Genetic Stability Research Articles

Pharmacokinetic and Environmental Risk Assessment of Prime-2-CoV, a Non-Replicating Orf Virus-Based Vaccine against SARS-CoV-2

Viral vector vaccines represent a substantial advancement in immunization technology, offering numerous benefits over traditional vaccine modalities. The Orf virus (ORFV) strain D1701-VrV is a particularly promising candidate for vaccine development due to its distinctive attributes, such as a good safety profile, the ability to elicit both humoral and cellular immunity, and its favorable genetic and thermal stability. Despite ORFV’s theoretical safety advantages, such as its narrow host range and limited systemic spread post-inoculation, a critical gap persists between these theoretical benefits and the empirical evidence regarding its in vivo safety profile. This discrepancy underscores the need for comprehensive preclinical validations to bridge this knowledge gap, especially considering ORFV’s use in humans. Our research introduces Prime-2-CoV, an innovative ORFV-based vaccine candidate against COVID-19, designed to elicit a robust immune response by expressing SARS-CoV-2 Nucleocapsid and Spike proteins. Currently under clinical trials, Prime-2-CoV marks the inaugural application of ORFV in human subjects. Addressing the aforementioned safety concerns, our extensive preclinical evaluation, including an environmental risk assessment (ERA) and detailed pharmacokinetic studies in rats and immunocompromised NOG mice, demonstrates Prime-2-CoV’s favorable pharmacokinetic profile, negligible environmental impact, and minimal ERA risks. These findings not only affirm the vaccine’s safety and efficacy but also pioneer the use of ORFV-based therapeutics, highlighting its potential for wider therapeutic applications.

Advancing Hemidesmus indicus propagation and conservation: Somatic embryogenesis, histology, metabolite assessment and genetic stability

Hemidesmus indicus L. R. Br. is a commonly traded ethnomedicinal plant, and a method has been developed to produce somatic embryos (SEs) from calli grown from the leaves of the plant. With a maximum callus induction frequency of 91.66 %, pink-red friable proembryogenic calli were generated on Murashige and Skoog (MS) and 5.0 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) from the leaf explant (LE). With the aim of achieving a high success rate of 95.00 % for the induction of SEs, with a yield of 91.33 SEs per 100 mg of callus, the proembryogenic calli were transferred to fresh culture medium containing MS and 5.0 µM 6-benzylaminopurine (BA). This induction medium allowed the embryos to develop to the torpedo stage, but their further growth into emblings required a different culture medium. ½ MS with 5.0 µM BA and 1.0 µM gibberellic acid (GA3) resulted in 99.16 % maturation and 39.66 emblings regeneration out of 40 immature embryoids, respectively. With a maximum survival rate of 91.67 %, healthy emblings were successfully acclimatized using Soilrite. DNA-based molecular markers (RAPD and ISSR) were used to confirm the genetic similarities of the in vitro grown plants. Histological examinations were performed to determine the ontogeny, morphology, and development of somatic embryoids. Gas chromatography and mass spectrometry (GCMS–) were performed for both the mother and the in vitro generated plant to analyse the root for secondary metabolites. In the physiological and biochemical studies, enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) were found to increase in vitro derived H. indicus plants during different acclimatization periods (0, 7, 14, 21 and 28 days). With increasing acclimatization time, net photosynthetic rate (PN) and total chlorophyll show a decrease at first, then an increase. The protocol provides high callus induction rates, efficient somatic embryogenesis, successful maturation and regeneration, robust acclimatization, genetic fidelity, detailed insight into development, and screening of secondary metabolites.

