Biotechnology Research Research Articles

Engineering a thermophilic luciferase variant from Photuris pennsylvanica into a mesophilic-like enzyme for expanded applications potential.

Luciferase, known for its exceptional catalytic bioluminescent properties, has been widely utilized in diverse applications within biotechnology and medical research. Currently, enhancing thermostability and catalytic activity is a primary focus for optimizing luciferase modifications to further expand its detection range and accuracy. This study revealed a highly thermostable luciferase variant from Photuris pennsylvanica, Ppe146-1H2, which inherently exhibits thermophilic enzyme characteristics that are not conducive for optimal catalytic performance in practical applications. Building upon structural analysis, this research engineered Ppe146-1H2 into Ppe146-LGR via the residue substitutions I422L, D435G, and I519R. Ppe146-LGR retained notably thermostability, exhibiting a melting temperature (Tm value) of 75.3±0.3°C. Additionally, the variant demonstrated efficient catalytic activity at moderate temperatures, exhibiting 3.8 and 3.7-fold higher catalytic efficiencies towards D-luciferin and ATP at 37°C compared to Ppe146-1H2. Overall, Ppe146-LGR displayed mesophilic-like catalytic activity and thermophilic-like thermostability simultaneously. In addition to enhanced catalytic properties, Ppe146-LGR emitted longer-wavelength light (580nm) and operated optimally at near-neutral pH, coordinating with the current demands of luciferase applications. Through validation via rapid bacterial detection and reporter gene assays, it has been demonstrated that Ppe146-LGR holds promise as a valuable tool in the field of bioluminescence technology.

Advancements in sequencing technologies:

Single-cell Sequencing (SCS) technologies, methods for analyzing genetic material at the single-cell level, offer extensive insights into cellular heterogeneity. This has broadened oncology research by enabling the exploration of functional and genetic diversity within tissues of different cell types. Furthermore, SCS facilitates the study of complex biological processes like metastasis tracking and tumor microenvironment analysis. However, the implementation of SCS methods is furrowed by a lack of clinical accessibility and high application costs. This review examines the development of SCS technologies, analyzing trends in throughput, accessibility, and cost of various commercial platforms, by focusing on the domain of cancer research and precision medicine. Despite the significant advancements offered by third-generation sequencing platforms, which provide high accuracy, versatility, and throughput for sequencing single-cell genetic information, these methods face challenges such as high error rates, insufficient funding, and complex data analysis. Furthermore, we’ve determined that the advancements of the previous decade have enabled personalized medicine and in-depth analysis of cellular heterogeneity, revolutionizing fields like medicine, biotechnology, and biological research. We anticipate our assay indicating extensive advancements in healthcare through the adoption of precision medicine concerning individual genomes and helping to demonstrate the promise of advancement in general understandings of complex biological systems. Furthermore, our research indicates that efforts to overcome technical, analytical, and cost-related challenges are essential in future clinical application, distribution, and growth of SCS methods.

Surface Plasmon Resonance Biosensors: Advances, Applications, and Future Prospects

In recent years, surface plasmon resonance (SPR) biosensors have gained importance as analytical methods for biomolecular interaction studies in real-time and without the need for labels. This review encompasses the principle of SPR, the kinds of SPR biosensors, and their diverse applications ranging from medical diagnostics, environmental monitoring, food safety, and biotechnological researches. Recent advances in the development of SPR-based biosensors, including their multiple abilities and portable SPR devices, are also discussed. Furthermore, the discussion focuses on current challenges to SPR biosensors with regard to sensitivity and specificity. Besides, the future directions of research and development are also discussed, which include the possibility of novel enzyme-based SPR biosensors for direct biomarker detection. These advances clearly demonstrate that SPR biosensors, which revolutionize modern detection and monitoring techniques, are becoming increasingly significant in a number of scientific and clinical applications. As SPR technology continues to evolve, its potential to contribute to emerging areas, such as personalized medicine and environmental sustainability, is becoming increasingly apparent.

