Production Of Specific Proteins Research Articles

Skin fragility in autoimmune blistering diseases of the skin

In many disorders, skin and/or mucosal changes are dominated by blisters, wounds, or erosions. While these changes can be observed during infective, traumatic, metabolic, or inflammatory processes, these are normally clinical hallmarks of the disruption of the cytoarchitectural stability of the skin. Several proteins, such as those located in the dermal-epidermal junction zone and forming the hemidesmosomes, or those forming epidermal desmosomes are crucial for the maintenance of skin integrity. Defective function may be genetically determined due to impaired or absent production of specific proteins (i.e., epidermolysis bullosa) or due to autoimmune disorders that lead to the production of autoreactive antibodies targeting desmosomal or hemidesmosomal skin antigens. The latter group of diseases are classically named autoimmune blistering diseases of the skin. These can be divided in the pemphigoid group, where antigens are components of the hemidesmosomes, and pemphigus group, where desmosomal proteins are targeted. In this review, we provide ashort vademecum of autoimmune skin disorders that are associated with skin fragility.

RiboScreenTM Technology Delivers a Ribosomal Target and a Small-Molecule Ligand for Ribosome Editing to Boost the Production Levels of Tropoelastin, the Monomeric Unit of Elastin.

Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.

Orchestrated centers for the production of proteins or "translation factories".

The mechanics of how proteins are generated from mRNA is increasingly well understood. However, much less is known about how protein production is coordinated and orchestrated within the crowded intracellular environment, especially in eukaryotic cells. Recent studies suggest that localized sites exist for the coordinated production of specific proteins. These sites have been termed "translation factories" and roles in protein complex formation, protein localization, inheritance, and translation regulation have been postulated. In this article, we review the evidence supporting the translation of mRNA at these sites, the details of their mechanism of formation, and their likely functional significance. Finally, we consider the key uncertainties regarding these elusive structures in cells. This article is categorized under: Translation Translation > Mechanisms RNA Export and Localization > RNA Localization Translation > Regulation.

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody–producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation.Key points• Proteome profiling of recombinant CHO cells under mild TM treatment.• Identified protein clusters are associated with the unfolded protein response (UPR).• The compared cell lines revealed noticeable disparities in protein expression levels.

Rethinking Aspergillosis in the Era of Microbiota and Mycobiota.

Aspergillosis encompasses a wide range of clinical conditions based on the interaction between Aspergillus and the host. It ranges from colonization to invasive aspergillosis. The human lung provides an entry door for Aspergillus. Aspergillus has virulence characteristics such as conidia, rapid growth at body temperature, and the production of specific proteins, carbohydrates, and secondary metabolites that allow A. fumigatus to infiltrate the lung's alveoli and cause invasive aspergillosis. Alveolar epithelial cells play an important role in both fungus clearance and immune cell recruitment via cytokine release. Although the innate immune system quickly clears conidia in immunocompetent hosts, A. fumigatus has evolved multiple virulence factors in order to escape immune response such as ROS detoxifying enzymes, the rodlet layer, DHN-melanin and toxins. Bacterial co-infections or interactions can alter the immune response, impact Aspergillus growth and virulence, enhance biofilm formation, confound diagnosis, and reduce treatment efficacy. The gut microbiome's makeup influences pulmonary immune responses generated by A. fumigatus infection and vice versa. The real-time PCR for Aspergillus DNA detection might be a particularly useful tool to diagnose pulmonary aspergillosis. Metagenomics analyses allow quick and easy detection and identification of a great variety of fungi in different clinical samples, although optimization is still required particularly for the use of NGS techniques. This review will analyze the current state of aspergillosis in light of recent discoveries in the microbiota and mycobiota.

Sex-related immunity: could Toll-like receptors be the answer in acute inflammatory response?