Zinc oxide and silver effects on the growth, pigment content and genetic stability of chrysanthemums propagated by the node culture method

ABSTRACT This article describes benefits of the application of zinc oxide submicron particles (ZnO SMPs), zinc oxide nanoparticles (ZnO NPs) and ZnO NPs combined with silver NPs (ZnO + Ag NPs) in chrysanthemum micropropagation. Single node explants of Chrysanthemum × morifolium (Ramat.) Hemsl. ‘UTP Burgundy Gold (UBG)’ and ‘UTP Pinky Gold (UPG)’ were inoculated on the Murashige and Skoog (MS) medium and treated with 100 mg · L−1, 200 mg · L−1, or 400 mg · L−1 ZnO SMPs, ZnO NPs (1.5% H2O), ZnO NPs (6% H2O), ZnO + 0.1% Ag NPs (1.5% H2O), ZnO + 0.1% Ag NPs (6% H2O), ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O). Generally, the tested materials stimulated the growth and development of plantlets. In ‘UBG’, the most prominent treatments affecting increases in the number of leaves, micropropagation coefficient, shoot length and shoot FW/DW weight included 400 mg · L−1 ZnO SMPs and 100 mg · L−1 ZnO NPs (6% H2O). In ‘UPG’, the treatments with 200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) and 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) were the most successful. The latter treatment stimulated an intensive development of root systems in the two studied cultivars. High values of leaf area, perimeter and width were reported in both cultivars for 400 mg · L−1 ZnO + 1% Ag NPs (6% H2O). As compared to the control, the treated plants were characterised by a similar or, most often, lower content of chlorophylls and carotenoids. The randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT) marker system analyses of the 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated chrysanthemums confirmed their genetic fidelity with the control plants. The obtained results can be implemented in the commercial large-scale production of chrysanthemums.

Optimizing suitable solvent for phenylpropanoid extraction and antioxidant activities in Agastache rugosa hairy roots

Agrobacterium-mediated hairy roots (HRs) can induce genetic stability, rapid growth, and the synthesis of bioactive compounds in plant roots. Agastache rugosa is otherwise called Korea Mint and several studies have reported that this plant has been used for the treatment of various diseases due to the presence of a variety of bioactive compounds. A. rugosa HRs are rich in secondary metabolites than the seedling roots, and the HRs extract might be more useful in pharmacology, especially in cosmetology. This study aimed to select the suitable solvent for the extraction of phenylpropanoid compounds, total phenolic (TP), flavonoid (TF), and antioxidant activities (DPPH, ABTS scavenging activity, and reducing power assay). In this study, we extracted the A. rugosa HRs with three different extracts of solvent (water, MeOH, and EtOH) of A. rugosa HRs and analyzed the phenylpropanoid compounds, TP, TF, and antioxidant activities. The result showed that 70% MeOH extracts showed the highest activities in all assays, followed by the 70% EtOH, and water extracts. In addition, 70% of MeOH extracts showed the highest TP and TF (46.14±0.25 GAE mg/g DW and 65.46±1.41 QE mg/g DW, respectively) contents, which was 1.96- and 1.76- times higher than that of the water extracts. The phenylpropanoids in A. rugosa HR extracts were identified by using HPLC, and the results showed that 70% MeOH and EtOH showed the highest contents. Regarding these results, we can conclude that 70% MeOH is the optimal solvent to extract the A. rugosa HRs for the highest phenylpropanoid, TP, TF content, and antioxidant activities. This study might be useful for producing useful compounds at an industrial scale.

Genetic diversity and stability analysis based on agro-morphological traits among rice genotypes developed through marker-assisted backcrossing

Background: The genetic diversity and stability analysis based on agro-morphological traits among rice genotypes developed through SSR marker-assisted backcrossing was evaluated at college of agricultural science (CAS), Ebonyi State niversity, Abakaliki, Nigeria.   Methods: In this study, 9 rice accessions sourced from the Iinstitute of tropical agriculture and food security (ITAFoS), universiti Putra, Malaysia and Ebonyi, Nigeria; were evaluated for yield stability performance. The experiments were laid out in randomized complete block design (RCBD) with three replications each for two seasons. Results: Result of the evaluated rice accessions showed highly marked morphological variability based on the mean performance and were grouped into three main clusters. Cluster I had CP, cluster II contained Line4, Line5, Line6, and Line8, and cluster III had Line1, Line7, Line12, and Putra-1. The first, 3 principal components were identified and retained for having the Eigen-value >1. The first PC had an eigenvalue of (6.30) with 52.5% proportion of which plant height accounted most for this variation with (0.359) whereas panicle length contributed the least (-0.13). For stability analysis, CP had highest values for most of the parameters. High heritability value was recorded for all nine traits which range between 71.04% and 95.06%. The highest heritability value (95.06%) was found in Days 50% heading while grain width had the lowest (71.04%). Conclusions: It could be concluded that CP, Putral-1, and line7 were the most stable in terms of yield across the two seasons of this study; hence, recommended for parental materials for hybridization.

