Biological Production Research Articles

Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440

Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.

Hydrocarbons in waters and bottom sediments of the coastal areas of the Caucasian sector of the Black Sea (2021–2023)

The current level of aliphatic and polycyclic aromatic hydrocarbons was determined in suspended particulate matter of waters (mainly in surface waters) and in upper layer of bottom sediments of coastal areas in the Caucasian sector of the Black Sea of the Russian Federation (September 2021, May and September 2022, March 2023). IR method was used to determine the aliphatic hydrocarbons, and the method of high performance liquid chromatography was employed for PAHs. The average concentration of aliphatic hydrocarbons was ≈20 μg/L and for polycyclic aromatic hydrocarbons ≈130 ng/L in suspended particulate matter in open surface waters of Gelendzhik and Golubaya bays. Their content naturally increased as they approached the coast. Despite the decrease in hydrocarbon concentrations in surface waters in recent years, the Kerch Strait and Novorossiysk remain the most polluted areas as before. Higher concentrations in the waters of the Taman Peninsula may be caused by the seepage of hydrocarbons from the sedimentary layer. Hydrocarbons are accumulated in bottom sediments, that leads to their rising portion in total organic carbon (e.g., aliphatic hydrocarbons up to 14.2 % in the Tuapse region and 13.1 % in the Novorossiysk region). Along with contamination from anthropogenic sources, natural processes also affect the hydrocarbon levels such as high biological productivity of the area and fluid flows from the sediment layer.

Productivity of the Bakchar bog plant communities in the initial stages of pyrogenic succession (southern taiga of Western Siberia)

The results of a study on the productivity of plant communities after a crown fire in a drained oligotrophic bog in the southern taiga of Western Siberia are presented. A quantitative assessment of biological productivity in areas with varying degrees of pyrogenic disturbance is given. The reserves of the grass-shrub and moss layers in the most disturbed area in the second year of restoration amounted to 1220 g/m2, production was 324 g/m2 per year; in the third year, reserves increased to 1635 g/m2 and production increased to 1038 g/m2 per year due to the growth of blueberries, while the production in the control site with high reserves of living phytomass (2200 g/m2) amounted to 745 g/m2 per year. There was a change in the structure of the phytomass, compaction of the deposit by 34%, and an increase in the mortmass of vascular plants by three times. The living mass of roots in the disturbed area concentrated in the upper layer of 0–10 cm, while it was distributed in the control area in the layer of 10–30 cm. It was found that the roots of dead pines retain the morphological characteristics of living ones during the first two years and pass into the fraction of dead roots on the third year of pyrogenic succession. Carbon losses during burning of the top layer of tow up to 20 cm amounted to 3016 g C/m2, 500 g/m2 of which can be buried as a layer of coal in peat.

Customized 3D-Printed Mesh, Membrane, Bone Substitute, and Dental Implant Applied to Guided Bone Regeneration in Oral Implantology: A Narrative Review.

Background: The new frontiers of computer-based surgery, technology, and material advances, have allowed for customized 3D printed manufacturing to become widespread in guided bone regeneration (GBR) in oral implantology. The shape, structural, mechanical, and biological manufacturing characteristics achieved through 3D printing technologies allow for the customization of implant-prosthetic rehabilitations and GBR procedures according to patient-specific needs, reducing complications and surgery time. Therefore, the present narrative review aims to elucidate the 3D-printing digital radiographic process, materials, indications, 3D printed manufacturing-controlled characteristics, histological findings, complications, patient-reported outcomes, and short- and long-term clinical considerations of customized 3D printed mesh, membranes, bone substitutes, and dental implants applied to GBR in oral implantology. Methods: An electronic search was performed through MEDLINE/PubMed, Scopus, BioMed Central, and Web of Science until 30 June 2024. Results: Three-dimensionally printed titanium meshes and bone substitutes registered successful outcomes in vertical/horizontal bone defect regeneration. Three-dimensionally printed polymeric membranes could link the advantages of conventional resorbable and non-resorbable membranes. Few data on customized 3D printed dental implants and abutments are available, but in vitro and animal studies have shown new promising designs that could improve their mechanical properties and tribocorrosion-associated complications. Conclusions: While 3D printing technology has demonstrated potential in GBR, additional human studies are needed to evaluate the short- and long-term follow-up of peri-implant bone levels and volumes following prosthetic functional loading.