An increasing number of studies have highlighted the existence of a sex-specific immune response, wherein men experience a worse prognosis in cases of acute inflammatory diseases. Initially, this sex-dependent inflammatory response was attributed to the influence of sex hormones. However, a growing body of evidence has shifted the focus toward the influence of chromosomes rather than sex hormones in shaping these inflammatory sex disparities. Notably, certain pattern recognition receptors, such as Toll-like receptors (TLRs), and their associated immune pathways have been implicated in driving the sex-specific immune response. These receptors are encoded by genes located on the X chromosome. TLRs are pivotal components of the innate immune system, playing crucial roles in responding to infectious diseases, including bacterial and viral pathogens, as well as trauma-related conditions. Importantly, the TLR-mediated inflammatory responses, as indicated by the production of specific proteins and cytokines, exhibit discernible sex-dependent patterns. In this review, we delve into the subject of sex bias in TLR activation and explore its clinical implications relatively to both the X chromosome and the hormonal environment. The overarching objective is to enhance our understanding of the fundamental mechanisms underlying these sex differences.

Rapid Extraction of Prefrontal Cortex and Hippocampus for Molecular Analysis in Rats

Memory functions in laboratory animals are a crucial area of research in neuroscience, with a particular focus on identifying drugs that enhance various aspects of memory, particularly long-term declarative memory. This field involves studying cognition and memory from behavioral and molecular perspectives. Researchers evaluate changes in genes, enzymes, and proteins to discover the neurobiological mechanisms underlying memory formation and retrieval. The hippocampus and prefrontal cortex are key brain regions associated with memory and cognition, undergoing synaptogenesis following the acquisition of new information or skills. The production of specific proteins plays a crucial role in consolidating and storing memories, necessitating precise analysis of these brain regions to understand the molecular mechanisms involved in memory processes fully. However, the rapid degradation of these proteins by cellular enzymatic activities highlights the need for efficient and timely extraction techniques to ensure precise and reliable analysis. This methodological study aimed to introduce a rapid and accurate method for extracting the prefrontal cortex and hippocampus in rats.

Molecular farming- importance of stewarding food crops engineered to produce transferred food allergens and non-food substances

Developers are looking for new ways to produce proteins and other substances for food, pharmaceutical and industrial use by genetically engineering food crops to produce the desired substance of interest (i.e., molecular farming). Developers should be aware of the food safety concerns, legality and potential liability, and loss of consumer confidence that could arise if food or other plant materials from these crops were to inadvertently enter the food supply and compromise safety. At the earliest stages of product development, developers should consider whether it is feasible to consistently steward their crops and resulting plant materials from development through disposal to ensure they do not enter the food or feed supply in a way that would be unlawful. Developers should engage FDA’s foods program when considering their stewardship program. While molecular farming holds promise for the economical production of specific proteins and other substances at a large scale, it is important for developers to consider the efforts needed to protect the food supply from the crops used for molecular farming- particularly when the crop chosen for molecular farming is a crop traditionally used for human or animal food.

The Effect of Molasses Concentration on the Growth of Yeast Saccharomyces Cereviceae in Making Single Cell Proteins the Effect of the concentrate on of Waste Molasses on the Growth of Yeast Saccharomyces Cereviceae in the Making of Single Cell Proteins

The aim of the research was to determine the optimal molasses concentration for the growth of Saccharomyces Cereviciae yeast and the protein content of Saccharomyces cerevisiae yeast in making PST. This research used an experimental method with a quantitative approach and used a Completely Randomized Design (CRD) with 5 treatments and 3 replications. The concentrations in this study were 10%, 15%, 20%, 25% molasses and control. The results obtained showed that the protein levels obtained ranged from 13-47%. Apart from that, the highest specific growth rate (SGR) and protein productivity were found in the M1 treatment with 10% molasses.