JFH1-based Core-NS2 genotype variants of HCV with genetic stability in vivo and in vitro: Important tools in evaluation of virus neutralization.

HCV infection continues to be a major global health burden, despite effective antiviral treatments. The urgent need for a protective vaccine is hindered by the scarcity of suitable HCV permissive animal models tractable in vaccination and challenge studies. Currently, only antibody neutralization studies in infectious cell culture systems or studies of protection by passive immunization of human-liver chimeric mice offer the possibility to evaluate the effect of vaccine-induced antibodies. However, differences between culture-permissive and in vivo-permissive viruses make it a challenge to compare analyses between platforms. To address this problem, we aimed at developing genotype-specific virus variants with genetic stability both in vitro and in vivo. We demonstrated infection of human-liver chimeric mice with cell culture-adapted HCV JFH1-based Core-NS2 recombinants of genotype 1-6, with a panel of 10 virus strains used extensively in neutralization and receptor studies. Clonal re-engineering of mouse-selected mutations resulted in virus variants with robust replication both in Huh7.5 cells and human-liver chimeric mice, with genetic stability. Furthermore, we showed that overall, these virus variants have similar in vitro neutralization profiles as their parent strains and demonstrated their use for in vivo neutralization studies. These mouse-selected HCV recombinants enable triage of new vaccine-relevant antibodies in vitro and further allow characterization of protection from infection in vivo using identical viruses in human-liver chimeric mice. As such, these viruses will serve as important resources in testing novel antibodies and can thus guide strategies to develop an efficient protective vaccine against HCV infection.

Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology. In comparison to plasmid-based multi-copy expression, the utilization of chromosomal multi-copy genes offers increased stability of expression level, diminishes the metabolic burden on host cells, and enhances overall genetic stability. In this study, we developed the “BacAmp”, a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis, which was achieved by employing a combination of repressor and non-natural amino acids (ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination. When the reversible switch is turned on, genome editing and gene amplification can be achieved. Subsequently, the reversible switch was turned off therefore stabilizing the gene copy number. The stabilized gene amplification system marked by green fluorescent protein, achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations. When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid (NeuAc) synthesis, the copy number of the critical gene increased to an average of 7.7, which yielded a 1.3-fold NeuAc titer. Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B. subtilis.

An efficient in vitro organogenesis protocol for the endangered relic tree species Bretschneidera sinensis and genetic fidelity assessment using DNA markers.

Bretschneidera sinensis is a monotypic species of rare and tertiary relic trees mainly distributed in China. B. sinensis is a potentially valuable horticultural plant, which has significant ornamental and research value, and is a crucial tool for the study of phylogeography. The artificial cultivation of B. sinensis is of great scientific value and practical significance. In this study, we developed a direct organogenesis process of B. sinensis using mature zygotic embryos as initial materials. The highest sterile germination induction (54.5%) from the mature zygotic embryo was obtained in a Murashige and Skoog (MS) medium with 2.0 mg·L-1 6-benzylaminopurine (6-BA) and 0.2 mg·L-1 α-naphthaleneacetic acid (NAA). The highest percentage of shoot regeneration (90.37%) was attained using 1.0 mg·L-1 6-BA and 0.01 mg·L-1 NAA in the MS medium. The Woody Plant Medium (WPM) had the greatest adventitious shoot elongation rate of 93.33%. The most optimized rooting rate was 88.89% in a half-strength MS medium containing 2.0 mg·L-1 indole-3-butyric acid (IBA) and 1.0 mg·L-1 NAA. The genetic fidelity of in vitro regenerated plantlets was assessed using inter-simple sequence repeats and random amplified polymorphic DNA molecular markers, confirming the genetic uniformity and stability of regenerated B. sinensis plantlets. Our research presents an effective in vitro propagation system for B. sinensis, laying the groundwork for its germplasm conservation and large-scale production while maintaining high genetic integrity.