Toxicity And Antioxidant Activities of Endophytic Bacteria from Butterfly Pea (<i>Clitoria ternatea</i> Linn.)

Endophytic bacteria are bacteria that live in healthy plant tissues without causing damage. Several studies have reported that endophytic bacteria can produce active compounds similar to those secreted by their host and which potentially have medicinal value. Butterfly pea (Clitoria ternatea L.) was noted to be able to produce antioxidants and have toxicity potential from its compounds. Therefore, endophytic bacteria from butterfly pea have great potential to have antioxidant activity along with evaluating the toxicity level of the selected bacteria. This study aimed to determine the number of isolates, to characterize, and test the toxicity and antioxidant activities of endophytic bacteria from butterfly pea. Toxicity level was tested using the Brine Shrimp Lethality Test (BSLT) method while levels of antioxidants were tested using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method. A total of fifteen endophytic bacteria were obtained and successfully purified. Based on the morphological observations, Gram staining, and biochemical test results, isolate EBT13 was determined to belong to the genus Bacillus. Isolate EBT13 was categorized as highly toxic, with the highest toxicity value with other bacterial isolates with an LC50 of 84 ppm and antioxidant activity with an IC50 value of 44.32 ppm. Based on the phylogenetic tree of 16S rRNA gene analysis, EBT13 belongs to the genus Bacillus, it forms a sister group with Bacillus pumilus with a bootstrap value of 100%. This study advances our knowledge of plant-microbe interactions by identifying a highly toxic, antioxidant-producing strain of bacteria of butterfly pea. The results have significance for the development of cytotoxic chemicals and natural antioxidants, which could advance biotechnological research and boost therapeutic purpose.

Estimates of Genetic Variability on Agro-Morphological Parameters of Newly Selected Maize [<i>Zea mays L.</i>] Landraces in Nigeria

Agro-morphological characterizations offer resilient and strong means for the precise characterization of germplasm to be used in breeding programs. Here, agro-morphological parameters were analyzed to figure out the genetic variability within 23 maize accessions commonly grown in the maize producing states. A total of 12 important agro-morphological traits were determined in the field trails at Agas research farm, kwara state Nigeria during 2023 planting season. The experiment was conducted in a complete randomized design with three replications. The highest plant height was recorded in the Accession NG03 (187.45), thenumber of leaves per plant was highest in the accessions BA-02 and KW-03 with the mean value (17.27) respectively. The length of cob was highest in the accessions NG-03 and JG-04 (21.60). The length of husk was lowest in the accession JG-04 (24.40). The accession NG-03 can serve as a breeding tool in breeding programmes as it showed highest in some morphological parameters. Other agro-morphological parameters showed significant differences as revealed by ANOVA statistically. This study revealed some accessions with diverse morphological traits that might be used as promising parents for maize in current and future biotechnology research and breeding programmes.

Genome-wide identification and diversity of FAD2, FAD3 and FAE1 genes in terms of biotechnological importance in Camelina species