Rational Design of Untranslated Regions to Enhance Gene Expression

How to improve gene expression by optimizing mRNA structures is a crucial question for various medical and biotechnological applications. Previous efforts focus largely on investigation of the 5′ UTR hairpin structures. In this study, we present a rational strategy that enhances mRNA stability and translation by engineering both the 5′ and 3′ UTR sequences. We have successfully demonstrated this strategy using green fluorescent protein (GFP) as a model in Escherichia coli and across different expression vectors. We further validated it with luciferase and Plasmodium falciparum lactate dehydrogenase (PfLDH). To elucidate the underlying mechanism, we have quantitatively analyzed both protein, mRNA levels and half-life time. We have identified several key aspects of UTRs that significantly influence mRNA stability and protein expression in our system: (1) The optimal length of the single-stranded spacer between the stabilizer hairpin and ribosome binding site (RBS) in the 5′ UTR is 25–30 nucleotide (nt) long. An optimal 32% GC content in the spacer yielded the highest levels of GFP protein production. (2) The insertion of a homodimerdizable, G-quadruplex structure containing RNA aptamer, “Corn”, in the 3′ UTR markedly increased the protein expression. Our findings indicated that the carefully engineered 5′ UTRs and 3′ UTRs significantly boosted gene expression. Specifically, the inclusion of 5 × Corn in the 3′ UTR appeared to facilitate the local aggregation of mRNA, leading to the formation of mRNA condensates. Aside from shedding light on the regulation of mRNA stability and expression, this study is expected to substantially increase biological protein production.

Engineered CD4 T cells for in vivo delivery of therapeutic proteins

The CD4 T cell, when engineered with a chimeric antigen receptor (CAR) containing specific intracellular domains, has been transformed into a zero-order drug-delivery platform. This introduces the capability of prolonged, disease-specific engineered protein biologics production, at the disease site. Experimental findings demonstrate that CD4 T cells offer a solution when modified with a CAR that includes 4-1BB but excludes CD28 intracellular domain. In this configuration, they achieve ~3X transduction efficiency of CD8 T cells, ~2X expansion rates, generating ~5X more biologic, and exhibit minimal cytolytic activity. Cumulatively, this addresses two main hurdles in the translation of cell-based drug delivery: scaling the production of engineered T cell ex vivo and generating sufficient biologics in vivo. When programmed to induce IFNβ upon engaging the target antigen, the CD4 T cells outperforms CD8 T cells, effectively suppressing cancer cell growth in vitro and in vivo. In summary, this platform enables precise targeting of disease sites with engineered protein-based therapeutics while minimizing healthy tissue exposure. Leveraging CD4 T cells' persistence could enhance disease management by reducing drug administration frequency, addressing critical challenges in cell-based therapy.

Microbial community respiration kinetics and their dynamics in coastal seawater

Oxygen (O2) concentrations in coastal seawater have been declining for decades and models predict continued deoxygenation into the future. As O2 declines, metabolic energy use is progressively channelled from higher trophic levels into microbial community respiration, which in turn influences coastal ecology and biogeochemistry. Despite its critical role in deoxygenation and ecosystem functioning, the kinetics of microbial respiration at low O2 concentrations in coastal seawater remain uncertain and are mostly modeled based on parameters derived from laboratory cultures and a limited number of environmental observations. To explore microbial responses to declining O2, we measured respiration kinetics in coastal microbial communities in Hong Kong over the course of an entire year. We found the mean maximum respiration rate (Vmax) ranged between 560 ± 280 and 5930 ± 800 nmol O2 L−1 h−1, with apparent half-saturation constants (Km) for O2 uptake of between 50 ± 40 and 310 ± 260 nmol O2 L−1. These kinetic parameters vary seasonally in association with shifts in microbial community composition that were linked to nutrient availability, temperature, and biological productivity. In general, coastal communities in Hong Kong exhibited low affinities for O2, yet communities in the dry season had higher affinities, which may play a key role in shaping the relationship between community size, biomass, and O2 consumption rates through respiration. Overall, parameters derived from these experiments can be employed in models to predict the expansion of deoxygenated waters and associated effects on coastal ecology and biogeochemistry.

