Biotechnology Research Research Articles

Induced pluripotent stem-cell-derived corneal epithelium for transplant surgery: a single-arm, open-label, first-in-human interventional study in Japan

The loss of corneal epithelial stem cells from the limbus at the edge of the cornea has severe consequences for vision, with the pathological manifestations of a limbal stem-cell deficiency (LSCD) difficult to treat. Here, to the best of our knowledge, we report the world's first use of corneal epithelial cell sheets derived from human induced pluripotent stem cells (iPSCs) to treat LSCD. This non-randomised, single-arm, clinical study involved four eyes of four patients with LSCD at the Department of Ophthalmology, Osaka University Hospital. They comprised a woman aged 44 years with idiopathic LSCD (patient 1), a man aged 66 years with ocular mucous membrane pemphigoid (patient 2), a man aged 72 years with idiopathic LSCD (patient 3), and a woman aged 39 years with toxic epidermal necrosis (patient 4). Allogeneic human iPSC-derived corneal epithelial cell sheets (iCEPSs) were transplanted onto affected eyes. This was done sequentially in two sets of HLA-mismatched surgeries, with patients 1 and 2 receiving low-dose cyclosporin and patients 3 and 4 not. The primary outcome measure was safety, ascertained by adverse events. These were monitored continuously throughout the 52-week follow-up period, and during an additional 1-year safety monitoring period. Secondary outcomes, reflective of efficacy, were also recorded. This study is registered with UMIN, UMIN000036539 and is complete. Patients were enrolled between June 17, 2019 and Nov 16, 2020. We had 26 adverse events during the 52-week follow-up period (consisting of 18 mild and one moderate event in treated eyes, and seven mild non-ocular events), with nine recorded in the additional 1-year safety monitoring period. No serious adverse events, such as tumourigenesis or clinical rejection, occurred during the whole 2-year observational period. At 52 weeks, secondary measures of efficacy showed that the disease stage had improved, corrected distance visual acuity was enhanced, and corneal opacification had diminished in all treated eyes. Corneal epithelial defects, subjective symptoms, quality-of-life questionnaire scores and corneal neovascularisation mostly improved or were unchanged. Overall, the beneficial efficacy outcomes achieved for patients 1 and 2 were better than those achieved for patients 3 and 4. iCEPS transplantation for LSCD was found to be safe throughout the study period. A larger clinical trial is planned to further investigate the efficacy of the procedure. The Japan Agency for Medical Research and Development, the Ministry of Education, Culture, Sports, Science, and Technology-Japan, and the UK Biotechnology and Biological Sciences Research Council.

Process Synthesis, Design and Techno-Economic Assessment of Malonic Acid Production

This work focuses on the design and techno-economic evaluation of an industrial facility for the production of malonic acid. The raw material utilized is commercial glucose syrup with a concentration of 95%. Based on a patent of Lygos, Inc., an innovative biotechnology research company, this study presents a comprehensive synthesis, design, and simulation framework for the production of malonic acid through oligosaccharide fermentation. An integrated process flowsheet is proposed and simulated using SuperPro Designer™. The analysis indicates that for an installation capacity of about 8000 MT/yr of the final product with a purity of 99.5%, the production cost is estimated at USD 7.92/kg. A comprehensive study of the capacity’s impact on economics reveals that this cost could decrease to as low as USD 6.05/kg. A parametric analysis and optimization conducted at the flowsheet level identifies opportunities for further reducing production costs, laying the groundwork for a potential decrease in the product’s selling price.

Split Probe-Induced Protein Translational Amplification for Nucleic Acid Detection.

