Higher Protein Expression Levels Research Articles

Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is an important zoonotic pathogen, which causes central nervous system symptoms in humans and reproductive disorders in swine. It has led to severe impacts on human health and the swine industry; however, there is no medicine available for treating yet. Therefore, vaccination is the best preventive measure for this disease. In the study, a modified mRNA vaccine expressing the prM and E proteins of the JEV P3 strain was manufactured, and a mouse model was used to assess its efficacy. The mRNA encoding prM and E proteins showed a high level of protein expression in vitro and were encapsulated into a lipid nanoparticle (LNP). Effective neutralizing antibodies and CD8+ T-lymphocytes-mediated immune responses were observed in vaccinated mice. Furthermore, the modified mRNA can protect mice from a lethal challenge with JEV and reduce neuroinflammation caused by JEV. This study provides a new option for the JE vaccine and lays a foundation for the subsequent development of a more efficient and safer JEV mRNA vaccine.

A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.Graphical

Citrus sudden death-associated virus as a new expression vector for rapid in planta production of heterologous proteins, chimeric virions, and virus-like particles

The more we understand the strategies used by viruses for protein expression, the more possibilities we have to exploit viruses as expression vectors for heterologous protein production. Advances in the development of virus-based expression systems have been possible due to generation of many virus infectious clones, especially those derived from plant viruses, which have the capability for rapid and high-level transient expression of proteins in plant cells, a robust and low-cost bioreactor. In this work, we generated new replicative virus expression vectors based on a previously constructed citrus sudden death-associated virus (CSDaV) infectious cDNA clone. These vectors were generated to express the reporter green fluorescent protein (GFP) in Nicotiana benthamiana leaves by taking advantage of the expression strategies used by CSDaV to produce its structural proteins. We show that higher amounts of GFP can be produced from a coat protein (CP)-independent CSDaV-based vector, compared to levels of GFP expressed from a widely used non-replicative vector (pEAQ series); or GFP can be produced in fusion with the major CSDaV CP (CPp21) to be incorporated into chimeric virions. However, GFP-recombinant CSDaV virions do not appear uniformly assembled, but more likely as mosaic particles. Cryo-electron microscopy analysis from this work revealed the structures of the wild-type and the GFP-recombinant CSDaV virions, but it was not able to reveal where exactly the GFP is displayed in the chimeric virions. We show though that the incorporation of GFP-CPp21 fusion protein into virions occurs solely due to its interaction with free/non-fused CPp21, independent of other viral proteins. Therefore, individual co-expression of GFP-CPp21 and CPp21 in the same plant cells leads to the production of chimeric virus-like particles (VLPs), while GFP-CPp21 fusion protein itself is not able to self-assemble into VLPs. The new CSDaV-based expression vectors may provide an alternative platform for use in molecular farming, either for production of heterologous proteins or as scaffold for heterologous protein display.

Immune-Related RNA-Binding Protein-Based Signature With Predictive and Prognostic Implications in Patients With Lung Adenocarcinoma.

Background: Dysregulation of RNA-binding proteins (RBPs) in cancers is associated with immune and cancer development. Here, we aimed to profile immune-related RBPs in lung adenocarcinoma (LUAD) and construct an immune-related RBP signature (IRBPS) to predict the survival and response to immunotherapy. Methods: A correlation analysis was performed to establish a co-expression network of RBPs and immune-related genes (IRGs) to characterize immune-related RBPs in the TCGA–LUAD cohort (n = 497 cases). Then, a combination of the Random survival forest (RSF) and Cox regression analysis was performed to screen the RBPs and establish IRBPS. This was followed by independent validation of IRBPS in GSE72094 (n = 398 cases), GSE31210, (n = 226 cases), and GSE26939 (n = 114 cases). Differences between the low- and high-risk groups were compared in terms of gene mutations, tumor mutation burden, tumor-infiltrating lymphocytes, and biomarkers responsive to immunotherapy. Results: DDX56, CTSL, ZC3H12D, and PSMC5 were selected and used to construct IRBPS. The high-risk scores of patients had a significantly worse prognosis in both training and testing cohorts (p < 0.0001 and p < 0.05, respectively), and they tended to be older and have an advanced TNM stage. Furthermore, IRBPS was a prognostic factor independent of age, gender, smoking history, TNM stage, and EGFR mutation status (p = 0.002). In addition, high-risk scores of IRBPS were significantly correlated with tumor-infiltrating lymphocytes (p < 0.05). They also had a high level of PD-L1 protein expression (p < 0.01), number of neoantigens (p < 0.001), and TMB (p < 0.001), implying the possible prediction of IRBPS in the immunotherapy of LUAD. Conclusion: The currently established IRBPS encompassing immune-related RBPs might serve as a promising tool to predict survival, reflect the immune microenvironment, and predict the efficacy of immunotherapy among LUAD patients.

Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus.

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family that damages the immune system of pigs, resulting in the death or slaughter of millions of animals worldwide. Recent modern techniques in ASFV vaccination have highlighted the potential of viral replicon particles (RPs), which can efficiently express foreign proteins and induce robust cellular and humoral immune responses compared with the existing vaccines. In this study, we established a Semliki Forest virus (SFV) vector by producing replication-defective viral particles. This vector was used to deliver RPs expressing ASFV antigens. SFV-RPs expressing ASFV p32 (SFV-p32) and p54 (SFV-p54) were tested in baby hamster kidney (BHK-21) cells. Proteins expression was evaluated via western blotting and indirect immunofluorescence, while immunogenicity was evaluated in BALB/c mice. The resulting RPs exhibited high levels of protein expression and elicited robust humoral and cellular immune responses.

The requirement of ubiquitin C-terminal hydrolase L1 in mouse ovarian development and fertility†.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

Variation in ORF3a Protein of SARS‐CoV‐2 Decreases The Severity of Host Cell Damage

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) primarily targets the respiratory system. However, direct SARS‐CoV‐2 infection and viral protein expression have also been reported in other organs, which potentially contributes to multi‐organ dysfunction and increased mortality in COVID‐19. Since the original Wuhan‐type genome was sequenced, several genetic variants of SARS‐CoV‐2 have emerged with differing pathophysiological properties such as the levels of transmissibility, disease severity, and mortality. We previously reported that the protein encoded by open reading frame 3a (ORF3a), a critical protein for SARS‐CoV‐2 replication and release, is found in the mitochondria of host cells and could increase oxidative stress and apoptotic signaling. ORF3a‐Q57H is a highly recurrent variation and the most commonly found variant of ORF3a. Interestingly, the Q57H variant is associated with increased transmissibility, but lower mortality.AimTo investigate the impact of the ORF3a‐Q57H variant on host cell damage.MethodsPlasmids carrying ORF3a‐Q57H were generated by PCR‐based site mutagenesis using Wuhan‐type ORF3a (ORF3a‐WT) as a template. Whole cell lysates were prepared from HEK293T cells and H9c2 cardiac myoblasts expressing ORF3a‐WT or the mutant ORF3a‐Q57H and used for biochemical assays. Live cell imaging for assessing subcellular localization of ORF3a/ORF3a‐Q57H, mitochondrial reactive oxygen species (mROS), and caspase 3 activity in H9c2 cells was performed by confocal microscopy.ResultsIn whole cell lysates, we found that ORF3a‐Q57H exhibits significantly higher protein expression compared to ORF3a‐WT. However, there is no significant difference in the ability of mitochondrial trafficking between ORF3a‐WT and ORF3a‐Q57H assessed by live cell imaging using GFP‐tagged ORF3a/ORF3a‐Q57H with mitochondria‐targeted DsRed. Next, we investigated the effects of ORF3a expression on apoptotic and mitophagy signaling by quantifying the caspase 3 activity and LC3A/B ratio. We found that ORF3a‐Q57H has significantly lower apoptosis and mitophagy signaling compared to WT despite its higher protein expression levels. ER‐stress signaling was not activated in either ORF3a‐WT or ORF3a‐Q57H, as assessed by markers including Glucose‐regulated protein 78/94 and C/EBP‐homologous protein. Lastly, live cell imaging using the mitochondrial superoxide‐sensitive dye Mitosox Red revealed that ORF3a‐WT significantly increases mROS levels, but ORF3a‐Q57H expression does not.ConclusionSARS‐CoV‐2‐ORF3a‐Q57H causes lower oxidative stress and cell damage compared to the Wuhan‐type variant, which could help explain the decreased mortality associated with the ORF3a‐Q57H variant. These results provide novel insights on how genetic variations of SARS‐CoV‐2 influence the pathophysiology and clinical severity of COVID‐19.