Microalgae bio-reactive façade: System thermal–biological optimization

This article explores numerically the biotechnological performances of microalgae biofaçade. The model computes the system’s thermal behavior using a radiative-convective approach accounting for location on Earth and actual weather data. In a coupled manner, it simulates the microalgae culture behavior, i.e. light-driven growth and cell pigment content acclimation. In addition, it features refinement such as wavelength-dependent biomass optical properties and thermal-modulated biological rates. Thanks to this model, operation strategies and design possibilities were evaluated using actual weather data for a biofaçade module deployed in Marseille in 2023. Investigations revealed that a semi-batch mode of operation, while simplistic, is the most efficient way to operate a biofaçade if sole biological production is considered (about 18.0 ± 0.9 kg per year, 2.44 ± 0.12 g/L output concentration). However, if intended as an office glazing, turbidostat mode of operation should be preferred for aesthetic and visual comfort reasons (about 19.1 ± 1.1 kg per year, 0.64 ± 0.07 g/L output concentration). System optimization also confirmed the experimental observation that the system could be prone to overheating. Nevertheless, while overheating can be mitigated by increasing the reservoir thickness, this strategy is detrimental to the average output concentration. Finally, location-specific optimization revealed that a standard biofaçade module could be deployed over France, and system performances are derived for the whole country thanks to the weather forecast agency data.

Manufacturing Process Affects Coagulation Kinetics of Ortho-R, an Injectable Chitosan-Platelet-Rich Plasma Biomaterial for Tissue Repair.

Ortho-R (ChitogenX Inc., Kirkland, QC, Canada) is an injectable combination drug-biologic product that is used as an adjunct to augment the standard of care for the surgical repair of soft tissues. The drug product comprises lyophilized chitosan, trehalose and calcium chloride, and it is dissolved in platelet-rich plasma (PRP), a blood-derived biologic, prior to injection at the surgical site where it will coagulate. The first step of the Ortho-R manufacturing process involves dissolving the chitosan in hydrochloric acid. The purpose of this study was to investigate the effect of increasing the amount of acid used to dissolve the chitosan on final drug product performance, more specifically, on the chitosan-PRP coagulation kinetics. Chitosans were solubilized in hydrochloric acid, with concentrations adjusted to obtain between 60% and 95% protonation of the chitosan amino groups. Freeze-dried Ortho-R was solubilized with PRP, and coagulation was assessed using thromboelastography (TEG). The clotted mixtures were observed with histology. Clot reaction time (TEG R) increased and clot maximal amplitude (TEG MA) decreased with protonation levels as pH decreased. Chitosan distribution was homogeneous in chitosan-PRP clots at the lowest protonation levels, but it accumulated toward the surface of the clots at the highest protonation levels as pH decreased. These changes in coagulation kinetics, clot strength and chitosan distribution induced by high protonation of the chitosan amino groups were partially reversed by adding sodium hydroxide to the dissolved chitosan component in order to decrease pH. Careful control of manufacturing processes is critical, and it is important to consider the impact of each manufacturing step on product performance.

