Production of Alginate Oligosaccharides (AOSs) Using Enhanced Physicochemical Properties of Immobilized Alginate Lyase for Industrial Application.

Alginate lyase (AL) is a polysaccharide-degrading enzyme that can degrade alginate by hydrolyzing glycosidic bonds and produces unsaturated alginate oligosaccharides (AOSs). These AOSs have wide therapeutic and nutraceutical applications. However, to produce alginate oligosaccharides in a cost-effective manner is challenging due to the low availability and high cost of this degrading enzyme. Immobilization of the enzyme facilitates industrial applications owing to its stability, reusability, and cost-effectiveness. This study was focused on the enhancement of the properties of alginate lyase and improvement of the production of AOS. Alginate lyase was immobilized on magnetic nanoparticles (NPs) using glutaraldehyde as the crosslinker. The study showed that the maximum binding achieved between NPs and protein in the enzyme was 71% at a ratio of 1:150 NP:protein. As a result of immobilization, the optimum activity of free enzyme which was obtained at 37 °C and pH 7.4 changed to 45 °C and pH 9. Furthermore, the enzyme was thermostable at 45 °C for 3 h with up to 50% reusability for six consecutive cycles. Storage stability after 15 days showed ~67% relative hydrolysis of alginate. The free alginate lyase (25 IU) showed 76% raw biomass (seaweed) hydrolysis which is higher compared to 63% provided by the immobilized enzyme. As a result of efficient hydrolysis, AOSs with molecular weight profile of 370-1040 kDa were produced and detected using HPLC.

Oleaginous Microbial Lipids' Potential in the Prevention and Treatment of Neurological Disorders.

The products of oleaginous microbes, primarily lipids, have gained tremendous attention for their health benefits in food-based applications as supplements. However, this emerging biotechnology also offers a neuroprotective treatment/management potential for various diseases that are seldom discussed. Essential fatty acids, such as DHA, are known to make up the majority of brain phospholipid membranes and are integral to cognitive function, which forms an important defense against Alzheimer's disease. Omega-3 polyunsaturated fatty acids have also been shown to reduce recurrent epilepsy seizures and have been used in brain cancer therapies. The ratio of omega-3 to omega-6 PUFAs is essential in maintaining physiological function. Furthermore, lipids have also been employed as an effective vehicle to deliver drugs for the treatment of diseases. Lipid nanoparticle technology, used in pharmaceuticals and cosmeceuticals, has recently emerged as a biocompatible, biodegradable, low-toxicity, and high-stability means for drug delivery to address the drawbacks associated with traditional medicine delivery methods. This review aims to highlight the dual benefit that lipids offer in maintaining good health for disease prevention and in the treatment of neurological diseases.

Therapeutic TNF Inhibitors Exhibit Differential Levels of Efficacy in Accelerating Cutaneous Wound Healing.

Adalimumab but neither etanercept nor certolizumab-pegol has been reported to induce a wound-healing profile in vitro by regulating macrophage differentiation and matrix metalloproteinase expression, which may underlie the differences in efficacy between various TNF-α inhibitors in impaired wound healing in patients with hidradenitis suppurativa, a chronic inflammatory skin disease. To examine and compare the efficacy of various TNF inhibitors in cutaneous wound healing in vivo, a human TNF knock-in Leprdb/db mouse model was established to model the impaired cutaneous wound healing as seen in hidradenitis suppurativa. The vehicle group exhibited severe impairments in cutaneous wound healing. In contrast, adalimumab significantly accelerated healing, confirmed by both histologic assessment and a unique healing transcriptional profile. Moreover, adalimumab and infliximab showed similar levels of efficacy, but golimumab was less effective, along with etanercept and certolizumab-pegol. In line with histologic assessments, proteomics analyses from healing wounds exposed to various TNF inhibitors revealed distinct and differential wound-healing signatures that may underlie the differential efficacy of these inhibitors in accelerating cutaneous wound healing. Taken together, these data revealed that TNF inhibitors exhibited differential levels of efficacy in accelerating cutaneous wound healing in the impaired wound-healing model in vivo.

Development of sustainable downstream processing for nutritional oil production.

