Higher Protein Expression Levels Research Articles

Endosialin-positive CAFs promote hepatocellular carcinoma progression by suppressing CD8+ T cell infiltration

Background and aimsEndosialin, also known as tumor endothelial marker1 or CD248, is a transmembrane glycoprotein that is mainly expressed in cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC). Our previous study...

High Glycolytic Activity Signature Reveals CCNB2 as a Key Therapeutic Target in Triple-Negative Breast Cancer.

Aerobic glycolysis and the cell cycle are well-established tumor hallmarks. Understanding their relationship could help to unravel the pathogenic mechanisms of breast cancer (BC) and suggest potential new strategies for treatment. Glycolysis-related genes (GRGs) were downloaded from the Reactome database and screened using univariate Cox analysis. The consensus clustering method was employed to identify a glycolytic activity signature (GAS) using the Gene Expression Omnibus (GEO) dataset. A nomogram risk prediction model was constructed using coefficients from univariate Cox analysis. Immune cell infiltration was evaluated using single-sample gene set enrichment analysis (ssGSEA) and the ESTIMATE algorithm. Gene co-expression modules were created using weighted correlation network analysis (WGCNA) to identify hub genes. Gene expression in three BC cell lines was quantified using Quantitative Reverse Transcriptase Polymera (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) data was used to examine the relationship between GAS and hub genes. The sensitivity of different groups to cell cycle-related clinical drugs was also examined. BC with high GAS (HGAS) showed high tumor grade and recurrence rate. HGAS was a prognostic indicator of worse overall survival (OS) in BC patients. HGAS BC showed more abundant immune cells and significantly higher expression of immunomodulators compared to BC with low GAS (LGAS). HGAS BC also showed enhanced cell cycle pathway, with high mRNA and protein expression levels of Cyclin B2 (CCNB2), a key component of the cell cycle pathway. Importantly, scRNA-seq analysis revealed that elevated CCNB2 expression was positively correlated with HGAS in triple-negative BC (TNBC). This was validated in clinical samples from TNBC patients. High expression of CCNB2 was found in three BC cell lines, and was also an indicator of poor prognosis. HGAS BC showed high sensitivity to several cell cycle-related clinical drugs, with 9 of these also showing activity in BC with high CCNB2 expression. HGAS was associated with enhanced cell cycle pathway and immune activity in BC. These results suggest that CCNB2 is a potential key therapeutic target in BC patients.

Acute ocular hypertension in the living human eye: Model description and initial cellular responses to elevated intraocular pressure

This initial methods study presents the initial immunohistochemical and transcriptomic changes in the optic nerve head and retina from three research-consented brain-dead organ donors following prolonged and transient intraocular pressure (IOP) elevation. In this initial study, research-consented brain-dead organ donors were exposed to unilateral elevation of IOP for 7.5 h (Donor 1), 30 h (Donor 2), and 1 h (Donor 3) prior to organ procurement. Optic nerve tissue and retinal tissue was obtained following organ procurement for immunohistological and transcriptomic analysis.Optic nerve sections in Donor 1 exposed to 7.5-hours of unilateral sub-ischemic IOP elevation demonstrated higher levels of protein expression of the astrocytic marker, glial fibrillary acidic protein (GFAP), within the lamina cribrosa with greatest expression inferior temporally in the treated eye compared to control. Spatial transcriptomic analysis performed on optic nerve head tissues from Donor 2 exposed to 30 h of unilateral IOP elevation demonstrated differential transcription of mRNA across laminar and scleral regions. Immunohistochemistry of retinal sections from Donor 2 exhibited higher GFAP and IBA1 expression in the treated eye compared with control, but this was not observed in Donor 3, which was exposed to only 1-hour of IOP elevation. While there were no differences in GFAP protein expression in the retina following the 1-hour IOP elevation in Donor 3, there were higher levels of transcription of GFAP in the inner nuclear layer, and CD44 in the retinal ganglion cell layer, indicative of astrocytic and Müller glial reactivity as well as an early inflammatory response, respectively.We found that transcriptomic differences can be observed across treated and control eyes following unilateral elevation of IOP in brain dead organ donors. The continued development of this model affords the unique opportunity to define the acute mechanotranscriptomic response of the optic nerve head, evaluate the injury and repair mechanisms in the retina in response to IOP elevation, and enable correlation of in vivo imaging and functional testing with ex vivo cellular responses for the first time in the living human eye.

