Sructural rearrangements of NTRK genes: characteristics, methods of detection and targeted therapy for cancer

Background. The first-generation trk inhibitors, larotrectinib and entrectinib, were approved by the u.s. Food and drug administration (Fda) for the treatment of advanced solid tumors harboring NTRK gene fusions in November 2018 and in august 2019, respectively. The purpose of the study was to present upto-date data on the structure and functions of ntrk genes, the frequency of occurrence of rearrangements with their participation, the consequences of their occurrence at the cellular level, methods of detecting such rearrangements, as well as targeted drugs used in the presence of chimeric NTRK genes. Material and methods. A systemic literature search was conducted in pubmed ncbi, Web of science, scopus databases. Results. The products of NTRK genes are receptors for neurotrophins, and their high expression is normally observed only in a narrow range of tissue types. Intrachromosomal or interchromosomal rearrangements lead to a significant increase in the level of expression of the chimeric gene regulated by the strong promoter of the partner gene. The high transcriptional activity of such a gene, along with the constant activation of the kinase activity of the protein product, leads to the activation of metabolic pathways responsible for cell escape from apoptosis and disruption of the regulation of the cell cycle. The occurrence of chimeric NTRK genes varies between different types of tumors, with the highest (up to 90 %) in rare cancers (secretory carcinoma of the breast, secretory carcinoma of the salivary glands, congenital mesoblastic nephroma, children’s fibrosarcoma). Larotrectinib and entrectinib are highly effective targeted drugs in suppressing the growth of a tumor carrying NTRK rearrangements, regardless of the type of tumor. In this regard, the introduction of new high-precision methods for the detection of chimeric NTRK genes, as well as the study of the mechanisms of the development of resistance with the assumption of ways to overcome it, seems relevant. Conclusion. Rearrangements of NTRK genes are quite common in various types of oncology and are an effective target for modern targeted drugs.

Cosmetic Potential of a Recombinant 50 kDa Protein

Collagen and its derivative proteins have been widely used as a major component for cosmetic formulations as a natural ingredient and moisturizer. Most commercially available collagens are animal-derived collagen type I and other forms of collagen, such as type III collagen, are far less prevalent in animals, making extraction and purification extremely difficult and expensive. Here, we report the production of a 50 kDa protein produced in yeast that is 100% identical to the N-terminus of the human type III collagen. This recombinant protein has a larger molecular weight than most incumbent recombinant collagen proteins available for personal care applications. We report the industrialization of both the fermentation and purification processes to produce a final recombinant protein product. This final protein product was shown to be safe for general applications to human skin and compatible with common formulation protocols, including ethanol-based formulations. This recombinant collagen type III protein was also shown to uniquely stimulate both collagen type I and type III production and secretion by primary human dermal fibroblasts. The unique combination of biostimulation, compatibility with beauty product formulations and demonstrated commercial production, make this novel recombinant type III collagen a good candidate for broad application in the cosmetics industry.

Coupled protein quality control during nonsense mediated mRNA decay

Translation of mRNAs containing premature termination codons (PTCs) can result in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance path-way responsible for detecting and degrading PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on an intact ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, and suggested a lack of dependence on the canonical ribosome-quality control (RQC) pathway. Finally, one of the strongest hits in our screens was the E3 ubiquitin ligase CNOT4, a member of the CCR4-NOT complex, which is involved in initiating mRNA degradation. We show that CNOT4 is involved in NMD coupled protein degradation, and its role depends on a functional RING ubiquitin ligase domain. Our results demonstrate the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a framework for identifying and characterizing factors involved in this process.

Glycosylation-on-a-Chip: A Flow-Based Microfluidic System for Cell-Free Glycoprotein Biosynthesis

In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.

Association between a SLC23A2 Gene Variation, Plasma Vitamin C Levels, and Risk of Different Diseases

Background: Vitamin C is an important plasma water-soluble antioxidant that plays an essential role in the absorption of iron, detoxification of exogenous compounds, and remaking vitamin E for the protection of lipid membranes. In addition, vitamin C is essential in the synthesis of collagen. Vitamin C concentrations of plasma are determined by dietary intake and genetic factors. Ascorbic acid is the functional form of vitamin C, which is transported into the cell through sodium vitamin C transporters (SVCTs). There are two forms of SVCTs which are SVCT1 encoded by the SLC23A1 gene and SVCT2 encoded by the SLC23A2. The SLC23A2 gene locus on human chromosome 20P12. It expresses in most human tissues, except lung and skeletal muscle that it is important in regulating the intracellular concentration of ascorbic acid to protect the cell from oxidative stress and promote type 1 collagen maturation. Maintaining proper concentrations of plasma and cellular vitamin C concentration is important for the normal metabolic function of the body and preventing several diseases. In the contrast, a low concentration of vitamin C caused by SLC23A2 variation can cause several chronic diseases. Our systematic review discusses four diseases related to the variation of SLC23A2 gene and plasma vitamin C levels which are glaucoma, acute coronary syndrome among women, gastric cancer, and HPV16-associated head and neck cancer. Methods: By using NCBI databases, specifically GenBank to analyze DNA sequence and mRNA sequence of SLC23A2 gene. GenBank file format was helpful to extract an accession number of the gene, number of amino acids, number of exons and introns, and length of nucleotides. FASTA format was also useful to retrieve the nucleotide sequence and get the function of the protein. BLAST was used to compare the protein product of the SLC23A2 gene between humans and Macaca mulatta (Rhesus monkey). Results: the accession number of the SLC23A2 gene was NC_000020.11, the number of exons found was 18, and the gene was located in chromosome 20. This gene encodes one of the two required transporters, and the encoded protein accounts for tissue-specific uptake of vitamin C. This gene had an official symbol of SLC23A1. And they found a significant association between the single-nucleotide polymorphism (SNP) rs1279683 (A > G) in SLC23A2 and an increased risk of POAG in homozygous G allele (GG) carriers. Also, POAG patients with this SNP appear to have a significantly lower level of plasma vitamin C compared to other genotypes. Finally, many organisms have the same gene, such as dogs, mice, rats, and chickens. Conclusion: there is a significant association between SLC23A2 gene mutation, increased risk for vitamin C deficiency, and several diseases. SNP in the SLC23A2 gene was significantly associated with a higher risk of POAG in GG allele carriers as well as lower plasma vitamin C concentration.