Nutritional oils (mainly omega-3 fatty acids) are receiving increased attention as critical supplementary compounds for the improvement and maintenance of human health and wellbeing. However, the predominant sources of these oils have historically shown numerous limitations relating to desirability and sustainability; hence the crucial focus is now on developing smarter, greener, and more environmentally favourable alternatives. This study was undertaken to consider and assess the numerous prevailing and emerging techniques implicated across the stages of fatty acid downstream processing. A structured and critical comparison of the major classes of disruption methodology (physical, chemical, thermal, and biological) is presented, with discussion and consideration of the viability of new extraction techniques. Owing to a greater desire for sustainable industrial practices, and a desperate need to make nutritional oils more available; great emphasis has been placed on the discovery and adoption of highly sought-after 'green' alternatives, which demonstrate improved efficiency and reduced toxicity compared to conventional practices. Based on these findings, this review also advocates new forays into application of novel nanomaterials in fatty acid separation to improve the sustainability of nutritional oil downstream processing. In summary, this review provides a detailed overview of the current and developing landscape of nutritional oil; and concludes that adoption and refinement of these sustainable alternatives could promptly allow for development of a more complete 'green' process for nutritional oil extraction; allowing us to better meet worldwide needs without costing the environment.

Repurposing mucosal delivery devices for live attenuated tuberculosis vaccines.

Tuberculosis (TB) remains one of the most lethal infectious diseases globally. The only TB vaccine approved by the World Health Organization, Bacille Calmette-Guérin (BCG), protects children against severe and disseminated TB but provides limited protection against pulmonary TB in adults. Although several vaccine candidates have been developed to prevent TB and are undergoing preclinical and clinical testing, BCG remains the gold standard. Currently, BCG is administered as an intradermal injection, particularly in TB endemic countries. However, mounting evidence from experimental animal and human studies indicates that delivering BCG directly into the lungs provides enhanced immune responses and greater protection against TB. Inhalation therapy using handheld delivery devices is used for some diseases and allows the delivery of drugs or vaccines directly into the human respiratory tract. Whether this mode of delivery could also be applicable for live attenuated bacterial vaccines such as BCG or other TB vaccine candidates remains unknown. Here we discuss how two existing inhalation devices, the mucosal atomization device (MAD) syringe, used for influenza vaccines, and the Respimat® Soft Mist™ inhaler, used for chronic obstructive pulmonary disease (COPD) therapy, could be repurposed for mucosal delivery of live attenuated TB vaccines. We also outline the challenges and outstanding research questions that will require further investigations to ensure usefulness of respiratory delivery devices that are cost-effective and accessible to lower- and middle-income TB endemic countries.

Epithelial dysfunction is prevented by IL-22 treatment in a Citrobacter rodentium-induced colitis model that shares similarities with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal epithelial barrier leading to breach of barrier immunity. Here we identified similar protein expression changes between IBD and Citrobacter rodentium-infected FVB mice with respect to dysregulation of solute transporters as well as components critical for intestinal barrier integrity. We attribute the disease associated changes in the model to the emergence of undifferentiated intermediate intestinal epithelial cells. Prophylactic treatment with IL-22.Fc in C. rodentium-infected FVB mice reduced disease severity and rescued the mice from lethality. Multi-omics and solute analyses revealed that IL-22.Fc treatment prevented disease-associated changes including disruption of the solute transporter machinery and restored proper physiological functions of the intestine, respectively. Taken together, we established the disease relevance of the C. rodentium-induced colitis model to IBD, demonstrated the protective role of IL-22 in amelioration of epithelial dysfunction and elucidated the molecular mechanisms with IL-22's effect on intestinal epithelial cells.

Integration of the Exogenous Tuning of Thraustochytrid Fermentation and Sulfur Polymerization of Single-Cell Oil for Developing Plant-like Oils.

In this study, we have demonstrated a bioprocessing approach encompassing the exogenous addition of low-molecular-weight compounds to tune the fatty acid (FA) profile in a novel thraustochytrid strain to produce desirable FAs. Maximum lipid recovery (38%, dry wt. biomass) was obtained at 1% Tween 80 and 0.25 mg/L of Vitamin B12. The transesterified lipid showed palmitic acid (C16, 35.7% TFA), stearic acid (C18, 2.1% TFA), and oleic acid (C18:1, 18.7% TFA) as the main components of total FAs, which are mainly present in plant oils. Strikingly, D-limonene addition in the fermentation medium repressed the production of polyunsaturated fatty acid (PUFAs). Sulfur-polymerization-guided lipid separation revealed the presence of saturated (SFAs, 53% TFA) and monounsaturated fatty acids (MUFAs, 46.6% TFA) in thraustochytrid oil that mimics plant-oil-like FA profiles. This work is industrially valuable and advocates the use of sulfur polymerization for preparation of plant-like oils through tuneable thraustochytrid lipids.