Air-sea CO2 exchange in the western Pacific influenced by monsoon and giant diatom (Ethmodiscus rex) blooms during the last deglaciation

The concentration of atmospheric CO2 increased rapidly during the last deglaciation due to CO2 outgassing from oceans. However, records of deglacial surface seawater pCO2-sw are sparse, hindering our understanding of the process and mechanism of air-sea CO2 exchange and its influence on glacial-interglacial climate change. Here we reconstructed surface seawater pCO2-sw for the last deglacial period using carbon isotope composition (δ13C) of giant diatom (Ethmodiscus rex) frustules from deep-sea sedimentary core collected in the Philippine Sea, western Pacific. Results showed that air-sea CO2 was fluctuating in the western Pacific during the last deglaciation. The gradients of air-sea CO2 are dominated by monsoon and biological productivity. The enhanced East Asian winter Monsoon and shallow thermocline during late Heinrich Stadial 1 maintained equilibrium in the air-sea CO2 exchange balance. During the Bølling period, enhanced East Asian Summer Monsoon has been observed to accelerate the dissolution of eolian-dust and promoted the growth of Ethmodiscus rex, which has been linked to increased primary productivity and, consequently, the uptake of atmospheric CO2 in the western Pacific. During the Allerød period, continued enhancement of EASM allowed the Philippine Sea to act as a weak CO2 source releasing CO2 to the atmosphere. During the Younger Dryas period, as the EASM weaken and the EAWM strengthen, ΔpCO2(sw-atm) decreased. Our findings highlight the tropical ocean’s role in deglacial air-sea CO2 exchange and provide insights into the monsoonal and biological drivers of the processes.

Diatom silicon isotope ratios in Quaternary research: Where do we stand?

Silicon stable isotope ratios (expressed as δ30Si) in biogenic silica have been widely used as a proxy for past and present biogeochemical cycling in both marine and lacustrine settings, in particular for nutrient utilization reconstructions. Yet an analysis of publication trends suggests a significant decline in the application of δ30Si to Quaternary science questions in the last five years. At the same time as δ30Si proxy applications have decreased, we are learning more about its complexities: an expanding body of work is highlighting biases, caveats or complications involved in the application of δ30Si-based approaches to the sediment record. These include the demonstration of species-specific silicon isotope fractionation factors (i.e. ‘vital effects’) or the potential for Fe or other trace metals to influence silicon isotope fractionation. Others have inferred the potential of biogenic silica dissolution to alter an initial δ30Si value, or questioned the preservation of the initial δ30Si through early diagenetic processes more generally. Another challenge receiving more attention is centered around deconvolving a δ30Si-value into a signal reflecting biological productivity and a signal reflecting changes in the δ30Si of dissolved silicon driven by whole-system and/or circulation changes. Finally, a number of studies focus on analytical difficulties, especially during sample preparation related to achieving and demonstrating a contaminant free biogenic silica. These challenges lead us to posit that the Quaternary science community is moving away from silicon isotope proxies because they are losing confidence in their reliability and usefulness. Here, focusing on the diatoms – the dominant biosilicifiers in both lakes and the ocean – we synthesize progress in understanding nuances and caveats of δ30Si-based proxies in order to answer whether the fall-off in δ30Si-based Quaternary research is warranted. We suggest that with some simple steps that can be readily implemented, and with the closing of key knowledge gaps, there is no reason to believe silicon isotopes do not have a promising future in the Quaternary sciences.

Selecting the best-value biosimilar in emerging countries

The aim of biosimilars is to alleviate the financial burden of biological medicinal products. A most relevant challenge for emerging countries is how to select the best option available. In most cases, price is the major determinant, as budgets are chronically scarce. However, initial savings due to price reductions can be overridden if there is a lack of supply due to product shortages or withdrawals. These events can be prevented by a best-value strategy. According to the concept of best-value medicinal products, price is only one of the various criteria to be considered. The purpose of the present paper is to provide suggestions of criteria that can be useful for selecting the best-value biological in emerging countries. Six criteria, that are not limitative, have been selected as follows: standards of regulatory approval, quality of the product, good distribution practices, security of supply, pharmacovigilance, and price.