Expression of Autophagy-Related Proteins in Microlissencephaly Associated with a Novel Variant in the WDR81 Gene

Introduction: Microlissencephaly is a subtype of congenital microcephaly characterized by extreme microcephaly with simplified gyral pattern. Other brain malformations may accompany it. WDR81 encodes a multi-domain transmembrane protein that is predominantly expressed in the brain and is thought to play a role in endolysosomal trafficking and autophagy. Methods: We reported two siblings with microlissencephaly born to consanguineous Turkish parents and reviewed all previously reported patients with WDR81 variants presenting with severe microcephaly accompanied by congenital brain malformations. Whole-exome sequencing was performed on both siblings. Sanger DNA sequencing was performed on the patients’ parents. We also examined LC3, p62, and Beclin 1 mRNA and protein expression levels from blood samples of both siblings using real-time PCR and Western blotting, respectively. Results: Whole-exome sequencing revealed a novel biallelic c.4157+5G>A splice variant in the WDR81 gene in both siblings. In our study, LC3, p62, and Beclin 1 mRNA expression levels were high, LC3 protein expression level was also high and p62 and Beclin 1 protein expression levels were low. Conclusion: High LC3 and low p62 protein expression levels supported studies concluding that WDR81 inhibits autophagy through PI3KC3 inhibition, while low Beclin 1 protein expression level supported studies concluding that autophagy is suppressed in case of loss of function variants in the WDR81 gene. We suggest that due to its role in endolysosomal trafficking, WDR81-related diseases should be included in vesicular trafficking disorders.

Association between endometrial protein expression of IFN-γ and delayed conception after parturition in dairy cows.

The cytokine context present in the reproductive tract of cows is closely involved in normal uterine functions, including the estrous cycle and the establishment and maintenance of pregnancy. However, the roles of some cytokines in the uterus, and their relation with reproductive performance remain to be elucidated. Thus, this study aimed to examine the protein expression of several cytokines such as TNFα, IL-6, IL-8, IFNγ, IL-4, and TGF-β3 in endometrial biopsies previous to conception, to evaluate the possible association with delayed conception in dairy cows. Protein expression levels were evaluated by immunohistochemistry. Results showed that the protein expression levels of TNFα, IL-6, IL-4 and TGF-β3 were not associated with the parturition-conception interval, whereas the high protein expression levels of IFNγ were associated with the parturition-conception interval. Finally, the low protein expression of IL-8 showed a statistical tendency to be associated with delayed conception. This is the first report about the protein expression of IFN-γ in the endometrium of dairy cows and also, this cytokine could enhance the favorable conditions to achieve an early pregnancy.

The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages

BackgroundTumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization.MethodsEVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting.ResultsEVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells.ConclusionOur results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.

Prognostic Significance of Bcl-2 Expression in Non-germinal Center B-cell-Like Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphomas (DLBCLs) are a group of malignant neoplasms with extensive clinical and molecular heterogeneity. Several key genetic aberrations have been identified, such as those involving the MYC, BCL6, and BCL2 genes. Prior studies on the prognostic significance of Bcl-2 protein expression in DLBCL have been contradictory, with some suggesting it has an adverse effect, while others have shown no such association. Bcl-2 is known to be more highly expressed in the non-germinal centerB-cell-like (non-GCB) subtype compared to germinal centerB-cell-like (GCB) DLBCL. Non-GCB status is associated with a less favorable prognosis. This study aimed to investigate whether the expression of Bcl-2 protein in non-GCB DLBCL influences response to treatment, progression-free survival, or overall survival. In this retrospective study, we investigated whether there was a difference in the clinical outcomes of non-GCB DLBCL cases (n = 97) that were confirmed by immunochemistry to demonstrate high levels of Bcl-2 protein expression (>50% neoplastic cells stained) when compared to those who were deemed negative based on this criterion. Response to rituximab-based induction immunochemotherapy, five-year progression-freesurvival, and five-year overall survival were assessed. There was no statistically significant difference in response to treatment, five-year progression-freesurvival, or five-year overall survival between the patients who were positive for Bcl-2 (n = 70) compared to those who were considered Bcl-2 negative (n = 27). High levels of Bcl-2 protein expression do not appear to be of prognostic significance in non-GCB DLBCL and therefore Bcl-2 may not be a key therapeutic target in the treatment and improvement of clinical outcome in such cases.

