Efficient Expression Research Articles

Characterization of the function of Adenovirus L4 gene products and their impact on AAV vector production

Efficient manufacturing of recombinant adenovirus-associated viral vectors is critical to the successful development of genomic medicines. We attempted to optimize AAV vector production in a producer cell line platform. In this system, helper functions required for AAV replication and production are provided via infection with a replication-competent wild-type Adenovirus. To evaluate strategies for reduction of replication and packaging of adenovirus as well as to understand the interplay of recombinant AAV and the helper virus during AAV vector production, wild-type adenovirus was compared to a mutant (Ad5ts149) containing a temperature-sensitive mutation in the DNA polymerase gene. Infection of a producer cell line with Ad5ts149 at the restrictive temperature reduced recombinant AAV titer and altered the pattern of AAV protein expression. Further investigation revealed that the adenoviral late L4-22K/33K gene products regulated both AAV rep/cap gene transcription and splicing of the rep/cap transcripts. Furthermore, the L4-33K gene products were found to impact AAV production in both the producer cell line and transient transfection platforms. Optimization of Adenovirus L4-22K/33K expression to facilitate efficient expression and splicing of AAV rep/cap transcripts therefore represents a unique opportunity to optimize AAV vector production.

Transcriptomic and Bioinformatic Analysis of Histone H2A-genes in Lates calcarifer and Oreochromis niloticus upon Vibrio vulnificus Challenge

Histone H2A-derived antimicrobial peptides (AMPs) have been recognized for their antimicrobial properties and role in host defense mechanisms. The present study investigated the transcriptional responses of two histone H2A-derived AMPs, Lc-Histone from Lates calcarifer and GIFT-Histone from Oreochromis niloticus, in response to Vibrio vulnificus infection. The genes encoding these peptides were amplified and sequenced from fingerling stage fishes, followed by in silico analysis and phylogenetic characterization using ClustalW. Computational methods were used to assess their functional attributes, and in vivo bacterial challenge assay was employed to evaluate the efficacy. Sequence alignment identified conserved histone motifs, supporting their evolutionary conservation. The antimicrobial peptide database predicted membrane interaction potential, suggesting antimicrobial activity, alongside biofilm-active properties and efficient expression in heterologous protein systems. Expression profiling of Histone H2A-derived AMPs under gram-negative bacterial stimulation revealed distinct temporal dynamics, indicative of their innate immune response involvement. Significant gene expression variations were observed between Lates calcarifer and Oreochromis niloticus. These baseline findings enhance understanding of host defense mechanisms and bacterial interactions in aquaculture. The antimicrobial potential of histone H2A-derived AMPs highlights their significance as therapeutic agents and warrants further investigation into their mechanisms of action and optimal usage in aquaculture settings

Ethanol consumption during gestation promotes placental alterations in IGF-1 deficient mouse placentas

Background During pregnancy, the placenta is an extremely important organ as it secretes its own hormones, e.g. insulin-like growth factor 1 (IGF-1), to ensure proper intrauterine fetal growth and development. Ethanol, an addictive and widely used drug, has numerous adverse effects during pregnancy, including fetal growth restriction (FGR). To date, the molecular mechanisms by which ethanol triggers its toxic effects during pregnancy, particularly in the placenta, are not entirely known. For this reason, a murine model of partial IGF-1 deficiency was used to determine ethanol alterations in placental morphology and aspartyl/asparaginyl β-hydroxylase (AAH) expression. Methods Wild type (WT, Igf1 +/+) and heterozygous (HZ, Igf1 +/-) female mice were given 10% ethanol in water during 14 days as an acclimation period and throughout pregnancy. WT and HZ female mice given water were used as controls. At gestational day 19, pregnant dams were sacrificed, placentas were collected and genotyped for subsequent studies. Results IGF-1 deficiency and ethanol consumption during pregnancy altered placental morphology, and decreased placental efficiency and AAH expression in placentas from all genotypes. No differences were found in Igf1, Igf2, Igf1r and Igf2r mRNA expression in placentas from all groups. Conclusions IGF-1 deficiency and ethanol consumption throughout gestation altered placental development, suggesting the crucial role of IGF-1 in the establishment of an adequate intrauterine environment that allows fetal growth. However, more studies are needed to study the precise mechanism to stablish the relation between both insults.

Novel dual-pathogen multi-epitope mRNA vaccine development for Brucella melitensis and Mycobacterium tuberculosis in silico approach.

Brucellosis and Tuberculosis, both of which are contagious diseases, have presented significant challenges to global public health security in recent years. Delayed treatment can exacerbate the conditions, jeopardizing patient lives. Currently, no vaccine has been approved to prevent these two diseases simultaneously. In contrast to traditional vaccines, mRNA vaccines offer advantages such as high efficacy, rapid development, and low cost, and their applications are gradually expanding. This study aims to develop multi-epitope mRNA vaccines argeting Brucella melitensis and Mycobacterium tuberculosis H37Rv (L4 strain) utilizing immunoinformatics approaches. The proteins Omp25, Omp31, MPT70, and MPT83 from the specified bacteria were selected to identify the predominant T- and B-cell epitopes for immunological analysis. Following a comprehensive evaluation, a vaccine was developed using helper T lymphocyte epitopes, cytotoxic T lymphocyte epitopes, linear B-cell epitopes, and conformational B-cell epitopes. It has been demonstrated that multi-epitope mRNA vaccines exhibit increased antigenicity, non-allergenicity, solubility, and high stability. The findings from molecular docking and molecular dynamics simulation revealed a robust and enduring binding affinity between multi-epitope peptides mRNA vaccines and TLR4. Ultimately, Subsequently, following the optimization of the nucleotide sequence, the codon adaptation index was calculated to be 1.0, along with an average GC content of 54.01%. This indicates that the multi-epitope mRNA vaccines exhibit potential for efficient expression within the Escherichia coli(E. coli) host. Analysis through immune modeling indicates that following administration of the vaccine, there may be variation in immunecell populations associated with both innate and adaptive immune reactions. These types encompass helper T lymphocytes (HTL), cytotoxic T lymphocytes (CTL), regulatory T lymphocytes, natural killer cells, dendritic cells and various immune cell subsets. In summary, the results suggest that the newly created multi-epitope mRNA vaccine exhibits favorable attributes, offering novel insights and a conceptual foundation for potential progress in vaccine development.

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Chronic activation of corticospinal tract neurons after pyramidotomy injury enhances neither behavioral recovery nor axonal sprouting.

Modulation of neural activity is a promising strategy to influence the growth of axons and improve behavioral recovery after damage to the central nervous system. The benefits of neuromodulation likely depend on optimization across multiple input parameters. Here we used a chemogenetic approach to achieve continuous, long-term elevation of neural activity in murine corticospinal tract (CST) neurons. To specifically target CST neurons, AAV2-retro-DIO-hM3Dq-mCherry or matched mCherry control was injected to the cervical spinal cord of adult Emx1-Cre transgenic mice. Pilot studies verified efficient transgene expression in CST neurons and effective elevation of neural activity as assessed by cFos immunohistochemistry. In subsequent experiments mice were administered either DIO-hM3Dq-mCherry or control DIO-mCherry, were pre-trained on a pellet retrieval task, and then received unilateral pyramidotomy injury to selectively ablate the right CST. Mice then received continual clozapine via drinking water and weekly testing on the pellet retrieval task, followed by cortical injection of a viral tracer to assess cross-midline sprouting by the spared CST. After sacrifice at eight weeks post-injury immunohistochemistry for cFos verified elevated CST activity in hM3Dq-treated animals and immunohistochemistry for PKC-gamma verified unilateral ablation of the CST in all animals. Despite the chronic elevation of CST activity, however, both groups showed similar levels of cross-midline CST sprouting and similar success in the pellet retrieval task. These data indicate that continuous, long-term elevation of activity that is targeted specifically to CST neurons does not affect compensatory sprouting or directed forelimb movements.

Long-term nerve regeneration in diabetic keratopathy mediated by a novel NGF delivery system.

Diabetic keratopathy (DK) is a common chronic metabolic disorder that causes ocular surface complications. Among various therapeutic approaches, local delivery of nerve growth factor (NGF) remains the most effective treatment for DK. However, achieving a sustained therapeutic effect with NGF and the frequent drug delivery burden remain challenging during clinical practice. Here, we developed a novel adeno-associated virus (AAV)-based NGF delivery system that achieved one-year-long-lasting effects by a single injection. We refined the corneal stromal injection technique, resulting in reduced corneal edema and improved AAV distribution homogeneity. AAV serotype AAV.rh10 exhibited high tropism and specificity to corneal nerves. A dose of 2×109 vector genomes (vg) was determined to achieve efficient Ngf gene expression without inducing corneal immune responses. Moreover, NGF protein was highly expressed in trigeminal ganglion (TG) through a retrograde transport mechanism, indicating the capacity for repairing corneal nerve damage both at the root and corneal nerve endings. In a mouse DK model, a single injection of AAV-Ngf into the corneal stroma led to marked corneal nerve regeneration for over 5 months. Together, we provide a novel therapeutic paradigm for long-term effective treatment of DK and this therapeutic approach is superior to current DK therapies.

Development of a xylose-inducible and glucose-insensitive expression system for Parageobacillus thermoglucosidasius

Inducible expression systems are pivotal for governing gene expression in strain engineering and synthetic biotechnological applications. Therefore, a critical need persists for the development of versatile and efficient inducible expression mechanisms. In this study, the xylose-responsive promoter xylA5p and its transcriptional regulator XylR were identified in Parageobacillus thermoglucosidasius DSM 2542. By combining promoter xylA5p with its regulator XylR, fine-tuning the expression strength of XylR, and reducing the glucose catabolite repression on xylose uptake, we successfully devised a xylose-inducible and glucose-insensitive expression system, denoted as IExyl*. This system exhibited diverse promoter strengths upon induction with xylose at varying concentrations and remained unhindered in the presence of glucose. Moreover, we showed the applicability of IExyl* in P. thermoglucosidasius by redirecting metabolic flux towards riboflavin biosynthesis, culminating in a 2.8-fold increase in riboflavin production compared to that of the starting strain. This glucose-insensitive and xylose-responsive expression system provides valuable tools for designing optimized biosynthetic pathways for high-value products and facilitates future synthetic biology investigations in Parageobacillus.Key points• A xylose-inducible and glucose-insensitive expression system IExyl* was developed.• IExyl* was applied to enhance the riboflavin production in P. thermoglucosidasius• A tool for metabolic engineering and synthetic biology research in Parageobacillus strains.

New Plasmid Constructs for Production of Yeast Based Oral Vaccines

In order to avoid conventional vaccines, there is an urgent need to look for new ways of vaccine generation that can cut down the cost, time, and the need for special requirements during storage and distribution. The development of oral peptide/protein-based vaccines could be an ideal alternative. Five expression plasmids are designed in this work for the production of Saccharomyces cerevisiae-based oral vaccines. Therefore, in this work genetic cassette with different organizations of the required genetic components for efficient gene expression is constructed in the pYES2 plasmid. The construction of five new plasmids for surface display of heterologous recombinant proteins in S. cerevisiae would enable easy insertion of the gene of interest into an optimized genetic cassette consisting of a strong host promoter GAL1, HA-tag for detection of recombinant enzymes, GAS1 gene with/without binding sequence coded for GAS1 cell wall protein and signal sequence for directing the protein into the secretory pathway. After the construction of the genetic cassettes, E gene coded for envelope protein of Chikungunya and Dengue virus is inserted in the genetic cassettes. Genetic models pYES2HAGAS1EgeneChikungunya, pYES2HAGAS1 Egene Dengue, pYES2GAS1 Egene ChikungunyaHA, and pYES2GAS1EGeneDengueHA are constructed with different genetic organizations to display envelope protein of Chikungunya and Dengue virus in the cell surface of S. cerevisiae. In the pYES2SignalEGeneDengueHA construction, envelope protein will be secreted by S. cerevisiae into the media. The constructed vectors are specialized to regulate the expression and surface display of heterologous recombinant proteins in Saccharomyces cerevisiae and will be suitable for large-scale oral vaccine production.

Development of a Flow Through-Based Limited Digestion Approach for High-Throughput and High-Sequence Coverage Mapping of Therapeutic mRNAs.

Messenger RNAs (mRNAs) have rapidly emerged as a pivotal class of biotherapeutics with great promise in the prevention and treatment of various diseases. As with other biotherapeutics, the sequence accuracy and integrity of mRNAs are critical quality attributes (CQAs), influencing the translation efficiency and protein expression fidelity of mRNAs. Due to the generation of short and repetitive oligonucleotides, traditional sequence mapping methods, which utilize in-solution RNase T1 digestion followed by LC-MS/MS analysis, face challenges in achieving high sequence coverage. In this study, we developed a novel flow through (FT)-based strategy to achieve the limited RNase T1 digestion of therapeutic mRNAs, leading to improved mRNA sequence mapping by LC-MS/MS analysis. Compared with the traditional in-solution digestion methods, the FT-based digestion method could consistently generate an increased number of unique oligonucleotides with miscleavages, which significantly boosted the overall sequence coverage (over 93%) of therapeutic mRNAs of varying sizes. Moreover, the automated digestion workflow using the AssayMAP platform offers significant advantages in method reproducibility and throughput. The high throughput and high sequence coverage features of this method could facilitate its wide application in the development of mRNA-based therapeutics.

Multi-head Network based Students Behaviour Prediction with Feedback Generation for Enhancing Classroom Engagement and Teaching Effectiveness

Emotions are pivotal in the learning process, highlighting the importance of identifying students' emotional states within educational settings. While neural network models, particularly those rooted in deep learning, have demonstrated remarkable accuracy in detecting primary emotions like happiness, sadness, fear, disgust, and anger from facial expressions in videos, these emotions occur infrequently in learning environments. Conversely, cognitive emotions such as engagement, confusion, frustration, and boredom are significantly more prevalent, transpiring five times more frequently than basic emotions. However, unlike basic emotions which are relatively distinct, cognitive emotions present a subtler distinction, necessitating the utilization of more sophisticated models for accurate recognition. The proposed work presents an efficient Facial Expression Recognition (FER) model for monitoring the student engagement in a learning environment by considering their facial expressions like boredom, frustration, confusion and engagement. The proposed methodology includes certain pre-processing steps followed by facial expression recognition founded on Efficient-Net B3 CNN in which the learning parameters are optimized using Circle-Inspired Optimization Algorithm (CIOA). Finally, the post processing stage estimates the frame-wise group engagement level (GEL) of students based on certain expression labels. Based on the acquired results, it is noted that the suggested Efficient-Net B3 CNN-CIOA based FER model provides promising results in terms of accuracy by 99.5%, precision by 99.2%, recall by 99.5% and f1-score by 99.6%, when compared with some state-of-art facial expression recognition approaches. Also, the suggested approach computational complexity is very much less than the compared existing approaches.

Efficient enhancement of the antimicrobial activity of Chlamydomonas reinhardtii extract by transgene expression and molecular modification using ionizing radiation

BackgroundIonizing radiation has been used for mutagenesis or material modification. The potential to use microalgae as a platform for antimicrobial production has been reported, but little work has been done to advance it beyond characterization to biotechnology. This study explored two different applications of ionizing radiation as a metabolic remodeler and a molecular modifier to enhance the antimicrobial activity of total protein and solvent extracts of Chlamydomonas reinhardtii cells.ResultsFirst, highly efficient transgenic C. reinhardtii strains expressing the plant-derived antimicrobial peptides, AtPR1 or AtTHI2.1, were developed using the radiation-inducible promoter, CrRPA70Ap. Low transgene expression was significantly improved through X-irradiation (12–50 Gy), with peak activity observed within 2 h. Protein extracts from these strains after X-irradiation showed enhanced antimicrobial activity against the prokaryotic bacterium, Pseudomonas syringae, and the eukaryotic fungus, Cryptococcus neoformans. In addition, X-irradiation (12 Gy) increased the growth and biomass of the transgenic strains. Second, C. reinhardtii cell extracts in ethanol were γ-irradiated (5–20 kGy), leading to molecular modifications and increased antimicrobial activity against the phytopathogenic bacteria, P. syringae and Burkholderia glumae, in a dose-dependent manner. These changes were associated with alterations in fatty acid composition. When both transgenic expression of antimicrobial peptides and molecular modification of bioactive substances were applied, the antimicrobial activity of C. reinhardtii cell extracts was further enhanced to some extent.ConclusionOverall, these findings suggest that ionizing radiation can significantly enhance the antimicrobial potential of C. reinhardtii through efficient transgene expression and molecular modification of bioactive substances, making it a valuable source of natural antimicrobial agents. Ionizing radiation can act not only as a metabolic remodeler of transgene expression in microalgae but also as a molecular modifier of the bioactive substances.

Development and validation of a droplet digital PCR method for quantifying lentiviral vector infectious titer

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

Nanogene editing drug delivery systems in the treatment of liver fibrosis.

Liver fibrosis is a group of diseases that seriously affect the health of the world's population. Despite significant progress in understanding the mechanisms of liver fibrogenesis, the technologies and drugs used to treat liver fibrosis have limited efficacy. As a revolutionary genetic tool, gene editing technology brings new hope for treating liver fibrosis. Combining nano-delivery systems with gene editing tools to achieve precise delivery and efficient expression of gene editing tools that can be used to treat liver fibrosis has become a rapidly developing field. This review provides a comprehensive overview of the principles and methods of gene editing technology and commonly used gene editing targets for liver fibrosis. We also discuss recent advances in common gene editing delivery vehicles and nano-delivery formulations in liver fibrosis research. Although gene editing technology has potential advantages in liver fibrosis, it still faces some challenges regarding delivery efficiency, specificity, and safety. Future studies need to address these issues further to explore the potential and application of liver fibrosis technologies in treating liver fibrosis.

Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

A Novel Self-Amplifying mRNA with Decreased Cytotoxicity and Enhanced Protein Expression by Macrodomain Mutations.

The efficacy and safety of self-amplifying mRNA (saRNA) have been demonstrated in COVID-19 vaccine applications. Unlike conventional non-replicating mRNA (nrmRNA), saRNA offers a key advantage: its self-replication mechanism fosters efficient expression of the encoded protein, leading to substantial dose savings during administration. Consequently, there is a growing interest in further optimizing the expression efficiency of saRNA. In this study, in vitro adaptive passaging of saRNA is conducted under exogenous interferon pressure, which revealed several mutations in the nonstructural protein (NSP). Notably, two stable mutations, Q48P and I113F, situated in the NSP3 macrodomain (MD), attenuated its mono adenosine diphosphate ribose (MAR) hydrolysis activity and exhibited decreased replication but increased payload expression compared to wild-type saRNA (wt saRNA). Transcriptome sequencing analysis unveils diminished activation of the double-stranded RNA (dsRNA) sensor and, consequently, a significantly reduced innate immune response compared to wt saRNA. Furthermore, the mutant saRNA demonstrated less translation inhibition and cell apoptosis than wt saRNA, culminating in higher protein expression both in vitro and in vivo. These findings underscore the potential of reducing saRNA replication-dependent dsRNA-induced innate immune responses through genetic modification as a valuable strategy for optimizing saRNA, enhancing payload translation efficiency, and mitigating saRNA cytotoxicity.

Silicon Modifies Photosynthesis Efficiency and hsp Gene Expression in European Beech (Fagus sylvatica) Seedlings Exposed to Drought Stress.

Background: Climate change is leading to severe and long-term droughts in European forest ecosystems. can have profound effects on various physiological processes, including photosynthesis, gene expression patterns, and nutrient uptake at the developmental stage of young trees. Objectives: Our study aimed to test the hypothesis that the application of silica (SiO2) influences photosynthetic efficiency and gene expression in 1- to 2-year-old Fagus sylvatica (L.) seedlings. Additionally, we aimed to assess whether silicon application positively influences the structural properties of leaves and roots. To determine whether the plant physiological responses are genotype-specific, seedlings of four geographically different provenances were subjected to a one-year evaluation under greenhouse conditions. Methods: We used the Kruskal-Wallis test followed by Wilcoxon's test to evaluate the differences in silicon content and ANOVA followed by Tukey's test to evaluate the physiological responses of seedlings depending on treatment and provenance. Results: Our results showed a significantly higher Si content in the roots compared with the leaves, regardless of provenance and treatment. The most significant differences in photosynthetic performance were found in trees exposed to Si treatment, but the physiological responses were generally nuanced and provenance-dependent. Expression of hsp70 and hsp90 was also increased in leaf tissues of all provenances. These results provide practical insights that Si can improve the overall health and resilience of beech seedlings in nursery and forest ecosystems, with possible differences in the beneficial role of silicon application arising from the large differences in wild populations of forest tree species.

Multimodal Emotion Recognition Based on Facial Expressions, Speech, and Body Gestures

The research of multimodal emotion recognition based on facial expressions, speech, and body gestures is crucial for oncoming intelligent human–computer interfaces. However, it is a very difficult task and has seldom been researched in this combination in the past years. Based on the GEMEP and Polish databases, this contribution focuses on trimodal emotion recognition from facial expressions, speech, and body gestures, including feature extraction, feature fusion, and multimodal classification of the three modalities. In particular, for feature fusion, two novel algorithms including supervised least squares multiset kernel canonical correlation analysis (SLSMKCCA) and sparse supervised least squares multiset kernel canonical correlation analysis (SSLSMKCCA) are presented, respectively, to carry out efficient facial expression, speech, and body gesture feature fusion. Different from the traditional multiset kernel canonical correlation analysis (MKCCA) algorithms, our SLSKMCCA algorithm is a supervised version and is based on the least squares form. The SSLSKMCCA algorithm is implemented by the combination of SLSMKCCA and a sparse item (L1 Norm). Moreover, two effective solving algorithms for SLSMKCCA and SSLSMKCCA are presented in addition, which use the alternated least squares and augmented Lagrangian multiplier methods, respectively. The extensive experimental results on the popular public GEMEP and Polish databases show that the recognition rate of multimodal emotion recognition is superior to bimodal and monomodal emotion recognition on average, and our presented SLSMKCCA and SSLSMKCCA fusion methods both obtain very high recognition rates, especially for the SSLSMKCCA fusion method.

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.

Transduced Olfactory Mucosa Cells Expressing Nerve Growth Factor for the Therapy of Experimental Spinal Cord Cysts.

A new gene-cell construct expressing nerve growth factor (NGF) has been developed. After obtaining engineered adenovectors Ad5-RGD-CAG-NGF and Ad5-RGD-CAG-EGFP, transduction efficiency and transgene expression were studied and multiplicity of infection was determined. The efficacy of transduced human olfactory ensheathing cells expressing NGF in restoring motor activity in rats has been shown in a limited period of time. Improved rat hindlimb mobility and cyst size reduction after gene-cell construct transplantation were more likely due to the cellular component of the construct.