Nucleic acid detection is important in a wide range of applications, including disease diagnosis, genetic testing, biotechnological research, environmental monitoring, and forensic science. However, the application of nucleic acid detection in various fields is hindered by the lack of sensitive, accurate, and inexpensive methods. This study introduces a simple approach to enhance the sensitivity for the accurate detection of nucleic acids. Our approach combined a split-probe strategy with in vitro translational amplification of reporter protein for signal generation to detect nucleic acids with high sensitivity and selectivity. This approach enables target-mediated translational amplification of reporter proteins by linking split probes in the presence of a target microRNA (miRNA). In particular, the fluorescence split-probe sensor adopts a reporter protein with various fluorescence wavelength regions, enabling the simultaneous detection of multiple target miRNAs. Moreover, luminescence detection by merely altering the reporter protein sequence can substantially enhance the sensitivity of detection of target miRNAs. Using this system, we analyzed and quantified target miRNAs in the total RNA extracted from cell lines and cell-derived extracellular vesicles with high specificity and accuracy. This split-probe sensor has potential as a powerful tool for the simple, sensitive, and specific detection of various target nucleic acids.

Droplet-Based Microfluidic Photobioreactor as a Growth Optimization Tool for Cyanobacteria and Microalgae

Microalgae and cyanobacteria are photosynthetic microorganisms with significant biotechnological potential for the production of bioactive compounds, making them a promising resource for diverse industrial applications. This study presents the development and validation of a modular, droplet-based microfluidic photobioreactor (µPBR) designed for high-throughput screening and cultivation under controlled light conditions. The µPBR, based on polytetrafluoroethylene (PTFE) tubing and a 4-channel LED illumination system, enables precise modulation of light intensity, wavelength, and photoperiod, facilitating dose–response experiments. Synechococcus elongatus UTEX 2973 and Chlorella vulgaris were used to demonstrate the system’s capacity to support photosynthetic growth under various conditions. The results indicate that continuous illumination, particularly under blue and mixed blue-red light, promotes higher autofluorescence and chlorophyll a content in cyanobacteria Synechococcus elongatus UTEX2973, while Chlorella vulgaris achieved optimal growth under a 16:8 light-dark cycle with moderate light intensity. This µPBR offers not only a flexible, scalable platform for optimizing growth parameters but also allows for the investigation of highly resolved dose response screenings of environmental stressors such as salinity. The presented findings highlight its potential for advancing microalgal biotechnology research and applications.

Whole genome sequence of the denitrifying thermophile Geobacillus thermodenitrificans subsp. calidus DSM 22629T.

We present a full genome sequence for the thermophilic denitrifier Geobacillus thermodenitrificans subsp. calidus DSM 22629T (3,408,575 bp, 48.94% GC). This genome includes 3,615 predicted genes, including those needed to reduce nitrate to nitrogen gas. This organism and genome sequence provide valuable resources for future phylogenetic, genomic, and biotechnological research.

Peptipedia v2.0: a peptide sequence database and user-friendly web platform. A major update.

In recent years, peptides have gained significant relevance due to their therapeutic properties. The surge in peptide production and synthesis has generated vast amounts of data, enabling the creation of comprehensive databases and information repositories. Advances in sequencing techniques and artificial intelligence have further accelerated the design of tailor-made peptides. However, leveraging these techniques requires versatile and continuously updated storage systems, along with tools that facilitate peptide research and the implementation of machine learning for predictive systems. This work introduces Peptipedia v2.0, one of the most comprehensive public repositories of peptides, supporting biotechnological research by simplifying peptide study and annotation. Peptipedia v2.0 has expanded its collection by over 45% with peptide sequences that have reported biological activities. The functional biological activity tree has been revised and enhanced, incorporating new categories such as cosmetic and dermatological activities, molecular binding, and antiageing properties. Utilizing protein language models and machine learning, more than 90 binary classification models have been trained, validated, and incorporated into Peptipedia v2.0. These models exhibit average sensitivities and specificities of 0.877±0.0530 and 0.873±0.054, respectively, facilitating the annotation of more than 3.6 million peptide sequences with unknown biological activities, also registered in Peptipedia v2.0. Additionally, Peptipedia v2.0 introduces description tools based on structural and ontological properties and user-friendly machine learning tools to facilitate the application of machine learning strategies to study peptide sequences. Database URL: https://peptipedia.cl/.