Investigation of the effect of allithiamine-enriched feed on the poultry gut microbiome composition and resistome

Over the past 20–25 years, the poultry industry has evolved into a specific protein production system. However, the stress resulting from intensive rearing practices has led to numerous negative consequences, making the optimisation of livestock gut microbiome composition crucial for mitigating these effects. Advancements in modern molecular biology methods have brought attention to the impacts of nutrients on gut microbiota. In our study, we extensively investigated the changes induced by feed formulations rich in phytonutrients on the gastrointestinal microbiota of livestock using targeted 16S rRNA amplicon sequencing. Our objective is to examine how the developed feed prototype affects the composition of core microbiomes in raised poultry, community diversity, and the resilience of complex microbial networks. We seek correlations between biological livestock and environmental samples to identify which community constituents, in what proportions and occurrences, may play a role in the development of specific diseases. Based on our measurement results, it can be asserted that allithiamine positively modulated "beneficial" community constituents. Beyond the impact of allithiamine-enriched feed rich in phytonutrients, the composition of the microbial community in the poultry gastrointestinal tract is significantly influenced by the age of the birds. Furthermore, due to the presence of multi-drug-resistant pathogens in environmental samples from livestock facilities, appropriate transmission risk management measures are of paramount importance.

Streamlined in vitro screening system of synthetic signal peptides in Chinese hamster ovary cells for therapeutic protein production.

Increasing the screening efficiency and maintaining the N-terminal cleavage pattern are key factors in the development of an in vitro synthetic signal peptide screening system for high therapeutic protein production in Chinese hamster ovary (CHO) cells. This study improved the in vitro screening system of synthetic signal peptides in CHO cells for therapeutic protein production by modifying the expression vector. Incorporating a leaky stop codon with IgG transmembrane and cytoplasmic domains into the expression vector improved the proportion of high producers in establishing stable CHO cell pools. The selected signal peptides from stable CHO cell pools that were generated using degenerate codon-based oligonucleotides with a conserved polar carboxy-terminal domain in the native signal peptide showed similar N-terminal cleavage patterns to the native one. In addition, replacing native signal peptide with selected synthetic signal peptides did not influence the sialylated N-linked glycan formation and biological activity of therapeutic Fc-fusion glycoprotein in CHO cells. Thus, an in vitro synthetic signal peptide screening system can be used for therapeutic Fc-fusion glycoprotein production in CHO cells with an enhanced specific protein productivity while maintaining the N-terminal cleavage pattern similar to the native one.

Existing methods to improve immunotherapy's limitations: CAR-T cell therapy and checkpoint inhibitors

Immunotherapy is a form of treatment for cancer that works by provoking the immune system of the body into tracking down and destroying cancer cells. CAR-T-CT is a treatment that makes use of CAR-T cells (CARs), which are T cells (T-Cs) that have been modified in some way by the incorporation of engineered synthetic receptors. This therapy is designed to destroy cells that express a specific target antigen, and its primary objective is to do so by locating and killing those cells. Due to the fact that Immune CPI inhibits the production of specific proteins (checkpoint protein) on immune cells as well as on cancer cells thereby preventing the immune system from combating cancer, Immune CPI are used to treat cancer. By inhibiting the activity of these proteins, CPI is able to contribute to the process of stimulating the immune system to specifically target and destroy cancer cells. However, there are still some limitations that exist, and these have the potential to reduce the effectiveness of the treatment or produce some side effects that are life-threatening. This article makes a few suggestions for innovative steps that could be taken in order to both improve the current state of affairs and find answers to any problems that may arise in the near or distant future. The article makes these suggestions in order to improve the current state of affairs and find answers to any problems that may arise in the future.

Protein-Specific Signal Peptides for Mammalian Vector Engineering.

Expression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic pathway. In comparison to other genetic part-types, there is a relative paucity of characterized signal peptide components, particularly for mammalian cell contexts. In this study, we describe a toolkit of signal peptide elements, created using bioinformatics-led and synthetic design approaches, that can be utilized to enhance production of biopharmaceutical proteins in Chinese hamster ovary cell factories. We demonstrate, for the first time in a mammalian cell context, that machine learning can be used to predict how discrete signal peptide elements will perform when utilized to drive endoplasmic reticulum (ER) translocation of specific single chain protein products. For more complex molecular formats, such as multichain monoclonal antibodies, we describe how a combination of in silico and targeted design rule-based in vitro testing can be employed to rapidly identify product-specific signal peptide solutions from minimal screening spaces. The utility of this technology is validated by deriving vector designs that increase product titers ≥1.8×, compared to standard industry systems, for a range of products, including a difficult-to-express monoclonal antibody. The availability of a vastly expanded toolbox of characterized signal peptide parts, combined with streamlined in silico/in vitro testing processes, will permit efficient expression vector re-design to maximize titers of both simple and complex protein products.