Bioprospecting Indigenous Marine Microalgae for Polyunsaturated Fatty Acids Under Different Media Conditions.

Marine microalgae produce a number of valuable compounds that have significant roles in the pharmaceutical, biomedical, nutraceutical, and food industries. Although there are numerous microalgal germplasms available in the marine ecosystem, only a small number of strains have been recognized for their commercial potential. In this study, several indigenous microalgal strains were isolated from the coast of the Arabian Sea for exploring the presence and production of high-value compounds such as polyunsaturated fatty acids (PUFAs). PUFAs are essential fatty acids with multiple health benefits. Based on their high PUFA content, two isolated strains were identified by ITS sequencing and selected for further studies to enhance PUFAs. From molecular analysis, it was found both the strains were green microalgae: one of them was a Chlorella sp., while the other was a Planophila sp. The two isolated strains, together with a control strain known for yielding high levels of PUFAs, Nannochloropsis oculata, were grown in three different nutrient media for PUFA augmentation. The relative content of α-linolenic acid (ALA) as a percentage of total fatty acids reached a maximum of 50, 36, and 50%, respectively, in Chlorella sp., Planophila sp., and N. oculata. To the best of our knowledge, this is the first study in exploring fatty acids in Planophila sp. The obtained results showed a higher PUFA content, particularly α-linolenic acid at low nutrients in media.

Marine bioactives: from energy to nutrition.

Microalgae have been evaluated as promising resource for biodiesel production, but algal biofuel production is not yet commercially viable, which reflects the high energy costs linked with cultivation, harvesting, and dewatering of algae. As crude oil processing declines, microalgae biorefineries are being considered for producing bioactives such as enzymes, proteins, omega-3 oils, pigments, recombinant products, and vitamins, to offset the costs of biofuel production. We believe that producing algal bioactives through advanced manufacturing pathways, encompassing a biorefinery approach, would be effective, profitable, and economical.

Global status of lignocellulosic biorefinery: Challenges and perspectives.

The bioprocessing of lignocellulosic biomass to produce bio-based products under biorefinery setup is gaining global attention. The economic viability of this biorefinery would be inclined by the efficient bioconversion of all three major constituents of lignocellulosic biomass i.e. cellulose, hemicellulose, and lignin for value-added biochemicals and biofuels production. Although the lignocellulosic biorefinery setup has a clear value proposition, the commercial success at the industrial scale is still inadequate. This can be attributed mainly to irregular biomass supply chain, market uncertainties, and scale-up challenges. Global research efforts are underway by public and private sectors to get deeper market penetration. A comprehensive account of important factors, limitations, and propositions are worth consideration for the commercial success of lignocellulosic biorefineries. In this article, the importance of integration of lignocellulosic biorefineries with existing petrochemical refineries, the technical challenges of industrialization, SWOT analysis, and future directions have been reviewed.

Multiproduct biorefinery from marine thraustochytrids towards a circular bioeconomy.

Microalgal biotechnology research continues to expand due to largely unexplored marine environments and growing consumer interest in healthy products. Thraustochytrids, which are marine oleaginous protists, are known for their production of bioactives with significant applications in nutraceuticals, pharmaceuticals, and aquaculture. A wide range of high-value biochemicals, such as nutritional supplements (omega-3 fatty acids), squalene, exopolysaccharides (EPSs), enzymes, aquaculture feed, and biodiesel and pigment compounds, have been investigated. We discuss thraustochytrids as potential feedstocks to produce various bioactive compounds and advocate developing a biorefinery to offset production costs. We anticipate that future advances in cell manufacturing, lipidomic analysis, and nanotechnology-guided lipid extraction would facilitate large-scale cost-competitive production through these microbes.

Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed.

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn2+, Co2+, and Fe2+ increased the alginate degrading activity, but it was inhibited in the presence of Zn2+ and Cu2+. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.

Release of encapsulated bioactives influenced by alginate viscosity under in-vitro gastrointestinal model.