A strategy for successful dual-species protein expression of genes with non-optimal codon usage destined for bacterial and yeast cell factories.

Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins aimed for expression in S. cerevisiae are often mutagenized and pre-screened in E. coli before expression in yeast. This introduces artificial bottlenecks as the bacterial expression vector needs to be substituted for a yeast expression vector via sub-cloning, and the new library re-evaluated before a final screening in yeast. Here, we put forward a protein expression and engineering strategy that involves the use of a dual-host shuttle vector (pYB-Dual) designed with both a strong inducible yeast promoter (pGAL1), and a strong inducible bacterial promoter (pT7-RNAP), which allows for inducible protein expression in both species. Additionally, we demonstrate that by transforming the pYB-Dual vector into the E. coli strain Rosetta 2, which has elevated levels of 7 rare tRNAs, we can achieve high-level protein expression in both yeast and bacteria, even when using a mNeonGreen gene codon optimized for yeast. This dual expression vector is expected to remove bottlenecks during protein engineering of commercially important enzymes destined for high-titer expression in yeast.

Identifying Potential Drug Targets for Keloid: A Mendelian Randomization Study

Keloids are a skin fibrosis disease characterized by troublesome symptoms, a varying degree of recurrence and inevitable side effects from treatments. Thus, identifying their drug targets is necessary. A two-sample MR analysis was conducted using proteins from the intersection of the deCODE database and "The Druggable Genome and Support for Target Identification and Validation in Drug Development" as the exposure variable. The outcome variable was based on recently published GWAS of keloids. SMR and colocalization analysis was employed to distinguish pleiotropy from linkage. Candidate targets underwent drug target analysis. The primary findings were validated through scRNA-seq data, Western Blot and immunofluorescence staining on keloids. Seven proteins were identified as potential drug targets for keloids. Among these proteins, HHIP, NTM, KLKB1, and CRIPTO showed positive correlations with keloids, while PLXNC1, SCG3 and PDGF-D exhibited negative correlations. Combined with the scRNA-seq data, NTM, PLXNC1, and PDGF-D were found highly expressed in the fibroblasts. NTM showed a significant increase in keloids as compared to normal scars. In accordance with the analysis, higher levels of protein expression of NTM in keloids compared to normal skin was observed. The identified proteins may be appealing drug targets for keloids treatment with a special emphasis on NTM.

High-level expression of codon-optimized Taq DNA polymerase under the control of rhaBAD promoter

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.

High LGALS3 expression induced by HCP5/hsa-miR-27b-3p correlates with poor prognosis and tumor immune infiltration in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is widely recognized for its unfavorable prognosis. Increasing evidence has revealed that LGALS3 has an essential function in initiating and developing several malignancies in humans. Nevertheless, thorough analysis of the expression profile, clinical prognosis, pathway prediction, and immune infiltration of LGALS3 has not been fully explored in HCC.MethodsIn this study, an initial pan-cancer analysis was conducted to investigate the expression and prognosis of LGALS3. Following a comprehensive analysis, which included expression analysis and correlation analysis, noncoding RNAs that contribute to the overexpression of LGALS3 were subsequently identified. This identification was further validated using HCC clinical tissue samples. TIMER2 and GEPIA2 were employed to examine the correlation between LGALS3 and HCP5 with immunological checkpoints, cell chemotaxis, and immune infiltration in HCC. The R program was applied to analyze the expression distribution of immune score in in HCC patients with high and low LGALS3 expression. The expression profiles of immune checkpoints were also analyzed. Use R to perform GSVA analysis in order to explore potential signaling pathways.ResultsFirst, we conducted pan-cancer analysis for LGALS3 expression level through an in-depth analysis of public databases and found that HCC has a high LGALS3 gene and protein expression level, which were then verified in clinical HCC specimens. Meanwhile, high LGALS3 gene expression is related to malignant progression and poor prognosis of HCC. Univariate and multivariate analyses confirmed that LGALS3 could serve as an independent prognostic marker for HCC. Next, by combining comprehensive analysis and validation on HCC clinical tissue samples, we hypothesize that the HCP5/hsa-miR-27b-3p axis could serve as the most promising LGALS3 regulation mechanism in HCC. KEGG and GO analyses highlighted that the LGALS3-related genes were involved in tumor immunity. Furthermore, we detected a significant positive association between LGALS3 and HCP5 with immunological checkpoints, cell chemotaxis, and immune infiltration. In addition, high LGALS3 expression groups had significantly higher immune cell scores and immune checkpoint expression levels. Finally, GSVA analysis was performed to predict potential signaling pathways linked to LGALS3 and HCP5 in immune evasion and metabolic reprogramming of HCC.ConclusionsOur findings indicated that the upregulation of LGALS3 via the HCP5/hsa-miR-27b-3p axis is associated with unfavorable prognosis and increased tumor immune infiltration in HCC.