Distinctive High Expression of Antiretroviral APOBEC3 Protein in Mouse Germinal Center B Cells.

Tissue and subcellular localization and its changes upon cell activation of virus-restricting APOBEC3 at protein levels are important to understanding physiological functions of this cytidine deaminase, but have not been thoroughly analyzed in vivo. To precisely follow the possible activation-induced changes in expression levels of APOBEC3 protein in different mouse tissues and cell populations, genome editing was utilized to establish knock-in mice that express APOBEC3 protein with an in-frame FLAG tag. Flow cytometry and immunohistochemical analyses were performed prior to and after an immunological stimulation. Cultured B cells expressed higher levels of APOBEC3 protein than T cells. All differentiation and activation stages of freshly prepared B cells expressed significant levels of APOBEC3 protein, but germinal center cells possessed the highest levels of APOBEC3 protein localized in their cytoplasm. Upon immunological stimulation with sheep red blood cells in vivo, germinal center cells with high levels of APOBEC3 protein expression increased in their number, but FLAG-specific fluorescence intensity in each cell did not change. T cells, even those in germinal centers, did not express significant levels of APOBEC3 protein. Thus, mouse APOBEC3 protein is expressed at distinctively high levels in germinal center B cells. Antigenic stimulation did not affect expression levels of cellular APOBEC3 protein despite increased numbers of germinal center cells.

Estradiol reduced 5-HT reuptake by downregulating the gene expression of Plasma Membrane Monoamine Transporter (PMAT, Slc29a4) through estrogen receptor β and the MAPK/ERK signaling pathway

Estrogen deficiency-induced female depression is closely related to 5-hydroxytriptamine (5-HT) deficiency. Estradiol (17β-estradiol, E2) regulates the monoamine transporters and acts as an antidepressant by affecting 5-HT clearance through estrogen receptors and related signaling pathways at the genomic level, although the specific mechanisms require further exploration. The brain expresses higher levels of plasma membrane monoamine transporter (PMAT, involved in 5-HT reuptake of the uptake 2 system) than other uptake transporters. In this study, we found that estrogen-deficient ovariectomized (OVX) rats had high PMAT mRNA and protein expression levels in the hippocampus and estradiol significantly reduced these levels. Furthermore, estradiol inhibited PMAT expression and reduced 5-HT reuptake in neurons and astrocytes and estradiol regulated the PMAT expression mainly by affecting estrogen receptor β (ERβ) at the genomic level in astrocytes. Further experiments showed that estradiol also regulated PMAT expression through the MAPK/ERK signaling pathway and not through the PI3K/AKT signaling pathway. In conclusion, estradiol inhibited 5-HT reuptake by regulating PMAT expression at the genomic level through ERβ and the MAPK/ERK signaling pathway, highlighting the importance of PMAT in the antidepressant effects of estradiol through 5-HT clearance reduction.

Development of spirulina for the manufacture and oral delivery of protein therapeutics

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

Role of kinesin family member 14 in disease monitoring and prognosis in patients with gastrointestinal cancer.

Kinesin family member 14 (KIF14) is not only involved in numerous essential biological activities, such as cytokinesis and myelination, but also regulates several malignant behaviors and progression of cancer. However, its role in gastrointestinal cancer is rarely reported. Therefore, the present study aimed to investigate the association of KIF14 expression with disease-free survival (DFS) and overall survival (OS) times in patients with gastrointestinal cancer. A total of 101 patients with gastrointestinal cancer (36 patients with gastric cancer and 65 patients with colorectal cancer) were retrospectively reviewed, and their cancer samples were collected to detect the protein and mRNA expression levels of KIF14 using immunohistochemistry and reverse transcription-quantitative PCR, respectively. KIF14 protein expression was increased in cancer tissues compared with adjacent tissues (all P<0.001). The protein expression levels of KIF14 were positively associated with T stage (P<0.001), distant metastases (P=0.007) and TNM stage (P<0.001), while KIF14 mRNA expression was positively associated with T stage (P<0.001), lymph node metastasis (P=0.004), distant metastases (P=0.001) and TNM stage (P<0.001). High protein and mRNA expression levels of KIF14 were associated with worse DFS (P<0.001) and OS (P=0.016) times. In addition, high KIF14 protein expression independently predicted unfavorable DFS times (P=0.007). Subgroup analysis revealed that in patients with gastric cancer, KIF14 expression was associated with DFS and OS times, while in patients with colorectal cancer, KIF14 expression was only associated with DFS time, but not with OS time. In conclusion, KIF14 expression was not only associated with advanced pathological differentiation and TNM stage but was also associated with poor survival time in patients with gastrointestinal cancer. These results indicate the potential of KIF14 as a biomarker for gastrointestinal cancer prognosis.