The physicochemical properties of alginate can affect the release profile of encapsulated bioactives, but this is poorly understood. The influence of alginate viscosity (low- A1, medium- A2 and high- A3) and molecular weight (kDa) on the release of encapsulated bioactives (seaweed and spirulina powder) was investigated in an in-vitro gastrointestinal (GSI) model. Beads encapsulated with A2 at 1% (w/v) have overall higher release of bioactives (protein, phlorotannins and antioxidants) but A3 at 0.5% (w/v) was able to release and absorb similar amount of bioactives with ~10% difference with A2. The relative release of protein, phlorotannins and antioxidant was 96%, 111% and 43% respectively from A2 in gastric digestion. In contrast, protein (165%) and phlorotannins (234%) release was highest from A3 in intestinal phase. These results establish the importance of physicochemical properties of the encapsulating matrix on water retention capacity and their interaction with bioactive material to release into the system.

Enzyme systems of thermophilic anaerobic bacteria for lignocellulosic biomass conversion.

Economic production of lignocellulose degrading enzymes for biofuel industries is of considerable interest to the biotechnology community. While these enzymes are widely distributed in fungi, their industrial production from other sources, particularly by thermophilic anaerobic bacteria (growth Topt ≥ 60 °C), is an emerging field. Thermophilic anaerobic bacteria produce a large number of lignocellulolytic enzymes having unique structural features and employ different schemes for biomass degradation, which can be classified into four systems namely; 'free enzyme system', 'cell anchored enzymes', 'complex cellulosome system', and 'multifunctional multimodular enzyme system'. Such enzymes exhibit high specific activity and have a natural ability to withstand harsh bioprocessing conditions. However, achieving a higher production of these thermostable enzymes at current bioprocessing targets is challenging. In this review, the research opportunities for these distinct enzyme systems in the biofuel industry and the associated technological challenges are discussed. The current status of research findings is highlighted along with a detailed description of the categorization of the different enzyme production schemes. It is anticipated that high temperature-based bioprocessing will become an integral part of sustainable bioenergy production in the near future.

Combination of Balsamin and Flavonoids Induce Apoptotic Effects in Liver and Breast Cancer Cells.

Flavonoids such as naringenin, quercetin, and naringin are known to exhibit anticancer properties. In this study, we examined the effects of these flavonoids on cell viability and apoptotic pathways of cancer cells, either singly or in combination with the type 1 ribosome inactivating protein, Balsamin. Treatment with flavonoids (naringenin, quercetin, and naringin) plus Balsamin for 48 h reduced HepG2 and MCF-7 cell viability, increased the activation of caspase-3 and -8, and induced apoptosis through up-regulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes. Out of the three flavonoids tested, the Balsamin-Naringenin and Balsamin-Quercetin combinations appeared to be most effective compared to the Balsamin-Naringin combination. Balsamin combined with flavonoids also activated endoplasmic reticulum (ER)-stress-mediated apoptosis in breast cancer (MCF-7) cells, which was not activated by Balsamin treatment alone. These experimental results showed that Balsamin combined with flavonoids can reduce HepG2 and MCF-7 cells viability and induce apoptosis, which could be considered as a promising therapeutic approach to sensitize cells to Balsamin treatment, thereby improving its efficacy in breast or liver cancer therapy.

Marine Protists and Rhodotorula Yeast as Bio-Convertors of Marine Waste into Nutrient-Rich Deposits for Mangrove Ecosystems.

This paper represents a comprehensive study of two new thraustochytrids and a marine Rhodotorula red yeast isolated from Australian coastal waters for their abilities to be a potential renewable feedstock for the nutraceutical, food, fishery and bioenergy industries. Mixotrophic growth of these species was assessed in the presence of different carbon sources: glycerol, glucose, fructose, galactose, xylose, and sucrose, starch, cellulose, malt extract, and potato peels. Up to 14g DW/L (4.6gDW/L-day and 2.8gDW/L-day) of biomass were produced by Aurantiochytrium and Thraustochytrium species, respectively. Thraustochytrids biomass contained up to 33% DW of lipids, rich in omega-3 polyunsaturated docosahexaenoic acid (C22:6, 124mg/g DW); up to 10.2mg/gDW of squalene and up to 61µg/gDW of total carotenoids, composed of astaxanthin, canthaxanthin, echinenone, and ß-carotene. Along with the accumulation of these added-value chemicals in biomass, thraustochytrid representatives showed the ability to secrete extracellular polysaccharide matrixes containing lipids and proteins. Rhodotorula sp lipids (26% DW) were enriched in palmitic acid (C16:0, 18mg/gDW) and oleic acid (C18:1, 41mg/gDW). Carotenoids (87µg/gDW) were mainly represented by ß-carotene (up to 54µg/gDW). Efficient growth on organic and inorganic sources of carbon and nitrogen from natural and anthropogenic wastewater pollutants along with intracellular and extracellular production of valuable nutrients makes the production of valuable chemicals from isolated species economical and sustainable.