Exploring the role of the CapG gene in hypoxia adaptation in Tibetan pigs.

Introduction: The CapG gene, which is an actin-binding protein, is prevalent in eukaryotic cells and is abundantly present in various pathways associated with plateau hypoxia adaptation. Tibetan pigs, which have inhabited high altitudes for extended periods, provide an excellent research population for investigating plateau hypoxia adaptation. Results: This study focused on Tibetan pigs and Yorkshire pigs residing in Nyingchi, Tibet. The blood physiological data of Tibetan pigs were found to be significantly higher than those of Yorkshire pigs, including RBC, HGB, HCT, MCH, and MCHC. The SNP analysis of the CapG gene identified six sites with mutations only present in Tibetan pigs. Notably, the transcription factors at sites C-489T, C-274T, and A-212G were found to be altered, and these sites are known to be associated with hypoxia adaptation and blood oxygen transportation. The mRNA expression of the CapG gene exhibited highly significant differences in several tissues, with the target proteins predominantly higher in the Yorkshire pig compared to the Tibetan pig. Specifically, a notable difference was observed in the lung tissues. Immunohistochemistry analysis revealed high expression levels of CapG proteins in the lung tissues of both Tibetan and Yorkshire pigs, primarily localized in the cytoplasm and cell membrane. Conclusion: The CapG gene plays a significant role in regulating hypoxia adaptation in Tibetan pigs. This study provides a theoretical basis for the conservation and utilization of Tibetan pig resources, the breeding of highland breeds, epidemic prevention and control, and holds great importance for the development of the highland livestock economy.

Increased Expression Level of Human Blood Clotting Factor VIII Using NS0 Cell Line as a Host Cells.

Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields. The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest. We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.mL-1 Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA. A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030). The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry.

Abstract 5151: High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma

Abstract Introduction: CheckMate 914 (CM-914) Part A is a double-blind, phase III randomized trial of the Nivolumab (NIVO) plus Ipilimumab (IPI) vs placebo (PBO) in localized ccRCC. Our prior report from this study suggested a disease-free survival (DFS) benefit for NIVO+IPI among patients with Fuhrman grade 4, TNM stages PT2a and PT4, or sarcomatoid features, although the sample size was limited. It is known that high circulating KIM-1 is associated with worse DFS after nephrectomy. In this exploratory post hoc analysis, we investigated whether high KIM-1 may help identify a subset of patients who benefit from adjuvant NIVO+IPI. Methods: Patients (n=816) with RCC after nephrectomy were randomized in CM-914 Part A to receive NIVO+IPI or PBO as previously described. Assessment of KIM-1 levels was performed using enzyme linked immunoassay (ELISA) on pre-treatment (n=584) and matched on-treatment blood samples (n=584). We used pre-treatment tumor samples to assess PD-L1% tumor cell expression (%TC) in an PD-L1 IHC 28-8 pharm Dx assay. The association between biomarkers and DFS outcomes was investigated by Kaplan-Meier (KM) and Cox proportional hazards analysis. Results: Median baseline serum KIM-1 level was 102 (9.9 - 1055.7) pg/mL. Serum KIM-1 levels were higher in males vs females, ≥65 yrs vs <65 yrs, Asian vs white patients, and patients with partial vs radical nephrectomy. In the PBO arm, subjects with highest quartile of pre-treatment KIM-1 had significantly worse DFS than those from the three lower quartiles. In contrast, this DFS risk among the subjects within the highest KIM-1 quartile was mitigated with NIVO+IPI treatment. Among patients within the highest quartile of pre-treatment KIM-1, there was trend for better DFS for NIVO+IPI versus PBO, HR=0.6 (0.34-1.04). Increase in KIM-1 during study therapy was positively associated with higher DFS rate in both arms. Multivariable analysis showed that PD-L1 %TC was predictive at predefined PD-L1 cutoffs (>=1%, >=5%, and >=10%), associating with improved DFS compared to placebo. Subjects with high PD-L1 expression had a DFS benefit from NIVO+IPI independent of KIM-1. Conclusion: Circulating KIM-1 and tumor PD-L1 expression may enrich for benefit from IO therapy in adjuvant ccRCC and hence holds promise for informing risk stratification and patient inclusion in neoadjuvant or adjuvant clinical trials. Citation Format: Sai Vikram Vemula, Wenxin Xu, Yu Wang, Xiaowen Liu, Jorge Ruiz de Somocurcio, David McDermott, Jun Li, Rupal Bhatt, Chung-Wei Lee, Burcin Simsek, Saurabh Gupta, Robert Motzer. High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5151.

Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications.

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

Biomarker cystatin B expression correlates with pathogenesis in cervical cancer.

Cervical cancer (CC) is one of the most common gynecologic malignancies worldwide. Although rapid improvements have been made regarding its prevention and treatment, little is known about disease pathogenesis and the clinical relevance of reliable biomarkers. The present study evaluated the expression of cystatin B (CSTB) as a potential biomarker of CC. Tissue microarray analysis and immunohistochemical staining were performed to detect CSTB expression, while CSTB mRNA and protein expression levels of freshly isolated CC tissue were measured by quantitative real-time PCR and western blot, respectively. Bioinformatics were used to analyze the CSTB co-expression network and functional enrichments. We observed high CSTB mRNA and protein expression levels in CC tissues, which was confirmed by tissue microarray in a comparison with paired adjacent non-cancerous cervical tissue samples. CSTB gene enrichments and associations with co-expressed genes were also observed. Further analysis showed that elevated CSTB expression was associated with pathological progress in CC. Our data demonstrate that CSTB has the potential to be used as a tissue biomarker with clinical value in patients with CC, which may aid the development of intervention strategies.

High serum levels of leucine-rich α-2 glycoprotein 1 (LRG-1) are associated with poor survival in patients with early breast cancer

BackgroundLeucine-rich α-2 glycoprotein 1 (LRG-1) is a secreted glycoprotein that is mainly produced in the liver. Elevated levels of LRG-1 are found in a multitude of pathological conditions including eye diseases, diabetes, infections, autoimmune diseases, and cancer. In patients with early breast cancer (BC), high intratumoral LRG-1 protein expression levels are associated with reduced survival. In this study, we assessed serum levels of LRG-1 in patients with early BC and investigated its correlation with the presence of disseminated tumor cells (DTCs) in the bone marrow and survival outcomes.MethodsSerum LRG-1 levels of 509 BC patients were determined using ELISA and DTCs were assessed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. We stratified LRG-1 levels according to selected clinical parameters. Using the log-rank (Mantel–Cox) test and multivariate Cox regression analysis, Kaplan–Meier survival curves and prognostic relevance were assessed.ResultsMean serum levels of LRG-1 were 29.70 ± 8.67 µg/ml. Age was positively correlated with LRG-1 expression (r = 0.19; p < 0.0001) and significantly higher LRG-1 levels were found in patients over 60 years compared to younger ones (30.49 ± 8.63 µg/ml vs. 28.85 ± 8.63 µg/ml; p = 0.011) and in postmenopausal patients compared to premenopausal patients (30.15 ± 8.34 µg/ml vs. 26.936.94 µg/ml; p = 0.002). Patients with no DTCs showed significantly elevated LRG-1 levels compared to the DTC-positive group (30.51 ± 8.69 µg/ml vs. 28.51 ± 8.54 µg/ml; p = 0.004). Overall and BC-specific survival was significantly lower in patients with high serum LRG-1 levels (above a cut-off of 33.63 µg/ml) compared to patients with lower LRG-1 levels during a mean follow-up of 8.5 years (24.8% vs. 11.1% BC-specific death; p = 0.0003; odds ratio 2.63, 95%CI: 1.56—4.36). Multivariate analyses revealed that LRG-1 is an independent prognostic marker for BC-specific survival (p = 0.001; hazard ratio 2.61).ConclusionsThis study highlights the potential of LRG-1 as an independent prognostic biomarker in patients with early BC.