Fermenting Distiller's Grains by the Domesticated Microbial Consortium To Release Ferulic Acid.

The microbial consortium FA12 that can release ferulic acid (FA) by fermenting distiller's grains was screened from Daqu. Taibaiella, Comamonadaceae, and Ochrobacum were highly abundant in FA12 by 16S rRNA gene sequencing. In the process of long-term acclimation with distiller's grains as a medium, the biomass of FA12 remained stable, and the pH value of fermentation liquid was also relatively stable. Meanwhile, the activities of cellulase, xylanase, and feruloyl esterase secreted by FA12 were stable in the ranges of 0.2350-0.4470, 0.1917-0.3078, and 0.1103-0.1595 U/mL, respectively, and the release of FA could reach 133.77 μg/g. It is proven that the microbial consortium has good genetic stability. In addition, the structural changes of lignocellulose in distiller's grains before and after fermentation were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR), and the changes of distiller's grains weight and lignocellulose content before and after fermentation were also detected. These results all confirmed that FA12 had the function of degrading distiller's grains. In this study, we explored a method to use microbial communities to release FA from distiller's grains and degrade lignocellulose in the waste, which opened up a new way for the application of the high value of lost waste.

The Genome of Bifidobacterium longum subsp. infantis YLGB-1496 Provides Insights into Its Carbohydrate Utilization and Genetic Stability.

Bifidobacterium longum subsp. infantis YLGB-1496 (YLGB-1496) is a probiotic strain isolated from human breast milk. The application of YLGB-1496 is influenced by carbohydrate utilization and genetic stability. This study used genome sequencing and morphology during continuous subculture to determine the carbohydrate utilization characteristics and genetic stability of YLGB-1496. The complete genome sequence of YLGB-1496 consists of 2,758,242 base pairs, 2442 coding sequences, and a GC content of 59.87%. A comparison of carbohydrate transport and metabolism genes of Bifidobacterium longum subsp. infantis (B. infantis) showed that YLGB-1496 was rich in glycosyl hydrolase 13, 20, 25, and 109 gene families. During continuous subculture, the growth characteristics and fermentation activity of the strain were highly stable. The bacterial cell surface and edges of the 1000th-generation strains were progressively smoother and well-defined, with no perforations or breaks in the cell wall. There were 20 SNP loci at the 1000th generation, fulfilling the requirement of belonging to the same strain. The presence of genes associated with cell adhesion and the absence of resistance genes supported the probiotic characteristics of the strain. The data obtained in this study provide insights into broad-spectrum carbohydrate utilization, genomic stability, and probiotic properties of YLGB-1496, which provide theoretical support to promote the use of YLGB-1496.

Applying knowledge and experience from potato (Solanum tuberosum) to update genetic stability data requirements in the risk assessment for vegetatively propagated biotech crops.

Regulatory agencies require data on genetic stability as part of the safety assessment for biotech crops, even though the genetic stability of a plant is not necessarily an environmental, human or animal health safety concern. While sexual reproduction has the potential to introduce genomic variation in conventionally bred and biotech crops, vegetative propagation is genetically stable. In vegetatively propagated crops, meiosis does not occur thus limiting the number of homologous recombination events that could lead to chromosomal rearrangements in progeny plants. Genetic stability data is often, but should not be, an automatic requirement for the safety assessment of vegetatively propagated biotech crops. Genetic stability data from biotech potato events has demonstrated that vegetative propagation of potato tubers does not affect the stability of introduced DNA sequences or lead to loss of trait efficacy. The knowledge and experience gained from over 30years of assessing the safety of biotech crops can be used by regulatory authorities to eliminate data requirements that do not address environmental, food or feed safety concerns. As a first step, regulators should consider removing requirements for genetic stability as part of the safety review for vegetatively propagated biotech crops.

Resilience to periodic disturbances and the long-term genetic stability in Acropora coral

Climate change is restructuring natural ecosystems. The direct impacts of these events on biodiversity and community structure are widely documented, but the impacts on the genetic variation of populations remains largely unknown. We monitored populations of Acropora coral on a remote coral reef system in northwest Australia for two decades and through multiple cycles of impact and recovery. We combined these demographic data with a temporal genetic dataset of a common broadcast spawning corymbose Acropora to explore the spatial and temporal patterns of connectivity underlying recovery. Our data show that broad-scale dispersal and post-recruitment survival drive recovery from recurrent disturbances, including mass bleaching and mortality. Consequently, genetic diversity and associated patterns of connectivity are maintained through time in the broader metapopulation. The results highlight an inherent resilience in these globally threatened species of coral and showcase their ability to cope with multiple disturbances, given enough time to recover is permitted.

Assessment of genetic homogeneity of in-vitro propagated apple root stock MM 104 using ISSR and SCoT primers

Apple is an important fruit crop that is always in demand due to its commercial and nutraceutical value. Also, the requirement for quality planting material for this fruit crop for new plantations is increasing continuously. In-vitro propagation is an alternative approach, which may help to produce genetically identical high grade planting material. In this study, for the first time, an efficient and reproducible propagation protocol has been established for apple root stock MM 104 via axillary bud. Culturing axillary buds on Murashige and Skoog apple rootstock (MM 104) resulted in better in-vitro propagation. (MS) basal medium supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar. The axillary buds were established in MS basal medium with BA (5.0 µM), NAA (1.0 µM) and further used to establish invitro propagation protocol. Plant Growth Regulators (PGRs), BA (1.0 µM) in combination with NAA (1.0 µM) was found most efficient for shoot multiplication (100%) and produced 9.8 shoots/explants with an average shoot length of (2.4 ± cm). All the shoots produced roots in 0.1 µM IBA with a 5-day dark period. Acclimatization of in-vitro raised plantlets was obtained with vermiculite: perlite: sand: soil (2:2:1:1) resulting in 76% survival under field conditions. The study showed that the use of axillary bud is efficient for multiple-shoot production of apple rootstock (MM 104). This is the first comprehensive report on in-vitro growth of apple root stock MM 104 with an assessment of genetic stability using DNA fingerprinting profiles based on Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT). The genetic stability of in-vitro-produced plants, as determined by SCoT and ISSR primers, demonstrated genetic closeness to the mother plant.

Micropropagation, genetic fidelity and chromatographic analysis in Evolvulus alsinoides (L.): A potent multipurpose medicinal plant

Evolvulus alsinoides (L.) is an annually growing medicinal plant used in many popular systems as a source of traditional medicine. However, fresh plant material for medicinal uses and research purposes is critically unavailable year-round due to its short life cycle. A micropropagation method for E. alsinoides was developed, and its genetic diversity was described. Surface sterilized nodal explants were inoculated onto Murashige and Skoog (MS) medium supplemented with different plant growth regulators; the best response of multiple shoot induction was observed on MS media augmented with 5 μM kinetin (Kn) and 0.1 μM Indole-3-acetic acid (IAA), which gave a maximum number of 10.27 ± 0.0118 shoots. Further, the addition of 30 μM of adenine sulfate to the MS media enhanced shoot multiplication (18). The cloned shoots were accomplished to in vitro rooting on MS medium with 0.1 μM Indole-3-butyric acid (IBA) and 10 μM phloroglucinol, resulting in 16 healthy roots. The rooted plantlets were acclimatized in the shade house. The genetic stability of micropropagated plants was confirmed through molecular marker analysis. Gas chromatography-mass spectrometry (GC-MS) analysis of the tissues of clones revealed the contents of secondary metabolites. High-Performance Thin Layer Chromatography (HPTLC) revealed that squalene levels were highest in plants cultured in MS medium supplemented with adenine sulfate (378.89 ng/ 15 μl). The methods developed and defined are highly reproducible and can be used for commercial production of E. alsinoides valued for human health.

The biological characteristics of chicken embryo mesenchymal stem cells isolated from chorioallantoic membrane.

Mesenchymal stem cells (MSCs) derived from fetal membranes (FMs) have the potential to exhibit immunosuppression, improve blood flow, and increase capillary density during transplantation. In the field of medicine, opening up new avenues for disease treatment. Chicken embryo chorioallantoic membrane (CAM), as an important component of avian species FM structure, has become a stable tissue engineering material in vivo angiogenesis, drug delivery, and toxicology studies. Although it has been confirmed that chorionic mesenchymal stem cells (Ch-MSCs) can be isolated from the outer chorionic layer of FM, little is known about the biological characteristics of MSCs derived from chorionic mesodermal matrix of chicken embryos. Therefore, we evaluated the characteristics of MSCs isolated from chorionic tissues of chicken embryos, including cell proliferation ability, stem cell surface antigen, genetic stability, and in vitro differentiation potential. Ch-MSCs exhibited a broad spindle shaped appearance and could stably maintain diploid karyotype proliferation to passage 15 in vitro. Spindle cells were positive for multifunctional markers of MSCs (CD29, CD44, CD73, CD90, CD105, CD166, OCT4, and NANOG), while hematopoietic cell surface marker CD34, panleukocyte marker CD45, and epithelial cell marker CK19 were negative. In addition, chicken Ch-MSC was induced to differentiate into four types of mesodermal cells in vitro, including osteoblasts, chondrocytes, adipocytes, and myoblasts. Therefore, the differentiation potential of chicken Ch-MSC in vitro may have great potential in tissue engineering. In conclusion, chicken Ch-MSCs may be an excellent model cell for stem cell regenerative medicine and chorionic tissue engineering.

The high cost of movement in an arid working landscape for an endangered amphibian.

Connectivity is essential for the maintenance of genetic diversity and stability of wildlife populations. Drought and changing precipitation regimes have caused natural aquatic amphibian breeding habitats to disappear or become isolated and have led to the replacement of natural surface water with artificial livestock water tanks. Terrestrial movement is the only means of responding to aquatic threats in arid landscapes and to allow population connectivity. Aridity may present an impenetrable barrier in hydrologically fragmented environments. We used a facultatively paedomorphic and federally endangered salamander to assess the challenges of movement across arid working lands. Sonoran tiger salamanders (Ambystoma mavortium stebbinsi) are endemic to the San Rafael Valley of southeastern Arizona, United States of America, where they depend on livestock water tanks as breeding habitat. The ecology of this species' metamorphs outside of stock tanks is virtually unknown. To assess survival on the landscape during terrestrial movement we used radio-transmitters to track 78 adult metamorphosed salamanders over 2 years. Sonoran tiger salamanders moved up to 1 km from the tank edge, and average distances moved of over 400 m were higher than most Ambystoma species. However, during the study period, none reached neighboring stock tanks. We found high mortality due to predation and desiccation. Individuals that dispersed to terrestrial habitat in summer survived longer than individuals that dispersed in spring. High mortality suggests terrestrial movement is exceptionally risky and may contribute to isolated subpopulations and elevated levels of inbreeding. Conservation actions that improve and maintain artificial aquatic habitats as well as increase connectivity may improve long-term management for pond-breeding amphibians in arid regions.

Biochemical Changes in Embryogenic and Nonembryonic Callus of Glycyrrhiza glabra L. during Somatic Embryogenesis

The continuous extraction of glycyrrhizin from Glycyrrhiza glabra L. has led to the critical endangerment of this therapeutic plant. Glycyrrhizin, a triterpenoid saponin, is a vital secondary metabolite renowned for its diverse pharmacological benefits, including anti-inflammatory, immunomodulatory, antiallergic, antiulcer properties, and even efficacy against HIV. However, the relentless harvesting of roots for high-value glycyrrhizin results in the obliteration of the entire plant, posing a severe threat to its existence and causing a detrimental impact on biodiversity. To counteract this challenge and conserve the species, sustainable approaches are imperative. One such approach involves the utilization of in vitro regeneration through somatic embryogenesis for clonal proliferation and genetic modification. This report presents findings on SE-associated proteins in Glycyrrhiza glabra L., accomplished through the implementation of the discontinuous gel electrophoresis technique. The study focused on a comparative isozyme analysis employing non-embryogenic and embryogenic calli of G. glabra L. Remarkably, these isozymes were exclusively identified and exhibited affiliations with a spectrum of cellular functions. A substantial portion of these proteins are engaged in a diverse range of metabolic and stress-related functions. This study utilized crude enzyme/total protein extracts as a basis to evaluate the genetic stability of in vitro cultivated plant tissue. Acid phosphatase, esterase, and peroxidase were selected as indicators for this assessment. Intriguingly, esterase and acid phosphatase exhibited significant polymorphism between the segregated embryogenic callus (EC) and non-embryogenic callus (NEC) derived from the same G. glabra L. genotype. Comparative analyses encompassing esterase, acid phosphatase, and peroxidase activities, alongside the expression profiles of these isozymes throughout somatic embryogenesis (SE), were conducted using polyacrylamide gel electrophoresis. Notably, the isoenzyme patterns of calluses were assessed. The activity of isozymes within an embryogenic line of callus surpassed that within a non-embryogenic counterpart. However, no conspicuous alterations in quality were observed between the non-embryogenic and embryogenic calluses.

Novel Oral Poliovirus Vaccine 2 Safety Evaluation during Nationwide Supplemental Immunization Activity, Uganda, 2022.

Given its enhanced genetic stability, novel oral poliovirus vaccine type 2 was deployed for type 2 poliovirus outbreak responses under World Health Organization Emergency Use Listing. We evaluated the safety profile of this vaccine. No safety signals were identified using a multipronged approach of passive and active surveillance.

Technical Note on the quality of DNA sequencing for the molecular characterisation of genetically modified plants.

As part of the risk assessment (RA) requirements for genetically modified (GM) plants, according to Regulation (EU) No 503/2013 and the EFSA guidance on the RA of food and feed from GM plants (EFSA GMO Panel 2011), applicants need to perform a molecular characterisation of the DNA sequences inserted in the GM plant genome. This Technical Note to the applicants puts together requirements and recommendations for the quality assessment of the methodology, analysis and reporting when DNA sequencing is used for the molecular characterisation of GM plants. In particular, it applies to the use of Sanger sequencing and next-generation sequencing for the characterisation of the inserted genetic material and its flanking regions at each insertion site, the determination of the copy number of all detectable inserts and the analysis of the genetic stability of the inserts. This updated document replaces the EFSA 2018 Technical Note and reflects the current knowledge in scientific-technical methods for generating and verifying, in a standardised manner, DNA sequencing data in the context of RA of GM plants. It does not take into consideration the verification and validation of the detection method which remains under the remit of the Joint Research Centre (JRC).

HiBiT-tagged influenza A virus: a stable and efficient tool for antiviral reagent screening and vaccine evaluation

Influenza A viruses (IAVs) possess variable pathogenic potency causing great economic losses in the poultry industry worldwide and threatening public health. The control of IAV epidemics desperately necessitates an efficient platform for screening antiviral compounds and evaluating vaccine efficacy. In this study, we utilized the H9N2 subtype IAV as the working model. An 11-amino-acid HiBiT tag, derived from NanoLuc luciferase, was incorporated into the flexible linker region of the NS1 protein. Subsequently, the recombinant HiBiT-tagged virus was rescued. The recombinant virus exhibited high genetic stability and similar virological characteristics to the parental virus, both in vitro and in vivo. Of particular significance, the replication profile of the HiBiT-tagged virus can be easily measured using the Nano-Glo assay system, achieving an efficient screening platform. Based on this platform, we have developed assays with both convenience and efficiency for screening antiviral reagents, evaluating immunization efficacy, and measuring neutralizing antibodies.