Regulation and function of poised mRNAs in lymphocytes.

Pre-existing but untranslated or 'poised' mRNA exists as a means to rapidly induce the production of specific proteins in response to stimuli and as a safeguard to limit the actions of these proteins. The translation of poised mRNA enables immune cells to express quickly genes that enhance immune responses. The molecular mechanisms that repress the translation of poised mRNA and, upon stimulation, enable translation have yet to be elucidated. They likely reflect intrinsic properties of the mRNAs and their interactions with trans-acting factors that direct poised mRNAs away from or into the ribosome. Here, I discuss mechanisms by which this might be regulated.

Algorithm for determining the authenticity of biomedical cell preparations containing mesenchymal stem cells

The use of mesenchymal stem cells (MSCs), which have a pronounced immunomodulatory activity, is a promising direction in the development of biomedical cell preparations (BMCPs). In oncohematology, the use of BMCPs containing MSCs is aimed at supporting hematopoiesis during cotransplantation with hematopoietic stem cells (HSCs) and suppressing immune conflicts during allogeneic unrelated transplantation and severe autoimmune processes. An obligatory stage of registration of BMCPs is confirmation of the identity of the MSC cell line (CL), which includes the establishment of morphological characteristics, evaluation of the expression of specific markers and proteins, and confirmation of the genetic stability of CL during cultivation. Determination of markers of genetic stability is possible using various methods, however, according to the recommendations of the American National Standardization Institute, the standard is the analysis of short tandem repeats (STR analysis). The purpose of the study is to develop an algorithm for determining the authenticity of BMCPs containing MSCs, including STR analysis. Material and methods. Identification of MSC cells in BMCP was performed according to the criteria of the International Society for Cell Therapy. Viable cells were counted in a Goryaev chamber. Immunophenotypic characteristics of MSCs were determined by flow cytometry. The level of production of specific proteins was assessed using enzyme immunoassay. Genetic stability markers were identified by STR analysis. Results and discussion. The methods were tested in triplicate for ten BMCP samples to confirm the reproducibility and reliability of the results. The developed algorithm for determining the authenticity of BMCP has a high accuracy, as it includes the STR analysis technique, which makes it possible to identify 19 polymorphic STR markers located on different alleles. Using the method will allow BMCP manufacturers to go through the procedure of state registration of drugs.

Evaluation of the Cytokine Genes Expression in Vaccinated BALB/c Mice with pEGFP-C2-leoA DNA Vaccine

Introduction: Helicobacter pylori (H. pylori) causes successive changes in the stomach wall, which starts with inflammation of the stomach mucosa and in some cases leads to stomach cancer. The leoA gene, by encoding GTPase, plays a vital role in the pathogenicity of this bacterium in the gastric mucosa. The secretory vesicles produced by the leoA gene release poison and stimulate the immune system in the host's body. The aim of the present study was to evaluate the gene vaccine pEGFP-C2-leoA on the expression changes of cytokines such as IL6, IL4, and interferon-gamma in inflammation caused by Helicobacter pylori infection in a mouse model.
 Methods: In this interventional experimental study, recombinant plasmid (pEGFP-C2-leoA) was produced, propagated, and extracted through transformation into susceptible bacterial cells. Then, suitable concentrations of 1% chitosan nanoparticles solution were prepared for injection into the quadriceps muscle of BALB/c mice. Finally, the gene expression and changes of the mentioned cytokines were measured by Real-Time RT-PCR method, the obtained results were statistically analyzed by SPSS version 16 software; One way ANOVA test and subsequent LSD test, as well as independent t-test, were used to check the existence of correlation and the significance level of the data.
 Results: After the injection of the vaccine into the quadriceps muscle of mice during the treatment period, cytokines IFNᵧ (<0.038) and IL6 (<0.049) showed a significant increase in expression. On the other hand, cytokine IL4 and the leoA gene also showed a significant decrease in expression (>0.042).
 Conclusion: Based on the results, the leoA gene cloned in the expression vector pEGFP-C2 has the ability to express and produce the specific protein product of this gene in eukaryotic cells, and according to the results obtained in the animal model and the immune characteristic obtained in this research it is shown that the final construct pEGFP-C2-leoA has the necessary potential to investigate immunogenicity in a human model as a gene vaccine.