TL1A/DR3 Axis, A Key Target of TNF-a, Augments the Epithelial-Mesenchymal Transformation of Epithelial Cells in OVA-Induced Asthma.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A), a member of the TNF family, exists in the form of membrane-bound (mTL1A) and soluble protein (sTL1A). TL1A binding its only known functional receptor death domain receptor 3 (DR3) affects the transmission of various signals. This study first proposed that the TL1A/DR3 axis was significantly upregulated in patients and mice with both asthma and high TNF-a expression and in TNF-a-stimulated epithelial Beas-2B cells. Two independent approaches were used to demonstrate that the TL1A/DR3 axis of mice was strongly correlated with TNF-a in terms of exacerbating asthmatic epithelial–mesenchymal transformation (EMT). First, high expression levels of EMT proteins (e.g., collagen I, fibronectin, N-cadherin, and vimentin) and TL1A/DR3 axis were observed when mice airways were stimulated by recombinant mouse TNF-a protein. Moreover, EMT protein and TL1A/DR3 axis expression synchronously decreased after mice with OVA-induced asthma were treated with infliximab by neutralizing TNF-a activity. Furthermore, the OVA-induced EMT of asthmatic mice was remarkably improved upon the deletion of the TL1A/DR3 axis by knocking out the TL1A gene. TL1A siRNA remarkably intervened EMT formation induced by TNF-a in the Beas-2B cells. In addition, EMT was induced by the addition of high concentrations of recombinant human sTL1A with the cell medium. The TL1A overexpression via pc-mTL1A in vitro remarkably increased the EMT formation induced by TNF-a. Overall, these findings indicate that the TL1A/DR3 axis may have a therapeutic role for asthmatic with high TNF-a level.

Acetylshikonin suppresses diffuse large B-Cell Lymphoma cell growth by targeting the T-lymphokine-activated killer cell-originated protein kinase signalling pathway

ABSTRACT Diffuse large B-cell lymphoma (DLBCL) is one of the most common causes of cancer death worldwide, and responds poorly to the existing treatments. Thus, identifying novel therapeutic targets of DLBCL is urgently needed. In this study, we found that T-lymphokine-activated killer cell-originated protein kinase (TOPK) was highly expressed in DLBCL cells and tissues. Data from the GEPIA database also indicated that TOPK was highly expressed in DLBCL tissues. The high expression levels of proteins were identified via Western blots and immunohistochemistry (IHC). TOPK knockdown inhibited cell growth and induced apoptosis of DLBCL cells with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) and flow cytometry. Further experiments demonstrated that acetylshikonin, a compound that targeted TOPK, could attenuate cell growth and aggravate cell apoptosis through TOPK/extracellular signal-regulated kinase (ERK)-1/2 signaling, as shown by MTS, flow cytometry and Western blots. In addition, we demonstrated that TOPK modulated the effect of acetylshikonin on cell proliferation and apoptosis in U2932 and OCI-LY8 cells using MTS, flow cytometry and Western blots. Taken together, the present study suggests that acetylshikonin suppresses the growth of DLBCL cells by attenuating TOPK signaling, and the targeted inhibition of TOPK by acetylshikonin may be a promising approach for the treatment of DLBCL.

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products.Key points• A protein expression vector using double-antibiotic resistance genes was designed• Double antibiotic selection causes fewer colonies with more positive for phenotype• Coupling the new vector with FACS improves microalgal screening efficiency > 60%

Translational gene expression control in Chlamydia trachomatis.

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4 plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.