The Nutritional and Pharmacological Potential of New Australian Thraustochytrids Isolated from Mangrove Sediments.

Mangrove sediments represent unique microbial ecosystems that act as a buffer zone, biogeochemically recycling marine waste into nutrient-rich depositions for marine and terrestrial species. Marine unicellular protists, thraustochytrids, colonizing mangrove sediments have received attention due to their ability to produce large amounts of long-chain ω3-polyunsaturated fatty acids. This paper represents a comprehensive study of two new thraustochytrids for their production of valuable biomolecules in biomass, de-oiled cakes, supernatants, extracellular polysaccharide matrixes, and recovered oil bodies. Extracted lipids (up to 40% of DW) rich in polyunsaturated fatty acids (up to 80% of total fatty acids) were mainly represented by docosahexaenoic acid (75% of polyunsaturated fatty acids). Cells also showed accumulation of squalene (up to 13 mg/g DW) and carotenoids (up to 72 µg/g DW represented by astaxanthin, canthaxanthin, echinenone, and ß-carotene). Both strains showed a high concentration of protein in biomass (29% DW) and supernatants (2.7 g/L) as part of extracellular polysaccharide matrixes. Alkalinization of collected biomass represents a new and easy way to recover lipid-rich oil bodies in the form of an aqueous emulsion. The ability to produce added-value molecules makes thraustochytrids an important alternative to microalgae and plants dominating in the food, pharmacological, nutraceutical, and cosmetics industries.

Nano-immobilized cellulases for biomass processing with application in biofuel production.

Several researches have focused on the enzymatic pretreatment of lignocellulose biomass to produce fermentable sugars that can lead to ethanol production thus facilitating pathways for sustainable biofuel production. Enzymes are fundamental to the pretreatment process, however, are required in larger quantities during pretreatment process thus influencing biofuel production cost. Immobilization of enzymes to a suitable support/matrix could enhance its stability, and reusability thus containing cost. This chapter focuses on developing an advanced technology for immobilizing enzymes to nanomaterials; variety of nanomaterials used for immobilization, nature of enzyme/protein nanomaterial interactions, methods of enzyme immobilization, and factors affecting mode of interaction for achieving hydrolysis of microcrystalline cellulose and natural cellulosic substrate. The binding of enzyme (94%) to a nanomaterial was established by spectroscopy techniques. The kinetics study, conducted at optimum pH (pH 4) and temperature (50°C for free and 60°C immobilized enzyme), exhibited improvement in immobilized enzyme properties. The immobilized enzyme retained up to 50% of its enzyme activity in five consecutive cycles. This chapter advocates the use of nano-immobilized enzymes in biomass hydrolysis for biofuel production.

Automated Machine Learning Diagnostic Support System as a Computational Biomarker for Detecting Drug-Induced Liver Injury Patterns in Whole Slide Liver Pathology Images.

Drug-induced liver injury (DILI) is a challenging disease to diagnose, a leading cause of acute liver failure, and responsible for drug withdrawal from the market. There is no symptom, no biomarker or test for detection, no therapy, but discontinuation of the drug. Pharmaceutical companies spend huge money, time, and scientific research efforts to test DILI effects and drug efficacy. A preclinical diagnostic support system is designed and proposed for DILI detection and classification on liver biopsy histopathology images. Heterogeneity features and automated machine learning (AutoML) models were tested to classify DILI injury patterns on whole slide image. Fractal and lacunarity values were used to detect hepatocellular necrotic injury patterns caused on a rat liver (in vivo) by 10 drugs at four dose levels. Correlations between fractal and lacunarity values were statistically analyzed for the 10 drugs; the Pearson correlation (r = 0.9809), p-value (1.6612E-06), and R2 (0.9582) were found to be high in the case of carbon tetrachloride. The AutoML model was tested to understand the injury patterns on a subset of 1,277 histology images. The AutoML algorithm was able to classify necrotic injury patterns accurately with an average precision of 98.6% on a score threshold of 0.5.