Staying Out of Trouble: FDA Regulation of Orthobiologics and the Shoulder Surgeon

Food and Drug Administration (FDA) regulation of Orthobiologics can be challenging to interpret for the non-regulatory scientist. However, understanding how regulations apply to clinical use of these orthobiologic products is critical, as there are both ethical and legal ramifications to using orthobiologics in conflict with regulations. Recent FDA guidances have attempted to clarify these issues, although questions still remain regarding nuances in regulatory applications. FDA guidances, industry blogs, and relevant publications were searched for citations regarding recent orthobiologics and regulations. These sources were compiled into a current assessment of FDA regulations regarding the use of orthobiologics in the shoulder. Key to understanding these regulations is the FDA differentiation of human cellular and tissue based products (HCTPs) into 361 and 351 category products. While some controversy still exists, FDA has attempted to clarify these issues with several recent guidances. Of equal importance, FDA has ended enforcement discretion for many biologic products in June of 2021, creating a previously tolerated class of orthobiologics that now requires an Investigational New Drug (IND) application and subsequent Biologic License Application to legally market these orthobiologics. The Same Surgical Procedure Exception was further clarified in 2017 to exempt facilities from regulatory controls when specific guidelines are met. This article attempts to clarify the current thinking on FDA regulations and will allow the shoulder and elbow surgeon to stay within the current bounds of ethical and legal use of these products.

ANTICIPATING BIOSECURITY HAZARDS: DISTRIBUTION AND RECOGNITION OF BIOLOGICAL THREATS

Understanding the potential biological weapons that can be created, the technologies involved, and the entities capable of producing them is crucial for biosecurity. Equally important is comprehending the various targets a biological weapon might be aimed at, the deployment methods, and available detection strategies to counteract illegal biological threats. This chapter delves into these aspects, starting with methods of deployment. The Center for Disease Control and Prevention (CDC) categorizes disease transmission into direct and indirect modes. Direct transmission involves contact with an infected person or environmental reservoir, including droplet spread, proven highly effective in densely populated areas. Indirect transmission occurs through contaminated objects or vectors. Vehicle-based transmission involves contaminating inanimate objects like food, water, and biological products. Vector-based transmission involves organisms carrying and transmitting pathogens. Airborne transmission, receiving significant attention for its potential in biological attacks, involves pathogens suspended in the air, posing significant risks, especially in crowded areas. Biological weapons could target humans, agriculture, technology, and the environment. Attacks on humans, often the focus of biosecurity efforts, pose catastrophic outcomes. State-sponsored programs often focus on human pathogens like Bacillus anthracis and Yersinia pestis. Agroterrorism, targeting agriculture and livestock, can lead to economic losses and social instability. Attacks on technology, utilizing synthetic biology, have shown the potential to manipulate DNA to store and deploy malicious code, highlighting vulnerabilities in sequencing systems. While attacks on the environment remain theoretical, advancements in gene drives raise concerns about manipulating ecosystems. Effective biosecurity involves pre- and post-deployment screening techniques. Pre-deployment screening, particularly gene synthesis, focuses on preventing the acquisition of DNA fragments for weapon production. Current measures involve background checks and screening against pathogen lists, yet gaps exist, including inadequate international policies and the potential to hide malicious sequences within longer ones. Post-deployment detection relies on traditional molecular diagnostic methods, but synthetic pathogens may evade detection, necessitating ongoing advancements in detection techniques. Real-time sensors and biosensors offer promising avenues for quick and accurate pathogen recognition, although challenges remain in detecting novel pathogens against a natural background. In conclusion, understanding the range of biological weapons, their potential targets, deployment methods, and detection strategies is essential for effective biosecurity. Continued research and advancements in detection technologies are crucial to staying ahead of evolving biological threats, ensuring a safer and more secure future.

