatible with a wider selection of lipophilic active pharmaceutical ingredients (APIs) versus commercially available controls. Combined, this work demonstrates the potential benefits of expanding the array of available ACA materials in drug delivery. Novel ACAs have the potential to encapsulate a wider range of APIs in miniemulsion polymerisation processes and may also broaden PACA applicability in other fields.

Interlaboratory evaluation of a digital holographic microscopy-based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers.

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.

Standardization of an in vitro assay matrix to assess cytotoxicity of organic nanocarriers: a pilot interlaboratory comparison.

Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project "Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)" identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine.

Streptomyces tardus sp. nov.: A Slow-Growing Actinobacterium Producing Candicidin, Isolated From Sediments of the Trondheim Fjord.

Marine environments are home to an extensive number of microorganisms, many of which remain unexplored for taxonomic novelty and functional capabilities. In this study, a slow-growing Streptomyces strain expressing unique genomic and phenotypic characteristics, P38-E01 T , was described using a polyphasic taxonomic approach. This strain is part of a collection of over 8,000 marine Actinobacteria isolates collected in the Trondheim fjord of Norway by SINTEF Industry (Trondheim, Norway) and the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Strain P38-E01 T was isolated from the sediments of the Trondheim fjord, and phylogenetic analyses affiliated this strain with the genus Streptomyces, but it was not closely affiliated with other described species. The closest related type strains were Streptomyces daliensis YIM 31724 T (98.6%), Streptomyces rimosus subsp. rimosus ATCC 10970 T (98.4%), and Streptomyces sclerotialus NRRL ISP-5269 T (98.3%). Predominant fatty acids were C16:0 iso, C16:0, and Summed Feature 3, and the predominant respiratory quinones were MK-10(H6), MK-10(H4), and MK9(H4). The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and phosphoglycolipid. The whole-cell sugars were glucose, ribose, and in minor amounts, mannose. The cell wall peptidoglycan contained LL-diaminopimelic acid. The draft genome has a size of 6.16 Mb, with a %G + C content of 71.4% and is predicted to contain at least 19 biosynthetic gene clusters encoding diverse secondary metabolites. Strain P38-E01 T was found to inhibit the growth of the pathogenic yeast Candida albicans ATCC 90028 and a number of Gram-positive bacterial human and plant pathogens. Metabolites extracted from cultures of P38-E01 T were analyzed by mass spectrometry, and it was found that the isolate produced the antifungal compound candicidin. Phenotypic and chemotaxonomic signatures, along with phylogenetic analyses, distinguished isolate P38-E01 T from its closest neighbors; thus, this isolate represents a novel species of the genus Streptomyces for which the name Streptomyces tardus sp. nov. (P38-E01 T = CCM 9049 T = DSM 111582 T ) is proposed.

Streptomyces poriferorum sp. nov., a novel marine sponge-derived Actinobacteria species expressing anti-MRSA activity.

Marine sponges represent a rich source of uncharacterized microbial diversity, and many are host to microorganisms that produce biologically active specialized metabolites. Here, a polyphasic approach was used to characterize two Actinobacteria strains, P01-B04T and P01-F02, that were isolated from the marine sponges Geodia barretti (Bowerbank, 1858) and Antho dichotoma (Esper, 1794), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains P01-B04T and P01-F02 are closely related to Streptomyces beijiangensis DSM 41794T, Streptomyces laculatispora NRRL B-24909T, and Streptomyces brevispora NRRL B-24910T. The two strains showed nearly identical 16S rRNA gene sequences (99.93%), and the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness values were 99.96% and 99.6%, respectively, suggesting that these strains are affiliated with the same species. Chemotaxonomic and culture characteristics of both strains were also consistent with the genus Streptomyces, while phenotypic properties, genome-based comparisons, and phylogenomic analyses distinguished strains P01-B04T and P01-F02 from their closest phylogenetic relatives. In silico analysis predicted that the 8.9 Mb genome of P01-B04T contains at least 41 biosynthetic gene clusters (BGCs) encoding secondary metabolites, indicating that this strain could express diverse bioactive metabolites; in support of this prediction, this strain expressed antibacterial activity against Gram-positive bacteria including a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) EAMC30. Based on these results, the marine sponge-associated isolates represent a novel species of the genus Streptomyces, for which the name Streptomyces poriferorum sp. nov. is proposed, with P01-B04T (=DSM 111306T = CCM 9048T) as the type strain.