Identification and Functional Analysis of a Novel CTNNB1 Mutation in Pediatric Medulloblastoma

Simple SummaryWe have analyzed a panel of 88 pediatric medulloblastoma tumors for exon 3 mutations from the CTNNB1 gene and identified eight missense point-mutations and one in-frame deletion. We describe and functionally characterize a novel CTNNB1 in-frame deletion (c.109-111del, pSer37del, ΔS37) found in a pediatric patient with a classic medulloblastoma, WNT-activated grade IV (WHO 2016). To the best of our knowledge, this mutation has not been previously reported in medulloblastoma, and it is uncertain its role in the disease development and progression. Our analysis discloses gain-of-function properties for the new ΔS37 β-catenin variant.Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3 CTNNB1 mutations and were immunostained for nuclear β-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of β-catenin Ser37 residue (ΔS37). The ΔS37 β-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type β-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 β-catenin contributed to early medulloblastoma tumorigenesis.

MAC2 is a long-lasting marker of peripheral cell infiltrates into the mouse CNS after bone marrow transplantation and coronavirus infection.

Microglia are the primary resident myeloid cells of the brain responsible for maintaining homeostasis and protecting the central nervous system (CNS) from damage and infection. Monocytes and monocyte-derived macrophages arising from the periphery have also been implicated in CNS pathologies, however, distinguishing between different myeloid cell populations in the CNS has been difficult. Here, we set out to develop a reliable histological marker that can assess distinct myeloid cell heterogeneity and functional contributions, particularly in the context of disease and/or neuroinflammation. scRNAseq from brains of mice infected with the neurotropic JHM strain of the mouse hepatitis virus (JHMV), a mouse coronavirus, revealed that Lgals3 is highly upregulated in monocyte and macrophage populations, but not in microglia. Subsequent immunostaining for galectin-3 (encoded by Lgals3), also referred to as MAC2, highlighted the high expression levels of MAC2 protein in infiltrating myeloid cells in JHMV-infected and bone marrow (BM) chimeric mice, in stark contrast to microglia, which expressed little to no staining in these models. Expression of MAC2 was found even 6-10months following BM-derived cell infiltration into the CNS. We also demonstrate that MAC2 is not a specific label for plaque-associated microglia in the 5xFAD mouse model, but only appears in a distinct subset of these cells in the presence of JHMV infection or during aging. Our data suggest that MAC2 can serve as a reliable and long-lasting histological marker for monocyte/macrophages in the brain, identifying an accessible approach to distinguishing resident microglia from infiltrating cells in the CNS under certain conditions.

Making Use of Plant uORFs to Control Transgene Translation in Response to Pathogen Attack.

Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity. Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology. Nevertheless, enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects. Upstream open reading frames (uORFs) are translational control elements located in the 5 untranslated region (UTR) of eukaryotic mRNAs and may repress the translation of downstream genes. To investigate the function of three uORFs from the 5-UTR of ACCELERATED CELL 11 (uORFsACD11), we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene translation. Individual or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different levels. Importantly, uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves. When coupled with the PATHOGENESIS-RELATED GENE 1 (PR1) promoter, the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2 (AtLecRK-VI.2) during pathogen attack and enhance plant resistance to Phytophthora capsici. These findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack, while for ensuring lower levels of protein expression at normal conditions.

High FGFR1-4 mRNA Expression Levels Correlate with Response to Selective FGFR Inhibitors in Breast Cancer.

FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively. Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient-derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence. High FGFR1-4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1-4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1-4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs. Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1-4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

A Streptomyces-Based Cell-Free Protein Synthesis System for High-Level Protein Expression.

With the rapid development of cell-free biotechnology, more and more cell-free protein synthesis (CFPS) systems have been established and optimized for protein expression in vitro. Here, we aim to improve the productivity of a newly developed Streptomyces-based CFPS system. Protein translation in CFPS systems depends on the entire endogenous translation system from cell lysates. However, lysates might lack such translation-related elements, limiting the efficiency of protein translation and therefore the productivity of CFPS systems. To address this limitation, we sought to add protein translation related factors to CFPS reactions. By doing this, the protein yield of EGFP was significantly improved up to approximately 400μg/mL. In this chapter, we mainly describe the preparation of Streptomyces cell extracts, expression and purification of nine translation related factors, and optimization of the Streptomyces-based CFPS system for enhanced protein expression.