Cell Culture Media Optimization for Increased Production of Recombinant Proteins in Insect Cells

The insect cell/baculovirus expression system (IC-BEVS) has been at the forefront of biotechnologicalresearch, and has served as a tool to produce several human therapeutics such asCervavix ® (preventative vaccine against the human papilloma Virus), Flublok ® (preventativevaccine against seasonal influenza virus), Provenge ® (therapeutic treatment against prostatecancer) and Glybera ® (gene therapy treatment for lipoprotein lipase deficiency). IC-BEVS isan attractive alternative to mammalian cells for biomanufacturing as it offers advantages suchas easy adaptation to serum-free media, high levels of protein expression and post-translationalmodifications, and is appropriately scalable for manufacturing 2. Despite these advantages,the use of IC-BEVS to produce recombinant proteins can be costly and time-consuming.As the demand for new therapeutic increases, efficient, and robust methods to improvethe production and screening processes of recombinant proteins in this expression systemare necessary to respond to large-scale manufacturing needs. Research has shown that tailoredmedia supplementation and optimization is an efficient, and useful strategy to reachhigh density cultures and increase protein production in-vitro. Therefore, this project aimed toevaluate the effects of two macromolecules (glucose in high concentration, and glutathione)as potential cell culture additives or boosters to increase the production of therapeutic proteinsin the baculovirus/insect cell expression system. It was demonstrated that glutathioneaddition resulted in a more rapid, universal production of protein compared to minimal media,suggesting this to be a valuable addition to IC-BEVS media.

A simple and effective method to remove pigments from heterologous secretory proteins expressed in Pichia pastoris

Pichia pastoris is a popular yeast host for high-level heterologous expression of proteins on an industrial scale owing to its reliable expression, robust growth, high fermentation density, and easy genetic manipulation and cultivation at a relatively low cost. Of particular interest is its high secretion efficiency for small proteins including insulin, human serum albumin, vaccines, enzymes, and llama-derived heavy-chain only antibodies (nanobodies) for pharmaceutical and research applications. However, a recurring challenge in using P. pastoris heterologous secretory proteins is the co-purification of a sticky, yellow pigment which has been identified as a tetra-benzoyl disaccharide. Current methods for pigment removal involve crystallization of the heterologous secretory protein, active carbon absorption, and chromatography using cation exchange and hydrophobic interaction. Here, we present a simple and effective method to remove the yellow pigment, demonstrated with divalent nanobodies targeting SARS-CoV-2. The method entails capturing the nanobody on an affinity column and subsequent washing with the zwitterionic detergent lauryldimethylamine N-oxide (LDAO). We anticipate the method become generally useful to remove pigments from secretion proteins produced in P. pastoris, offering a practical solution to enhance the purity of heterologous proteins in various biotechnological applications.

A Prognostic Activity of Glutaredoxin 1 Protein (Grx1) in Colon Cancer.

Glutaredoxin 1 (Grx1) is an essential enzyme that regulates redox signal transduction and repairs protein oxidation by reversing S-glutathionylation, an oxidative modification of protein cysteine residues. Grx1 removes glutathione from proteins to restore their reduced state (protein-SH) and regulate protein-SSG levels in redox signaling networks. Thus, it can exert an influence on the development of cancer. To further investigate this problem, we performed an analysis of Grx1 expression in colon adenocarcinoma samples from the Polish population of patients with primary colon adenocarcinoma (stages I and II of colon cancer) and those with regional lymph node metastasis (stage III of colon cancer). Our study revealed a significant correlation between the expression of Grx1 protein through immunohistochemical analysis and various clinical characteristics of patients, such as histological grade, depth of invasion, angioinvasion, staging, regional lymph node invasion, and PCNA expression. It was found that almost 88% of patients with stage I had high levels of Grx1 expression, while only 1% of patients with stage III exhibited high levels of Grx1 protein expression. Furthermore, the study discovered that high levels of Grx1 expression were present in samples of colon mucosa without any pathological changes. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western blot.