BackgroundFalse flax, or gold-of-pleasure (Camelina sativa) is an oilseed that has received renewed research interest as a promising vegetable oil feedstock for liquid biofuel production and other non-food uses. This species has also emerged as a model for oilseed biotechnology research that aims to enhance seed oil content and fatty acid quality. To date, a number of genetic engineering and gene editing studies on C. sativa have been reported. Among the most common targets for this research are genes, encoding fatty acid desaturases, elongases, and diacylglycerol acyltransferases. However, the majority of these genes in C. sativa are present in multiple copies due to the allohexaploid nature of the species. Therefore, genetic manipulations require a comprehensive understanding of the diversity of such gene targets.ResultsHere we report the detailed analysis of FAD2, FAD3 and FAE1 gene diversity in five Camelina species, including hexaploid C. sativa and four diploids, namely C. neglecta, C. laxa, C. hispida var. hispida and var. grandiflora. It was established that FAD2, FAD3 and FAE1 homeologs in C. sativa retain very high conservancy, despite their allohexaploid inheritance. High sequence conservancy of the identified genes along with their different expression patterns in C. sativa suggest that subfunctionalization of these homeologs is mainly grounded on the transcriptional balancing between subgenomes. Finally, fatty acid composition of seed lipids in different Camelina species was characterized, suggesting potential variability in the activity of fatty acid elongation/desaturation pathways may vary among these taxa.ConclusionIt was shown that the FAD2, FAD3 and FAE1 genes retain high conservation, even in allohexaploid C. sativa after polyploidzation, in which the subfunctionalization of the described homeologs is mainly grounded on the expressional differences. The major differences in FA accumulation patterns within the seeds of different species were identified as well. These results provide a foundation for future precise gene editing, which would be based on targeting of particular FAD2, FAD3 and FAE1 gene copies in C. sativa that allow regulating the dosage of the mentioned genes, thus shaping the desired FA composition in cultivated false flax.

Bioinformatic Comparisons of Some Web-based PCR Primer Design Programs

Bioinformatics has become an indispensable tool for both basic and applied research in biotechnology in the life sciences. The polymerase chain reaction (PCR) is a laboratory method that can be used to quickly amplify a large number of identical copies of a specific DNA segment. In PCR, short synthetic DNA fragments known as primers are used to selectively amplify a specific section of the genome. For PCR to be as efficient and specific as possible, it is important to choose an effective primer sequence and use the correct concentration of primers. If the primer is not designed carefully, non-specific amplification and/or primer dimer formation may occur, which may prevent product formation. Currently, a number of different design tools are available on the internet to assist molecular geneticists in designing PCR primers under optimal conditions. In this study, out of 39 web-based PCR primer design programs, 7 accessible, freely available and widely used web-based PCR primer design programs (NCBI, Primer3, Biserach, Genscript and Primer3plus; Stitcher 2.0; and PrimerQest Tool) were compared using bioinformatics applications for genomic sequences. The advantages and disadvantages of the web-based PCR programs are discussed on the basis of the comparison results.

Biosynthesis of Bacteriochlorophylls and Bacteriochlorophyllides in Escherichia coli.

Photosynthesis, the most important biological process on Earth, converts light energy into chemical energy with essential pigments like chlorophylls and bacteriochlorophylls. The ability to reconstruct photosynthesis in heterotrophic organisms could significantly impact solar energy utilization and biomass production. In this study, we focused on constructing light-dependent biosynthesis pathways for bacteriochlorophyll (BChl) a and bacteriochlorophyllide (BChlide) d and c in the model strain Escherichia coli. The production of the starting compound, Mg protoporphyrin monomethylester, was optimized by screening the ribosome binding sites for the expression of each of the five genes. By fusing a maltose-binding protein and apolipoprotein A-I domain with the membrane protein BchF, the yield of 3-hydroxyethyl-Chlide a was increased by five-fold. Anaerobic cultivation of the engineered E. coli strains facilitated the reduction of the C7=C8 double bond by chlorophyllide a oxidoreductase, a critical step in BChl a synthesis. We further enhanced BChl a production by adjusting the isopropyl-β-d-thiogalactopyranoside concentration to optimize enzyme production and introducing an exogenous superoxide dismutase to combat oxidative stress. Additionally, fusing BciC with a RIAD tag resulted in an eight-fold increase in the production of 3-vinyl BChlide d. This study lays the foundation for the reconstitution of BChl-based photosynthetic apparatus in heterotrophic model organisms, offering promising avenues for future research and applications in biotechnology.