Comparative proteomics of whey proteins: New insights into quantitative differences between bovine, goat and camel species

Whey proteins are the leading proteins class in milk and play an essential role in the immune defense of neonatal mammals. The aim of this study was to analyze whey proteins in bovine, goat and camel milk by label free proteomics techniques. Finally, 840 proteins were identified, which considerably increasing the number of whey proteins identified in these species. The results of the PCA revealed significant differences in whey proteome patterns between bovine, goat and camel milk. Proteins such as PAEP, CST3, SERPING1, CTSB and GLG1 play an important role as markers in the classification of bovine, goat and camel milk. Statistical analysis showed that the relative abundances of many whey proteins such as ALB, LALBA, LTF and LPO were significantly different among different species. GO and KEGG functional analysis have shown that while the distribution of biological functions involved in whey proteins was relatively similar across species, they differed in terms of protein quantity. These data shed light on the quantitative differences and potential physiological functions of whey proteins across species, and may point the way to the production of specific functional whey proteins.

Comparison of milk fat globule membrane protein profile among bovine, goat and camel milk based on label free proteomic techniques

Milk fat globule membrane (MFGM) proteins, which are recognized to play a variety of biological roles; nevertheless, their composition and interspecies complexity remain unknown. This study aimed to identify and quantify proteins extracted from MFGM-enriched fractions of bovine, goat and camel milk by a label free proteomics approach. We identified 1579 proteins, substantially increasing the number of MFGM proteins identified for these species. The results of functional analysis showed that the identified MFGM proteins had similar functional annotations, with cellular processes, intracellular anatomical structures and binding being the predominant GO annotations. And most of the proteins were involved in KEGG pathways such as protein processing of ribosomes and endoplasmic reticulum. Subsequently, multiple statistical methods were used to compare the differences in MFGM protein quantification among different species. The results of principal component analysis showed that bovine, goat and camel milk MFGM proteins were significantly different, among which CSN1S1, XDH, LTF and PI4KA played an important role in taxonomic identification. Meanwhile the results of hierarchical clustering showed that the differences (p < 0.05) between bovine and goat milk were smaller than those of camel milk. These new data deepen the understanding of the protein composition of MFGM and its possible physiological roles, providing directions for the production of specific functional milk proteins.

Development of an in vitro screening system for synthetic signal peptide in mammalian cell-based protein production.

Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: • An in vitro screening system for synthetic signal peptide in mammalian cells was established • This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay • The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.

Advances in Nanoparticles for Effective Delivery of RNA Therapeutics.

RNA therapeutics, including messenger RNA (mRNA) and small interfering RNA (siRNA), are genetic materials that mediate the translation of genetic direction from genes to induce or inhibit specific protein production. Although the interest in RNA therapeutics is rising globally, the absence of an effective delivery system is an obstacle to the clinical application of RNA therapeutics. Additionally, immunogenicity, short duration of protein expression, unwanted enzymatic degradation, and insufficient cellular uptake could limit the therapeutic efficacy of RNA therapeutics. In this regard, novel platforms based on nanoparticles are crucial for delivering RNAs to the targeted site to increase efficiency without toxicity. In this review, the most recent status of nanoparticles as RNA delivery vectors, with a focus on polymeric nanoparticles, peptide-derived nanoparticles, inorganic nanoparticles, and hybrid nanoparticles, is discussed. These nanoparticular platforms can be utilized for safe and effective RNA delivery to augment therapeutic effects. Ultimately, RNA therapeutics encapsulated in nanoparticle-based carriers will be used to treat many diseases and save lives.