Prospecting Pharmacologically Active Biocompounds from the Amazon Rainforest: In Vitro Approaches, Mechanisms of Action Based on Chemical Structure, and Perspectives on Human Therapeutic Use

The Amazon rainforest is an important reservoir of biodiversity, offering vast potential for the discovery of new bioactive compounds from plants. In vitro studies allow for the investigation of biological processes and interventions in a controlled manner, making them fundamental for pharmacological and biotechnological research. These approaches are faster and less costly than in vivo studies, providing standardized conditions that enhance the reproducibility and precision of data. However, in vitro methods have limitations, including the inability to fully replicate the complexity of a living organism and the absence of a complete physiological context. Translating results to in vivo models is not always straightforward, due to differences in pharmacokinetics and biological interactions. In this context, the aim of this literature review is to assess the advantages and disadvantages of in vitro approaches in the search for new drugs from the Amazon, identifying the challenges and limitations associated with these methods and comparing them with in vivo testing. Thus, bioprospecting in the Amazon involves evaluating plant extracts through bioassays to investigate pharmacological, antimicrobial, and anticancer activities. Phenolic compounds and terpenes are frequently identified as the main bioactive agents, exhibiting antioxidant, anti-inflammatory, and antineoplastic activities. Chemical characterization, molecular modifications, and the development of delivery systems, such as nanoparticles, are highlighted to improve therapeutic efficacy. Therefore, the Amazon rainforest offers great potential for the discovery of new drugs; however, significant challenges, such as the standardization of extraction methods and the need for in vivo studies and clinical trials, must be overcome for these compounds to become viable medications.

Research work of Prof. De-Eknamkul, including the mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients

Androgenetic alopecia (AGA) is a genetically-predetermined disorder that affects many people around the world, leading to progressive hair loss. This baldness can negatively impact people’s perceptions of themselves and scientists are searching for a cure. There are only two US Food and Drug Administration-approved drugs used to treat AGA and they both have side effects. If a natural anti-AGA product could be developed with the ability to be effective minus negative side effects, this could be a game-changer for people with AGA. Professor Wanchai De-Eknamkul, Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University, Thailand, an expert in plant natural product biotechnology, is working to develop a natural anti-AGA product. He is a professor at the University’s Natural Product Biotechnology Research Unit, where research is underway to: elucidate the biosynthesis of bioactive natural products by searching for enzymes and genes involved in their biosynthetic pathways; discover bioactive natural compounds from medicinal plants by either enzyme-based or cell-based screening followed by studying their mechanism of actions; and develop high-performance thin-layer chromatography (HPTLC) as a tool for quick screening and identification of bioactive compounds. De-Eknamkul have been working to shed light on the genes and enzymes involved in the biosynthesis of the diterpenoid ‘plaunotol’ in Croton stellatopilosus. In another line of investigation, they are searching for natural compounds with anti-AGA and anti-grey hair activities and a further area of focus is the development of a technique for direct detection of compounds with anti-diabetes, anti-gonorrhoea and anti-AGA activities.

Photoinduced Transformations with Diverse Maleimide Scaffolds

Maleimides serve as crucial components in various synthetic processes and are of significant interest to researchers in bioorganic chemistry and biotechnology. Although thermal reactions involving maleimides have been studied extensively, light-mediated reactions with maleimides remain relatively underutilized. This review focuses on understanding the behavior of maleimides in their excited state, particularly their role as synthetic scaffolds for excited-state reactions. Specific emphasis is placed on the diverse photoreactions involving maleimides and photophysical evaluation from our research group.