Aflibercept Biosimilar MYL-1701P vs Reference Aflibercept in Diabetic Macular Edema

Biosimilars may be lower-cost alternatives to originator biologic products, potentially offering expanded access or reduced economic burden, but have not been evaluated with aflibercept in diabetic macular edema (DME). To compare efficacy and safety of MYL-1701P, an aflibercept biosimilar, with reference aflibercept (Eylea [Regeneron]) in DME. This was a double-masked, randomized clinical trial that included participants at 77 centers across the US, Europe, Japan, and India. Included in the analysis were individuals 18 years and older with type 1 or type 2 diabetes with central DME and best-corrected visual acuity (BCVA) letter score of 73 to 38 in the study eye using an Early Treatment Diabetic Retinopathy Study (ETDRS) chart. Study data were analyzed from October to December 2021. Formulations of MYL-1701P (0.5-mg vial) or reference aflibercept every 4 weeks for 5 consecutive intravitreal injections, followed by every 8 weeks through week 52. The primary outcome was the adjusted difference in least squares mean (SE) change from baseline BCVA letter score at week 8 with an equivalence margin of -3 to +3 letters. Secondary outcomes included change in central subfield thickness (CST), BCVA, number of injections over 52 weeks, incidence of adverse events (AEs), and antidrug antibodies (ADAs). A total of 355 participants (mean [SD] age, 62.2 [9.2] years; 216 male [60.8%]) were randomized to MYL-1701P (179 participants [50.4%]) and aflibercept (176 participants [49.6%]). At week 8, mean (SE) change in BCVA was 6.60 (0.55) letters vs 6.56 (0.55) letters in the MYL-1701P vs aflibercept groups. The adjusted mean difference of 0.04 letters (90% CI, -1.16 to 1.24 letters) met the primary outcome. At week 8, mean (SE) change in CST was -112 (7) μm vs -124 (7) μm in the MYL-1701P vs aflibercept groups (adjusted mean difference, 12 μm; 90% CI, -3 to 26 μm). The incidence of treatment-emergent AEs in the MYL-1701P and aflibercept arms were ocular (30.9% [55 of 178] vs 29.5% [52 of 176]), serious ocular (0.6% [1 of 178] vs 1.1% [2 of 176]), nonocular (65.2% [116 of 178] vs 65.3% [115 of 176]), and serious nonocular (16.9% [30 of 178] vs 11.9% [21 of 176]). The mean (SD) total number of injections was 8.4 (2.1) vs 8.7 (1.8) in the MYL-1701P vs aflibercept groups. The incidence of treatment-induced or treatment-boosted ADAs was 2.8% (5 of 177) vs 5.7% (10 of 176) in the MYL-1701P vs aflibercept arms. MYL-1701P demonstrated clinical equivalence in regard to efficacy, with comparable safety and immunogenicity, to reference aflibercept. These findings support use of MLY-1701P as an alternative to reference aflibercept. ClinicalTrials.gov Identifier: NCT03610646.

Methane capture, utilization, and sequestration technology based on biological ectoine production using Methylotuvimicrobium alcaliphilum 20Z

Methanotroph-based CH4 conversion technology is a promising alternative to conventional CH4 utilization methods that rely on energy-intensive CO2 capture processes. In this study, we introduce a novel CH4 capture, utilization, and sequestration (MCUS) technology as a proof-of-concept for eco-friendly CH4 conversion that is applicable to various methanotroph strains. We validated this hypothesis by evaluating ectoine production in Methylotuvimicrobium alcaliphilum 20Z. Cultivation experiments were conducted using pure O2 pulse injection to facilitate the separation of the CO2 generated during CH4 oxidation by concentrating it. To verify the MCUS concept systematically, we performed a model-based process analysis. The operational strategies for the reactors were developed using experiment-based kinetics and reactor models, followed by the design of a rigorous process flow diagram. Economic and environmental impact analyses were conducted to compare MCUS with air-based cultures and existing oxidative CH4 conversion technologies. The key advantage of MCUS is its significantly lower CO2 emissions (0.71 kg-CO2 eq/kg-CH4) than oxidative CH4 utilization (>2.75 kg-CO2 eq/kg-CH4). Moreover, the net present value per CH4 molecule was estimated at 0.7 $/kg-CH4, substantially higher than existing oxidative conversion methods (0.03 $/kg-CH4). Additionally, compared with air-based cultivation, the MCUS process exhibits superior economic and environmental performances. We used methanotrophs for CH4 “capture” and “utilization,” effectively leveraging “sequestration” to convert emissions into pure CO2. The proposed MCUS technology holds promise for practical applications of methanotrophs in producing various high-value-added products, emphasizing eco-friendly approaches.