Aymé–Gripp syndrome in a Russian patient with a newly detected mutation in the MAF gene

Aymé–Gripp syndrome is a rare autosomal dominant syndrome caused by mutations in the MAF gene and is characterized by a pronounced phenotypic polymorphism. The core of clinical signs consists of congenital cataracts, sensorineural hearing loss, specific dysmorphic facial features and intellectual disabilities. With varying frequency, patients have: radioulnar synostosis, Arnold–Chiari malformation, aseptic pericarditis, dental anomaly and osteoarthritis. The article presents the clinical and genetic characteristics of the first Russian patient with Aymé–Gripp syndrome caused by a newly identified mutation s.173C>A (p.Thr58Asn NM_005360.4) in a heterozygous state in the MAF gene. The influence of the lo calization and type of amino acid substitutions in the protein product of the gene on the severity and specificity of the clinical manifestations of the syndrome is discussed.

The cyclic dinucleotide 2′3′-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2′3′ cyclic guanosine monophosphate-adenosine monophosphate (2′3′-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis. Using RNA sequencing, we found that 2′3′-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis. Many of the antiviral genes induced by 2′3′-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2′3′-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2′3′-cGAMP signaling in animals.

Ingestion of an ample amount of meat substitute based upon a lysine-enriched, plant-based protein blend stimulates postprandial muscle protein synthesis to a similar extent as an isonitrogenous amount of chicken in healthy, young men

Plant-based proteins are considered to be less effective in their capacity to stimulate muscle protein synthesis when compared with animal-based protein sources, likely due to differences in amino acid contents. We compared the postprandial muscle protein synthetic response following the ingestion of a lysine-enriched plant-based protein product with an isonitrogenous amount of chicken. Twenty-four men (age: 24±5 y; BMI: 22.9±2.6 kg·m−2) participated in this parallel, double-blind, randomised controlled trial and consumed 40 g protein as a lysine-enriched wheat and chickpea protein product (Plant, n=12) or chicken breast fillet (Chicken, n=12). Primed, continuous intravenous L-[ring-13C6]-phenylalanine infusions were applied while repeated blood and muscle samples were collected over a 5h postprandial period to assess plasma amino acid responses, muscle protein synthesis rates, and muscle anabolic signalling responses. Postprandial plasma leucine and essential amino acid concentrations were higher following Chicken (P<0.001), while plasma lysine concentrations were higher throughout in Plant (P<0.001). Total plasma amino acid concentrations did not differ between interventions (P=0.181). Ingestion of both Plant and Chicken increased muscle protein synthesis rates from post-absorptive: 0.031±0.011 and 0.031±0.013 to postprandial: 0.046±0.010 and 0.055±0.015%∙h−1, respectively (P-time<0.001), with no differences between Plant and Chicken (P-interaction=0.068). Ingestion of 40 g protein in the form of a lysine-enriched plant-based protein product increases muscle protein synthesis rates to a similar extent as an isonitrogenous amount of chicken in healthy, young men. Plant-based protein products sold as meat replacers may be as effective as animal-based protein sources to stimulate postprandial muscle protein synthesis rates in healthy, young individuals.

A novel disease mechanism leading to the expression of a disallowed gene in the pancreatic beta-cell identified by non-coding, regulatory mutations controlling HK1

Gene expression is tightly regulated with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function. This silencing is largely controlled by non-coding elements and their disruption might cause human disease. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo mutations affecting a 42bp conserved region encompassed by a regulatory element in intron 2 of Hexokinase 1 (HK1), a pancreatic beta-cell disallowed gene. We demonstrated that these mutations resulted in expression of HK1 in the pancreatic beta-cells causing inappropriate insulin secretion and congenital hyperinsulinism. These mutations identify a regulatory region critical for cell-specific silencing. Importantly, this has revealed a new disease mechanism for non-coding mutations that cause inappropriate expression of a disallowed gene.

Codon-specific Ramachandran plots show amino acid backbone conformation depends on identity of the translated codon.

Synonymous codons translate into chemically identical amino acids. Once considered inconsequential to the formation of the protein product, there is now significant evidence to suggest that codon usage affects co-translational protein folding and the final structure of the expressed protein. Here we develop a method for computing and comparing codon-specific Ramachandran plots and demonstrate that the backbone dihedral angle distributions of some synonymous codons are distinguishable with statistical significance for some secondary structures. This shows that there exists a dependence between codon identity and backbone torsion of the translated amino acid. Although these findings cannot pinpoint the causal direction of this dependence, we discuss the vast biological implications should coding be shown to directly shape protein conformation and demonstrate the usefulness of this method as a tool for probing associations between codon usage and protein structure. Finally, we urge for the inclusion of exact genetic information into structural databases.