Biosensor applications in the monitoring of elderly patients

Nurse-based patient monitoring is prone to errors due to manual measurements and documentation, leading to potential inaccuracies in care. The use of biosensors offers a promising solution by enabling real-time and continuous monitoring of patient health. Categorizing patient care reports as critical or non-critical using mobile recording systems based on biosensor data can help prevent errors and improve care. The use of biosensors can significantly reduce morbidity and mortality, especially after emergencies and accidents. These devices improve the quality of care and increase the satisfaction of older people, their families and healthcare professionals. Wearable biosensors make it easier for older people to monitor their health, which can help reduce hospital admissions. Chronic diseases such as cardiovascular disease, cancer, diabetes, dementia and stroke pose challenges to healthcare delivery and interpretation of results. Integrating biosensors into health monitoring and measurement is an innovative approach to managing these chronic conditions more effectively. To improve self-management of chronic diseases in older people, it is essential to educate healthcare professionals and promote research in this area. As a result, the use of biosensors to monitor the daily activities and health parameters of elderly patients is expanding, highlighting the importance of multidisciplinary research in biotechnology, chemistry, engineering and nursing.

The Assembly of Miniaturized Droplets toward Functional Architectures.

Recent explorations of bioengineering have generated new concepts and strategies for the processing of soft and functional materials. Droplet assembly techniques can address problems in the construction of extremely soft architectures by expanding the manufacturing capabilities using droplets containing liquid or hydrogels including weak hydrogels. This Perspective sets out to provide a brief overview of this growing field, and discusses the challenges and opportunities ahead. The study highlights the recent key advances of materials and architectures from hitherto effective droplet-assembly technologies, as well as the applications in biomedical and bioengineering fields from artificial tissues to bioreactors. It is envisaged that these assembled architectures, as nature-inspired models, will stimulate the discovery of biomaterials and miniaturized platforms for interdisciplinary research in health, biotechnology, and sustainability.

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research

This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody’s single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody’s interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human β-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.

Structural study of the intrinsically disordered tardigrade damage suppressor protein (Dsup) and its complex with DNA

Studies of proteins, found in one of the most stress-resistant animals tardigrade Ramazzottius varieornatus, aim to reveal molecular principles of extreme tolerance to various types of stress and developing applications based on them for medicine, biotechnology, pharmacy, and space research. Tardigrade DNA/RNA-binding damage suppressor protein (Dsup) reduces DNA damage caused by reactive oxygen spices (ROS) produced upon irradiation and oxidative stresses in Dsup-expressing transgenic organisms. This work is focused on the determination of structural features of Dsup protein and Dsup-DNA complex, which refines details of protective mechanism. For the first time, intrinsically disordered nature of Dsup protein with highly flexible structure was experimentally proven and characterized by the combination of small angle X-ray scattering (SAXS) technique, circular dichroism spectroscopy, and computational methods. Low resolution models of Dsup protein and an ensemble of conformations were presented. In addition, we have shown that Dsup forms fuzzy complex with DNA.

Management of experimental workflows in robotic cultivation platforms

In the last decades, robotic cultivation facilities combined with automated execution of workflows have drastically increased the speed of research in biotechnology. In this work, we present the design and deployment of a digital infrastructure for robotic cultivation platforms. We implement a workflow management system, using Directed Acyclic Graphs, based on the open-source platform Apache Airflow to increase traceability and the automated execution of experiments. We demonstrate the integration and automation of experimental workflows in a laboratory environment with a heterogeneous device landscape including liquid handling stations, parallel cultivation systems, and mobile robots. The feasibility of our approach is assessed in parallel E. coli fed-batch cultivations with glucose oscillations in which different elastin-like proteins are produced. We show that the use of workflow management systems in robotic cultivation platforms increases automation, robustness and traceability of experimental data.