Exploring protein N-glycosylation in the green microalga Dunaliella salina

N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum where an oligosaccharide precursor is added to the newly synthesized protein and continues in the Golgi apparatus where the N-linked carbohydrate sequences are processed. Importantly, the most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly currently produced in mammalian cells which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered as efficient and eco-friendly alternative platforms for the production of biologics. However, unlike for C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man5GlcNAc2 oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii that gives rise to the synthesis of a non-canonical oligomannoside Man5GlcNAc2. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man5GlcNAc2.

Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013.

Biological production of commodity chemicals from abundant waste streams such as whey permeate represents an opportunity for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol promises to identify new targets that can be fine tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii for improved butanol production on lactose and ultimately in whey permeate.

Safety of Rituximab biosimilar (Riximyo®) following a single switch from the reference product in patients with Non-Hodgkin’s lymphoma: a retrospective study

Unlike small molecule drugs and generic products, the active component of biologics and biosimilars are not identical chemical entities. Despite bioequivalence, there is limited evidence in clinical practice (i.e. Phase IV post-marketing surveillance) regarding the safety of biosimilar rituximab and even less so for “switching therapy” with respect to safety. Drug substitution by switching aims to realise cost savings by changing therapy involving a reference (biologic) product to a biosimilar. A retrospective analysis of safety outcomes including treatment-emergent adverse effects (TEAEs), rates of death and discontinuation of therapy, for all patients that received switching therapy (from reference to biosimilar rituximab, n = 33) was compared to patients who did not did not switch therapy (received biosimilar rituximab only, n = 18) at an Australian metropolitan cancer centre, over a six-month period. There was no statistical significant differences for any safety outcomes examined. Switching therapy for patients receiving rituximab does not lead to poorer safety outcomes.

Ensuring Tree Protection, Growth and Sustainability by Microbial Isolates

Antimicrobial properties of the new strains of micro-organisms isolated from natural sources of various ecological niches in the Moscow region and the Republic of Tatarstan were studied. Antifungal activity of isolates was detected in a test culture of toxin-producing microscopic fungi that can cause animal and plant diseases: Aspergillus flavus, Candida albicans, Fusarium oxysporum and Penicillium spp. Of the 46 studied micro-organisms of genera Bacillus, Lactobacillus, Lactococcus and Streptomyces isolates, there are four strains (Bacillus subtilis, Lactobacillus plantarum, Propionibacterium freudenreichii and Streptomyces spp.) that showed an ability to produce biologically active metabolites with a pronounced antimicrobial potential against phytopathogenic fungi metabolites. Based on the selected four strains, a Bacterial product LRV composition has been created. Scots pine, pedunculate oak and small-leaved linden seedlings with single and double foliar treatment and Bacterial product LRV at a concentration of 10 mL/L led to an increase in the growth of the aboveground part by 31.8, 51.9 and 25.4%, respectively, and the underground part by 25.0, 37.2 and 25.7%, respectively, compared to the control. The weight of seedlings at the end of the study exceeded the control variant by an average of 26.0, 44.0 and 78.0%, respectively. Plant protection Bacterial product LRV use did not have a significant effect on the group of molds that caused the powdery mildew and Schütte disease damage to trees. The Biological product LRV provided plant protection from fungal diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides.