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Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of post-translational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains' secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion., (© 2021. The Author(s).)

Designing the expression cassettes with desired properties remains the most important consideration of gene engineering technology. One of the challenges for predictive gene expression is the modeling of synthetic gene switches to regulate one or more target genes which would directly respond to specific chemical, environmental, and physiological stimuli. Assessment of natural promoter, high-throughput sequencing, and modern biotech inventory aided in deciphering the structure of cis elements and molding the native cis elements into desired synthetic promoter. Synthetic promoters which are molded by rearrangement of cis motifs can greatly benefit plant biotechnology applications. This review gives a glimpse of the manual in vivo gene regulation through synthetic promoters. It summarizes the integrative design strategy of synthetic promoters and enumerates five approaches for constructing synthetic promoters. Insights into the pattern of cis regulatory elements in the pursuit of desirable "gene switches" to date has also been reevaluated. Joint strategies of bioinformatics modeling and randomized biochemical synthesis are addressed in an effort to construct synthetic promoters for intricate gene regulation.

CD25 is a marker molecule expressed on many T and B cell neoplasms and on activated T cells that mediate organ transplant rejection and many autoimmune diseases. Single-chain variable fragments (scFvs) have many advantages over whole antibodies for use in antibody-targeted immunotherapy due to their small size. Daclizumab is an Food and Drug Administration-approved humanized anti-CD25 antibody. We attempted to produce a daclizumab-derived scFv, designated as Dmab(scFv), in Pichia pastoris. The Dmab(scFv) gene was designed based on the variable regions of the heavy and light chains of daclizumab, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS115. Under optimized conditions (culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h), the yield of soluble recombinant Dmab(scFv) was approximately 80 mg l(-1). Flow cytometry, immunohistochemistry, and Western blotting revealed that the purified Dmab(scFv) could bind strongly to the membrane of CD25-positive cells, including SNT-8 cells, ConA-stimulated human peripheral blood mononuclear cells (hPBMCs), and rat splenocytes, but not to CD25-negative cells, including Raji cells, unstimulated hPBMCs, and rat splenocytes. These results suggest that Dmab(scFv) produced in P. pastoris is active and specific toward CD25-positive cells and has potential for use in CD25-targeted immunotherapy.

The Masculinizer gene, Masc, encodes a lepidopteran‐specific novel CCCH‐type zinc finger protein, which controls sex determination and dosage compensation in Bombyx mori. Considering the potential application of it in pest control, it is necessary to investigate the function of Masc gene in Hyphantria cunea, a globally invasive forest pest. In the present study, we identified and functionally characterized the Masc gene, HcMasc, in H. cunea. Sequence analysis revealed that HcMASC contained the conserved CCCH‐type zinc finger domain, nuclear localization signal, and male determining domain, in which the last was confirmed to be required for its masculinization in BmN cell line. However, expression data showed that unlike male‐biased expression in B. mori, HcMasc gene expresses in main all developmental stages or tissues in both sexes. Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR‐associated protein 9‐based disruption of the common exons 1 and 3 of the HcMasc gene resulted in imbalanced sex ratio and abnormal external genitalia of both sexes. Our results suggest that the HcMasc gene is required for both male and female sexual differentiation and dosage compensation in H. cunea and provide a foundation for developing better strategies to control this pest. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) represents a significant challenge in oncology, primarily due to its resistance to conventional therapies. Understanding the tumour microenvironment (TME) is crucial for developing new treatment strategies. This study focuses on the role of amyloid precursor protein (APP) in tumour‐associated macrophages (TAMs) within the ccRCC TME, exploring its potential as a prognostic biomarker. Basing TAM‐related genes, the prognostic model was important to constructed. Employing advanced single‐cell transcriptomic analysis, this research dissects the TME of ccRCC at an unprecedented cellular resolution. By isolating and examining the gene expression profiles of individual cells, particularly focusing on TAMs, the study investigates the expression levels of APP and their association with the clinical outcomes of ccRCC patients. The analysis reveals a significant correlation between the expression of APP in TAMs and patient prognosis in ccRCC. Patients with higher APP expression in TAMs showed differing clinical outcomes compared to those with lower expression. This finding suggests that APP could serve as a novel prognostic biomarker for ccRCC, providing insights into the disease progression and potential therapeutic targets. This study underscores the importance of single‐cell transcriptomics in understanding the complex dynamics of the TME in ccRCC. The correlation between APP expression in TAMs and patient prognosis highlights APP as a potential prognostic biomarker. However, further research is needed to validate these findings and explore the regulatory mechanisms and therapeutic implications of APP in ccRCC. [ABSTRACT FROM AUTHOR]

Cord blood (CB) CD34+ cells have the potential to be used to achieve artificial hematopoiesis because of their ability to expand and differentiate in multiple directions. However, the mechanism and molecular changes underlying such differentiation are still unclear. The differentiation of CB CD34+ cells is generally driven by subtle changes in gene expression. A crucial method for examining gene expression is quantitative real-time polymerase chain reaction, but the accuracy of the results is dependent on the use of reliable reference genes. Here, the transcription levels of 10 novel candidate reference genes (EIF4G2, DYNC1H1, LUC7L3, CD46, POLR1D, WSB1, GAPVD1, HGS, LGALS8, and RBM5) and 8 traditional reference genes (GAPDH, YWHAZ, ACTB, B2MG, TBP, HMBS, PPIA, HPRT1) in CB CD34+ cells under different oxygen concentrations were screened and evaluated by using the geNorm and NormFinder algorithms. Comprehensive analysis conducted by RefFinder online tool showed that TBP (a traditional reference gene) and EIF4G2 (a novel reference gene) had the most stable expression, whereas GAPDH and HMBS were the least suitable reference genes under these conditions. These results may serve as a basis for selecting reference genes with stable expression for more accurate normalization under different oxygen concentration stimulation during CB CD34+ cells differentiation. [ABSTRACT FROM AUTHOR]

Pulmonary inflammation involves complex changes of the immune cells, in which macrophages play important roles and their function might be influenced by metabolism. Slc38a6 acts as a carrier of nutrient for macrophages (Mφ) to exert the function. In this study, pneumonia patient blood was found up-regulated SLC38A6 expression, which correlated with monocytes number and white blood cell number. The similar result was also shown in LPS induced sepsis mice. To reveal the key role of Slc38a6, we used systemic and conditional knock-out mice. Either systemic or LyzCRE specific knock-out could alleviate the severity of sepsis mice, reduce the proinflammatory cytokine TNF-α and IL-1β expression in serum and decrease the monocytes number in bronchial alveolar lavage and peritoneal lavage via flow cytometry. In order to reveal the signal of up-regulated Slc38a6, the Tlr4 signal inhibitor TAK242 and TLR4 knock-out mice were used. By blocking Tlr4 signal in macrophages via TAK242, the expression of Slc38a6 was down-regulated synchronously, and the same results were also found in Tlr4 knock-out macrophages. However, in the overexpressed Slc38a6 macrophages, blocking Tlr4 signal via TAK242, 20% of the mRNA expression of IL-1β still could be expressed, indicating that up-regulated Slc38a6 participates in IL-1β expression process. Collectively, it is the first time showed that an amino acid transporter SLC38A6 up-regulated in monocytes/macrophages promotes activation in pulmonary inflammation. SLC38A6 might be a promising target molecule for pulmonary inflammation treatment. [ABSTRACT FROM AUTHOR]

The Qing-Tibet Plateau is characterized by low oxygen pressure, which is an important biomedical and ecological stressor. However, the variation in gene expression during periods of stay on the plateau has not been well studied. We recruited eight volunteers to stay on the plateau for 3, 7, and 30 days. Human Clariom D arrays were used to measure transcriptome changes in the mRNA expression profiles in these volunteers' blood. Analysis of variance (ANOVA) indicated that 699 genes were significantly differentially expressed in response to entering the plateau during hypoxic exposure. The genes with changes in transcript abundance were involved in the terms phosphoprotein, acetylation, protein binding, and protein transport. Furthermore, numerous genes involved in hematopoietic functions, including erythropoiesis and immunoregulation, were differentially expressed in response to hypoxia. This phenomenon may be one of reasons why the majority of people entering the plateau do not have excessive erythrocyte proliferation and are susceptible to infection. [ABSTRACT FROM AUTHOR]

The prostrate cultivars of ground-cover chrysanthemum have been used in landscape gardening due to their small stature, large crown width and strong branching ability. qRT-PCR is a rapid and powerful tool for gene expression analysis, while its accuracy highly depends on the stability of reference genes. The paucity of authentic reference genes presents a major hurdle in understanding the genetic regulators of prostrate architecture. Therefore, in order to reveal the regulatory mechanism of prostrate growth of chrysanthemum stems, here, stable reference genes were selected for expression analysis of key genes involved in shoot development and graviresponse. Based on transcriptome data, eleven reference genes with relatively stable expression were identified as the candidate reference genes. After the comprehensive analysis of the stability of these reference genes with four programs (geNorm, NormFinder, BestKeeper and RefFinder), we found that TIP41 was the most stable reference gene in all of the samples. SAND was determined as a superior reference gene in different genotypes and during the process of shoot development. The optimal reference gene for gravitropic response was PP2A-1. In addition, the expression patterns of LA1 and PIN1 further verified the reliability of the screened reference genes. These results can provide more accurate and reliable qRT-PCR normalization for future studies on the expression patterns of genes regulating plant architecture of chrysanthemums. [ABSTRACT FROM AUTHOR]

Plant microRNAs are small non-coding, endogenic RNA molecule (containing 20–24 nucleotides) produced from miRNA precursors (pri-miRNA and pre-miRNA). Evidence suggests that up and down regulation of the miRNA targets the mRNA genes involved in resistance against biotic and abiotic stresses. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful technique to analyze variations in mRNA levels. Normalizing the data using reference genes is essential for the analysis of reliable RT-qPCR data. In this study, two groups of candidate reference mRNAs and miRNAs in soybean leaves and roots treated with various abiotic stresses (PEG-simulated drought, salinity, alkalinity, salinity+alkalinity, and abscisic acid) were analyzed by RT-qPCR. We analyzed the most appropriate reference mRNA/miRNAs using the geNorm, NormFinder, and BestKeeper algorithms. According to the results, Act and EF1b were the most suitable reference mRNAs in leaf and root samples, for mRNA and miRNA precursor data normalization. The most suitable reference miRNAs found in leaf and root samples were 166a and 167a for mature miRNA data normalization. Hence the best combinations of reference mRNAs for mRNA and miRNA precursor data normalization were EF1a + Act or EF1b + Act in leaf samples, and EF1a + EF1b or 60s + EF1b in root samples. For mature miRNA data normalization, the most suitable combinations of reference miRNAs were 166a + 167d in leaf samples, and 171a + 156a or 167a + 171a in root samples. We identified potential reference mRNA/miRNAs for accurate RT-qPCR data normalization for mature miRNA, miRNA precursors, and their targeted mRNAs. Our results promote miRNA-based studies on soybean plants exposed to abiotic stress conditions. [ABSTRACT FROM AUTHOR]

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming of immune cells has been proven to be important for systemic lupus erythematosus (SLE). This study aims to understand the role of SLC7A5, an amino acid transporter, in SLE. We analyzed SLC7A5 mRNA expression of SLE patients compared to healthy controls using GEO database, and found that it was increased in CD4+ T cells and CD19+ B cells. We then confirmed the expression up-regulation using flow cytometry and found that the proportion of SLC7A5+ cells and its expression were increased in peripheral blood T and B cells from SLE patients. Importantly, SLC7A5 expression in T and B cells was positively correlated with blood urea nitrogen and serum creatinine. Therefore, we conclude that SLC7A5, up-regulating in circulating T and B cells, correlates with kidney function, suggesting its potential role in mediating renal damage in SLE, which provides novel insight into SLE pathogenesis and provides a potential biomarker for disease. [ABSTRACT FROM AUTHOR]

Abstract: Fibrinogen-like protein 2 (fgl2), a novel prothrombinase, is involved in microthrombosis. We examined fgl2 expression in the glomerular and tubulointerstitial capillaries and its correlation with microthromsis in rats with streptozocin-induced type 2 diabetic nephropathy. Our RT-PCR and immunoblotting analysis showed that fgl2 mRNA and protein levels were increased in microvascular endothelial cells of the glomeruli and renal interstitia at week 19 and became significantly elevated with the development of diabetic nephropathy (P < 0.01). Fgl2 was not or only weakly expressed in the renal tissues of normal rats. Furthermore, a direct significant correlation (r = 0.543, P < 0.01) was found between fgl2 expression and microthrombotic capillaries in the renal tissues. Enzyme linked immunosorbent assays (ELISA) additionally showed that circulating TNF-α levels in rats with type 2 diabetes were significantly elevated and closely correlated with fgl2 expression (r = 0.871, P < 0.01). Our results suggest that fgl2 may activate renal microthrombosis, thus contributing to glomerular hypertension and renal ischemia. [Copyright &y& Elsevier]

The domesticated silkworm is an economically important insect that is widely used as a lepidopteran insect model. Although somatic sex determination in the silkworm is well characterized, germline sex determination is not. Here, we used the transgenic-based CRISPR/Cas9 genome editing system to study the function of the Ovo gene in Bombyx mori. BmOvo is the homolog of a factor important in germline sex determination in Drosophila melanogaster. BmOvo mutants had abnormally shaped eggs that were disordered in the ovarioles, and gonad development was abnormal. Interestingly, wing discs and wings did not develop properly, and most of the mutants failed to eclose. Gene expression analyses by qRT-PCR showed that BmOvo gene was highly expressed in the wing disc and epidermis. Genes involved in the WNT signaling pathway and wing development genes BmWCP10 and BmE74 were downregulated in the BmOvo mutants when compared with wild-type animals. These results demonstrate that the BmOvo gene product plays an important role in wing metamorphosis. Thus, this study provides new insights into the multiple functions of BmOvo beyond germline sex determination. [ABSTRACT FROM AUTHOR]

Co-contamination of nanoplastics (NPs) and arsenic (As) in aquatic environments poses a serious threat to the growth of aquatic plants, but the molecular toxicity mechanism leading to this joint effect on submerged macrophytes is still unclear. Here, we investigated the physiological, transcriptomic, metabolomic responses, and organelle changes in submerged macrophyte, Vallisneria denseserrulata (V. denseserrulata), to single/combined exposure to NPs and As. Our results showed that co-exposure alters physiological traits in V. denseserrulata including chlorophyll, sugars, proteins, malondialdehyde, and antioxidant enzymes. The presence of NPs exacerbated As distribution 36.2–47.2% higher than the control in plant tissues, thus enhancing combined pollution to plants. Integration of physiological traits and differentially expressed genes via weighted correlation network analysis implicated stress-responsive candidate modules related to key enzymes, such as ribulose-bisphosphate carboxylase, alanine transaminase, aspartate aminotransferase, phosphofructokinase-1, and phenylalanine ammonia-lyase. Metabolomics profiling identified carbohydrates, amino acids, organic acids, and fatty acids. Conjoint transcriptome and metabolome analysis revealed that photosynthetic systems, energy conversion, and oxidative and antioxidant regulation are the key defensive response mechanisms for V. denseserrulata under NPs-As co-exposure. Taken together, our multi-omics study provided new molecular insights into submerged macrophytes tolerance mechanisms against combined NPs and As toxicity, highlighting potential targets for stress mitigation and biomonitoring in contaminated aquatic ecosystems. [Display omitted] • Macrophytes showed different omics responses to NPs, As, and NPs-As stress. • WGCNA revealed the relationship between plant traits and gene expression. • NPs and As stress mainly affected metabolites, e.g. carbohydrates, amino acids. • Conjoint analysis revealed key defensive response mechanisms under co-exposure. [ABSTRACT FROM AUTHOR]

Antibodies to human epidermal growth factor receptor 2 (HER2) are a key element of breast cancer therapy; however, they are expensive to produce and their availability is limited. A seed-specific expression system can be used to produce recombinant proteins. We report a seed-specific expression system for the manufacture of anti-HER2 ScFv-Fc in Arabidopsis thaliana , driven by the Phaseolus vulgaris β-phaseolin promoter. Recombinant anti-HER2 ScFv-Fc was successfully and specifically expressed in seeds, and identified by protein analysis. The highest protein accumulation level, with a maximum of 1.1% of total soluble protein, was observed in mature seeds. We also demonstrated the anti-tumor potency of the plant-derived antibody against SK-BR-3 cells. These results suggest that seed-expression systems could contribute to the manufacture of commercial antibodies such as anti-HER2 ScFv-Fc. [ABSTRACT FROM AUTHOR]

Highlights: [•] ESM-1 expression was detected under various immune states. [•] ESM-1 mRNA and ESM-1 protein expression was upregulated significantly in the AR group, when compared to CsA group and control group. [•] ESM-1 may be used as a new indicator for AR prediction and diagnosis. [ABSTRACT FROM AUTHOR]