Cyanobacteria: role in sustainable biomanufacturing and nitrogen fixation

AbstractCyanobacteria, renowned for their nitrogen‐fixing characteristics, are important for sustainable biomanufacturing and agricultural innovation. This review explores the synergy between cyanobacteria and nitrogen fixation, highlighting their potential to revolutionize biobased compound production and reduce the ecological impact of traditional nitrogen sources. It focuses on genetic enhancements and synthetic biology techniques, which transform these microorganisms into sustainable nitrogen providers. Current applications range from agricultural enhancement to cutting‐edge biotechnology, highlighting the important consequences of cyanobacterial nitrogen fixation. Challenges persist, however, requiring a meticulous analysis of ecological, regulatory, and scalability concerns. The untapped potential of cyanobacteria in nitrogen fixation promises a significant shift in biomanufacturing and environmental stewardship. The aim of this article is to inspire high‐impact research and transformative applications in biotechnology and sustainability.

Diversity of Mycotoxin-Producing Fungi in Leafy and Fruit Vegetables Sold in Port Harcourt Metropolis, Nigeria

Mycotoxins are secondary metabolites produced by fungi and are capable of causing diseases and even death in both humans and other animals. This study investigated the diversity of mycotoxin-producing fungi from leafy (Telfairia occidentalis and Bassica oleracea) and fruit (Solanum lycopersicum and Cucumis sativus) vegetables sold in Port Harcourt metropolis, Nigeria using molecular techniques. The fungi were isolated from vegetable samples showing signs of disease using standard methods. DNA extraction and Polymerase chain reaction were carried out at the molecular laboratory of Regional Centre of Biotechnology and Bioresources research, University of Port Harcourt. The isolates were screened for the presence of mycotoxigenic genes (nor1 (aflatoxin), tri6 (trichothecene), otanps (Ochratoxin A), fum13 (fumonisin) and ZEA (zearalenone)) using PCR technique. The isolates containing mycotoxins were identified using ITS gene sequences. Ten out of the eighteen isolates were positive for four (nor1, tri6, otanps and fum13) out of the five mycotoxigenic genes screened. The nine isolates included fungi belong to six genera: Meyerozyma, Pithomyces, Fusarium, Trametes, Penicillium and Aspergillus. The ten isolates were classified as Meyerozyma carbbica RCBBR_Sf13, Pithomyces chartarum RCBBR_Sf5, Fusarium falciforme RCBBR_Sf10, Trametes duplexa RCBBR_Sf17, Trametes versicolor RCBBR_Sf2a, Trametes duplexa RCBBR_Mf1, Fusarium longifundum RCBBR_Mf4, Penicillium soosanum RCBBR_Mf7, Aspergillus aflatoxiformans RCBBR_Sf9, Fusarium circinatum RCBBR_Sf3 based on their ITS gene sequences. Their ITS gene sequences have been deposited in GenBank under the accession numbers OR816039-OR816047, with the exception of Fusarium circinatum RCBBR_Sf3 which could not accession. This study has demonstrated that mycotoxin-producing fungi are diverse and widespread in leafy and fruit vegetables sold within Port Harcourt Metropolis. This raises both public health and food security concerns.

Characterization of genetic diversity in Eragrostis tef (zucc.) Trotter through microsatellite and morphological markers

Eragrostis tef (Zucc.) Trotter is Ethiopia's most significantly important grain crop; however, its productivity is quite low (1.7 tons per hectare) due to a variety of issues, including the poor yield potential of varieties produced thus far. To achieve future production improvements in tef, comprehensive study of the crop's genetic variability and variety, utilizing both genetic and morphological markers, necessary. The study was designed to assess the genetic diversity of 64 tef genotypes utilizing 10 Simple Sequence Repeats (SSRs) markers. This investigation took place at the Agricultural Biotechnology Research Center in Holeta, Ethiopia. Field phenotypic diversity evaluation was conducted at two distinct locations: Debre Zeit and Worabe, applied a simple lattice design during 2020/21 season. Molecular analysis of variance demonstrated a substantial proportion of variation among individuals (46 %), followed closely variation among individuals (43 %), with the least variation observed within population (11 %). Concurrently, analysis of morphological data, encompassing twenty phenotypic characteristics, revealed significant variation (P ≤ 0.01) among almost all the tested genotypes for recorded parameters, as indicated by combined analysis of variance across sites. These findings underscore the high diversity among the studied genotypes, suggesting a considerable potential for crop improvement through direct selection and intra-specific hybridization strategies. From the perspectives of both conservation and utilization of tef genetic materials, there is a pressing need for more extensive and systematic molecular level research. Promoting field trials and genotype-by-environment interaction research is crucial. These efforts will enhance our understanding of tef genetic and contribute to its effective conservation and utilization.

Determinants of Food Security in Tobacco and Sugarcane Production Zones, Migori County, Kenya

At 34%, food insecurity in Migori County is considered alarming while its measurement has posed challenges to academicians and researchers whilst many studies exist on food security determinants, none has been done in Migori entailing tobacco producers, sugarcane growers and non-growers in Kuria and Migori sub-counties. The study examined food security determinants guided by a consumer theory and measured by means of HFIAS modelled in ologit model. Data collection was done using an open data kit pre-loaded with a questionnaire and analyzed using stata software wherein results depicted 38.89% as severely insecure, 33.33% moderately insecure, 9.26% mildly insecure and 18.52% food secure while household size, off-farm income, capital sourcing was significant at p<0.001 in varying degrees and categories after ologit regression analysis. There is need for the national government to provide cash crop insurance and fixed contracts to prevent risks associated with price fluctuations as well as invest in innovative biotechnology research through formulation of policies to aid in ensuring capacity building through its stakeholders such as the national research institutes and other donor agencies.

Relics of interspecific hybridization retained in the genome of a drought-adapted peanut cultivar.

Peanut (Arachis hypogaea L.) is a globally important oil and food crop frequently grown in arid, semi-arid, or dryland environments. Improving drought tolerance is a key goal for peanut crop improvement efforts. Here we present the genome assembly and gene model annotation for 'Line8', a peanut genotype bred from drought tolerant cultivars. Our assembly and annotation are the most contiguous and complete peanut genome resources currently available. The high contiguity of the Line8 assembly allowed us to explore structural variation both between peanut genotypes and subgenomes. We detect several large inversions between Line8 and other peanut genome assemblies, and there is a trend for the inversions between more genetically diverged genotypes to have higher gene content. We also relate patterns of subgenome exchange to structural variation between Line8 homeologous chromosomes. Unexpectedly, we discover that Line8 harbors an introgression from A.cardenasii, a diploid peanut relative and important donor of disease resistance alleles to peanut breeding populations. The fully resolved sequences of both haplotypes in this introgression provide the first in situ characterization of A.cardenasii candidate alleles that can be leveraged for future targeted improvement efforts. The completeness of our genome will support peanut biotechnology and broader research into the evolution of hybridization and polyploidy.

Three-Dimensionally Printed Microsystems to Facilitate Flow-Based Study of Cells from Neurovascular Barriers of the Retina.

Rapid prototyping has produced accessible manufacturing methods that offer faster and more cost-effective ways to develop microscale systems for cellular testing. Commercial 3D printers are now increasingly adapted for soft lithography, where elastomers are used in tandem with 3D-printed substrates to produce in vitro cell assays. Newfound abilities to prototype cellular systems have begun to expand fundamental bioengineering research in the visual system to complement tissue engineering studies reliant upon complex microtechnology. This project used 3D printing to develop elastomeric devices that examined the responses of retinal cells to flow. Our experiments fabricated molds for elastomers using metal milling, resin stereolithography, and fused deposition modeling via plastic 3D printing. The systems were connected to flow pumps to simulate different flow conditions and examined phenotypic responses of endothelial and neural cells significant to neurovascular barriers of the retina. The results indicated that microdevices produced using 3D-printed methods demonstrated differences in cell survival and morphology in response to external flow that are significant to barrier tissue function. Modern 3D printing technology shows great potential for the rapid production and testing of retinal cell responses that will contribute to both our understanding of fundamental cell response and the development of new therapies. Future studies will incorporate varied flow stimuli as well as different extracellular matrices and expanded subsets of retinal cells.