Author Correction: High-throughput screening reveals higher synergistic effect of MEK inhibitor combinations in colon cancer spheroids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Engineering lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze the hydroxylation of glycosidic bonds found in the most abundant and recalcitrant polysaccharides on Earth. Since their discovery in 2010, these enzymes have received extensive attention in both fundamental and applied research due to their remarkable oxidative power and synergistic interplay with hydrolytic enzymes. The harsh and unnatural conditions used in industrial enzymatic saccharification processes and the sensitivity of LPMOs for damage induced by reactive oxygen species call for enzyme engineering to develop LPMOs to become robust industrial biocatalysts. Other engineering targets include improved catalytic activity, adjusted substrate specificity and the introduction of completely new activities. Reaching these targets not only requires appropriate methods for measuring enzyme activity, but also requires in-depth knowledge of the active site and the reaction mechanism, which is yet to be achieved in the LPMO field. Here we describe what has been done in the LPMO engineering field so far. Furthermore, we address the difficulties involved in properly assessing LPMO functionality, which are due to common side reactions taking place in LPMO reactions and which complicate screening methods.

Differences in sperm functionality and intracellular metabolites in Norwegian Red bulls of contrasting fertility.

In the dairy breeding industry, prediction of bull fertility in artificial insemination (AI) is important for efficient and economically sustainable production. However, it is challenging to identify bulls with superior fertility applying conventional in vitro sperm assays. In the present study, sperm functionality was investigated to identify a multivariate model that could predict fertility. Two groups of young Norwegian Red bulls were selected, one with inferior fertility (18 bulls) and one with superior fertility (19 bulls) based on non-return rate after 56 days (NR56). Frozen-thawed semen doses were analysed for sperm chromatin integrity, viability, acrosome integrity, motility, and ATP content. A targeted approach was used to study intracellular concentrations of amino acids and trace elements in viable sperm cells. Significant differences between the two groups of bulls were observed, both for sperm functional attributes and intracellular concentrations of metabolites. Pearson correlation analyses indicated a negative relationship between NR56 and chromatin integrity parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS). Several motility parameters correlated positively with NR56. The concentrations of cysteine and glutamic acid in sperm cells correlated negatively with NR56, while the concentrations of aspartic acid, leucine and serine showed a positive NR56-correlation. The sperm intracellular concentrations of the trace elements Fe, Al and Zn, correlated negatively with NR56. Correlations were observed between several sperm parameters and metabolites. Stepwise multiple regression analysis indicated that the best predictor of NR56 was a model containing %DFI, together with the intracellular sperm concentration of aspartic acid, Fe and Zn. This model explained 59% of the variability in NR56.

High-throughput screening reveals higher synergistic effect of MEK inhibitor combinations in colon cancer spheroids.

Drug combinations have been proposed to combat drug resistance, but putative treatments are challenged by low bench-to-bed translational efficiency. To explore the effect of cell culture format and readout methods on identification of synergistic drug combinations in vitro, we studied response to 21 clinically relevant drug combinations in standard planar (2D) layouts and physiologically more relevant spheroid (3D) cultures of HCT-116, HT-29 and SW-620 cells. By assessing changes in viability, confluency and spheroid size, we were able to identify readout- and culture format-independent synergies, as well as synergies specific to either culture format or readout method. In particular, we found that spheroids, compared to 2D cultures, were generally both more sensitive and showed greater synergistic response to combinations involving a MEK inhibitor. These results further shed light on the importance of including more complex culture models in order to increase the efficiency of drug discovery pipelines.

Global Assessment of Mycobacterium avium subsp. hominissuis Genetic Requirement for Growth and Virulence.

Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth and organized ∼3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection.IMPORTANCE Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the Mycobacterium avium complex (MAC), whereof >90% are due to Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention, and diagnostics, we need to understand the underlying mechanisms of infection. Here, we provide crucial insights into MAH's global genetic requirements for growth and infection. We find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis (Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment or translate to broader treatment of related mycobacterial diseases.

Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

A high-throughput drug combination screen of targeted small molecule inhibitors in cancer cell lines.

While there is a high interest in drug combinations in cancer therapy, openly accessible datasets for drug combination responses are sparse. Here we present a dataset comprising 171 pairwise combinations of 19 individual drugs targeting signal transduction mechanisms across eight cancer cell lines, where the effect of each drug and drug combination is reported as cell viability assessed by metabolic activity. Drugs are chosen by their capacity to specifically interfere with well-known signal transduction mechanisms. Signalling processes targeted by the drugs include PI3K/AKT, NFkB, JAK/STAT, CTNNB1/TCF, and MAPK pathways. Drug combinations are classified as synergistic based on the Bliss independence synergy metrics. The data identifies combinations that synergistically reduce cancer cell viability and that can be of interest for further pre-clinical investigations.

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library.

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.