Impact of proximity from chemical and pharmaceutical universities on startup founding: empirical evidence from the Visegrad countries

This paper examines the spatial distribution of chemical startups in the Visegrad Countries (Czech Republic, Slovakia, Poland, and Hungary), highlighting their potential to drive technological innovation by creating new products or services under conditions of high uncertainty. The study focuses on the proximity of these startups to medical or chemical universities and those with biotechnological research fields to better understand their geographical patterns and potential knowledge spillovers. Data were drawn from Crunchbase, a comprehensive startup database, resulting in a final sample of approximately 333 operational chemical, pharmaceutical, or biotechnological startups. Companies were identified using keyword-based searches, while startup locations and distances to the nearest medical universities were recorded. Statistical methods were applied to assess spatial patterns. Results indicate that these startups are frequently located in cities with biotechnological, chemical, or medical universities. Our findings highlight different types of startup activities and levels of financial support across the Visegrad countries, emphasising the role of chemical startups in fostering technological advancement and sustainable development.

Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.

Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.

Single-cell transcriptomics: a new frontier in plant biotechnology research.

Single-cell transcriptomic techniques have ushered in a new era in plant biology, enabling detailed analysis of gene expression at the resolution of individual cells. This review delves into the transformative impact of these technologies on our understanding of plant development and their far-reaching implications for plant biotechnology. We present a comprehensive overview of the latest advancements in single-cell transcriptomics, emphasizing their application in elucidating complex cellular processes and developmental pathways in plants. By dissecting the heterogeneity of cell populations, single-cell technologies offer unparalleled insights into the intricate regulatory networks governing plant growth, differentiation, and response to environmental stimuli. This review covers the spectrum of single-cell approaches, from pioneering techniques such as single-cell RNA sequencing (scRNA-seq) to emerging methodologies that enhance resolution and accuracy. In addition to showcasing the technological innovations, we address the challenges and limitations associated with single-cell transcriptomics in plants. These include issues related to sample preparation, cell isolation, data complexity, and computational analysis. We propose strategies to mitigate these challenges, such as optimizing protocols for protoplast isolation, improving computational tools for data integration, and developing robust pipelines for data interpretation. Furthermore, we explore the practical applications of single-cell transcriptomics in plant biotechnology. These applications span from improving crop traits through precise genetic modifications to enhancing our understanding of plant-microbe interactions. The review also touches on the potential for single-cell approaches to accelerate breeding programs and contribute to sustainable agriculture. This review concludes with a forward-looking perspective on the future impact of single-cell technologies in plant research. We foresee these tools becoming essential in plant biotechnology, spurring innovations that tackle global challenges in food security and environmental sustainability. This review serves as a valuable resource for researchers, providing a roadmap from sample preparation to data analysis and highlighting the transformative potential of single-cell transcriptomics in plant biotechnology.

Cloning of the Thermus thermophilus methionine aminopeptidase gene and confirmation of the enzyme functional activity

Background. Methionine aminopeptidases (MAPs) are a class of enzymes that catalyze the removal of the N-terminal initiator methionine from a polypeptide chain. Bacterial MAPs are considered as targets for the development of broad-spectrum antibacterial drugs, and using MAPs in biotechnology necessitates the search for new MAPs and the study of their functioning and inhibition mechanisms.The aim of the study. To identify methionine aminopeptidase in the Thermus thermophilus genome (Tt-MAP) and to confirm its functional activity.Materials and methods. To identify Tt-MAP, we analyzed the Thermus thermophilus genome in the GeneBank database. Modern genetic engineering techniques (polymerase chain reaction, restriction, transformation, heterologous expression) were used to clone the putative open reading frame (ORF) encoding Tt-MAP in the pHUE vector. Various chromatography techniques (affinity, ion exchange, and size-exclusion) were used to obtain a purified enzyme preparation. The fluorogenic substrate L-methionine 7-amino-4-methylcoumarin (Met-AMC) was used to confirm the specific functional aminopeptidase activity of the enzyme.Results. An ORF encoding MAP was identified in the Thermus thermophilus bacterium genome. Oligonucleotide primers were designed based on the nucleotide sequence. The ORF was cloned in the vector, and the recombinant enzyme was produced in E. coli cells. A method for purifying the enzyme to a homogeneous state was developed using a series of sequential chromatographies, allowing up to 30 mg to be obtained from 1 liter of culture. Using the fluorogenic substrate Met-AMC, the specific functional activity of the enzyme was demonstrated (the enzyme cleaves methionine from the substrate).Conclusion. We have identified the Thermus thermophilus MAP and tested its functional activity. It has been shown that the ORF product TTHA1670 encodes a methionine-specific aminopeptidase, i. e. methionine aminopeptidase. The enzyme can be used in various fields of biotechnology and scientific research.

Extraction of Genomic DNA from Different Plant Tissues through Phenol-chloroform Method

Background: DNA extraction is a fundamental technique in molecular biology that involves isolating DNA from biological samples for various downstream applications, including PCR and sequencing. Phenol-chloroform DNA extraction is an alternate method of the CTAB method for genomic DNA extraction. This method typically yields more DNA than the CTAB with high purity. It is useful for high molecular weight/long fragment DNA determinations. This study aims to extract DNA from plant tissues using the phenol-chloroform method and compare the response of different plant leaves to this method. Methods: In this study, we provide a step-by-step guide to DNA extraction from five different plant species, including Ficus capensis (Cape fig or Cape banyan), Ficus exaspirata (Forest Sandpaper), Mangifera indica (Mango), Gmelina arborea (Gamhar), and Bauhinia purpurea (Butterfly tree). The DNA isolation protocol involved several steps, such as grinding the plant tissue, adding NaCl solution, using a chloroform-isoamyl alcohol mixture to extract DNA, and further purification with phenol-chloroform-isoamyl alcohol. Isopropanol was used to precipitate the DNA, and ethanol was used to wash the DNA pellet to remove any remaining contaminants. Finally, the DNA pellet was dissolved in TE buffer for storage. Results: The results showed the successful implementation of the phenol chloroform method for isolating DNA from selected plant tissues. Overall, this study provides a comprehensive protocol for DNA extraction from plant tissue using readily available laboratory reagents and equipment. Conclusion: The protocol can be modified to accommodate different plant species and sample types and can facilitate further research in plant genetics and biotechnology.

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/.

Phytochemical Profiling and Biological Activities of Extracts from Bioreactor-Grown Suspension Cell Cultures of Schisandra henryi.

Plant biotechnology creates opportunities for the cultivation of plants regardless of their natural habitats, which are often protected or difficult to access. Maintaining suspension cell cultures in bioreactors is an advanced part of biotechnological research that provides possibilities for obtaining plant tissue on a large scale. In this study, the suspension culture cultivation of a Chinese endemic plant, Schisandra henryi, in a stirred tank bioreactor was elaborated for the first time. The phytochemical profile of the tissue extracts was determined with UHPLC-MS/MS for the lignans (fifteen dibenzocyclooctadiene lignans, one aryltetralin lignan, and two neolignans) and UHPLC-DAD-ESI-MS3 for the phenolic compounds (procyanidins and their derivatives and catechin). The maximum total lignan content of 1289 µg/100 g DW was detected for the extracts from suspensions cultured in a bioreactor for over 10 days. For the phenolic compounds, catechin was the dominant compound (390.44 mg/100 g DW). The biological activity of the extracts was tested too. To determine antioxidant potential we used DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Molybdenum reduction, and β-carotene bleaching tests. The inhibition activity of the S. henryi extract on the enzymes responsible for skin aging, hyaluronidase and tyrosinase, was assessed with spectrophotometry. The cytotoxic activity of the extracts was estimated on human ovarian SKOV-3, cervical HeLa, and gastric AGS cancer cells and non-cancer, normal fibroblasts by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results showed the great potential of the obtained cell biomass extracts. The results of the antioxidant tests indicated their strong ability to reduce the level of free radicals, similarly to that of ascorbic acid, as well as the weak capacity to protect lipids from oxidation. Moreover, anticancer potential, particularly on the cervical and gastric cancer cells, was confirmed too.

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.