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Mullen, A.C. et al. Master transcription factors determine cell-type-specific responses to TGF-β signaling. Cell 147, 565–576 (2011). Liu, C.L. et al. Single-nucleosome mapping of histone modifications in S. cerevisiae. PLoS Biol. 3, e328 (2005).

Shankaranarayanan, P., Mendoza-Parra, M.A., van Gool, W., Trindade, L.M. & Gronemeyer, H. Single-tube linear DNA amplification for genome-wide studies using a few thousand cells. Nat. Protoc. 7, 328–338 (2012). Thanos, D. & Maniatis, T. The high mobility group protein HMG I(Y) is required for NF-κB–dependent virus induction of the human IFN-β gene. Cell 71, 777–789 (1992). In addition to cell growth, which was analyzed via light scattering at λ = 620 nm, the use of a BioLector microbioreactor system for cultivation of expression cultures also enabled the online monitoring of target protein production through the fluorescence signal given by the intracellular assembly of GUS11 with the co-produced detector protein. Therefore, the fluorescence was measured with the eYFP filter: λ Ex = 508 nm (bandwidth 10 nm), λ Em = 532 nm (bandwidth 10 nm). Measurements were carried out at a time interval of 15 min. Determination of β-glucuronidase activity Bader G, Enkler L, Araiso Y, Hemmerle M, Binko K, Baranowska E, De Craene J-O, Ruer-Laventie J, Pieters J, Tribouillard-Tanvier D. Assigning mitochondrial localization of dual localized proteins using a yeast bi-genomic mitochondrial-split-GFP. Elife. 2020;9:e56649. Ramirez-Carrozzi, V.R. et al. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell 138, 114–128 (2009).

Kasana RC, Salwan R, Yadav SK. Microbial proteases: detection, production, and genetic improvement. Crit Rev Microbiol. 2011;37:262–76.

The third strategy entails using a physical approach to attach targeting moieties to the surface of NPs rather than chemical conjugation 107 (Fig. 2c). For instance, two types of targeted polystyrene NPs were synthesized by pre-adsorption versus chemical conjugation of anti-CD63 antibodies to their surfaces, and their targeting efficacies towards monocyte-derived dendritic cells with CD63 surface receptors, in the presence and absence of serum or plasma, were studied 107. Although, in the absence of serum or plasma, both targeted NPs demonstrated similar targeting capacity towards dendritic cells, the pre-absorbed antibody-coated NPs showed substantially higher targeting efficacy compared with chemically attached targeted NPs in the presence of serum or plasma. This is, at least in large part, because the pre-adsorption method allows attachment of antibodies with outward-facing recognition domains and better targeting outcomes compared with chemical conjugation. Conjugation to protein corona Veganski proteini VIVO LIFE Vivo Life Ritual so 100% rastlinske beljakovine narejene iz konopljinih, kvinojinih in grahovih beljakovin. Vsebujejo kar 20g beljakovin na merico. Na voljo so tri različice: okus čokolade, okus vanilije ali nevtralni nearomatiziran okus. The first strategy involves the use of protein-repellent coatings (such as zwitterionic compounds) on the surface of NPs to prevent and/or minimize corona formation 100, 101 (Fig. 2a). For example, silica NPs functionalized with cysteine (as a zwitterionic ligand) and conjugated with biotin (as a targeting molecule) prevented the formation of the protein corona, when compared with the same NP without cysteine functionalization 101. The zwitterionic coating also substantially improved the targeting capacity. Another relevant example is the use of antifouling polymers (such as polyethylene glycol or polyethylene oxide) 102, which reduce shielding by the protein corona on the basis of the characteristics of the specific coating (such as density, size, length and heterogeneity of the polymeric coating) 103, 104. Pre-coating strategy Postel EH, Berberich SJ, Flint SJ et al (1993) Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science 261:478–480 Many laboratories that study protein corona have expertise in characterizing and analysing the physicochemical properties of the protein corona in house, but they are not necessarily specialized in MS-based proteomics; therefore, they rely on core facilities for proteomic analysis of their samples. As different MS-based proteomics laboratories and/or core facilities may use different methods in their LC–MS/MS workflow, different instruments, equipment and commercial software, one can expect that the heterogenicity of the outcomes would be substantial 99, 119, 120, 121, 122. To shed more light on how much and to what extent LC–MS/MS characterization and analysis can affect protein corona outcomes, 17 identical aliquots of corona-coated polystyrene NPs were sent to 17 different laboratories/core facilities for proteomics analysis 123. The outcomes were surprising: out of 4,022 identified unique proteins in the protein corona layer, only 73 (1.8%) were shared across the laboratories and/or core facilities. It is noteworthy that the technical repeats from each of the core facilities revealed reproducible results, which emphasizes that using the identical sample preparation approach and instrumentation can provide reliable results. The observed heterogeneity across laboratories and/or core facilities, however, is an extremely important point, which needs to be seriously considered in nanomedicine literature, as any interpretation regarding the interactions of NPs with biological systems heavily relies on the composition of protein corona. To improve the reliability and robustness of protein corona data, the nanobio interface community should develop standard protocols on methodologies, analysis, reporting and interpretation of LC–MS/MS data. AI and the protein coronaIn vivo protein formation is a crucial part of cellular life. The process needs to adapt to growth conditions and is exploited for the production of technical and pharmaceutical proteins in microbes such as Escherichia coli. Accordingly, the elucidation of basic regulatory mechanisms controlling the in vivo translation machinery is of primary interest, not only to improve heterologous protein production but also to elucidate fundamental regulation regimens of cellular growth. Results

Mikkelsen, T.S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007). Implementation of standardized protocols on methodologies and analyses of protein corona can generate extensive multi-omic-based data sets that can be used to teach AI to prognosticate diseases using protein corona fingerprints and to predict protein corona formation on distinct NPs for the fundamental design of nanomedicines. Characterization and prediction of the protein corona are both important to understand the interactions of NPs in biological milieus, yet there is a large discrepancy between the two, as the former comprises substantially more reports than the latter 124, 125. This immense difference is attributed to the disadvantages of existing high-throughput techniques and instruments used to test the biocompatibility and biofouling of nanotechnologies. Protocols for protein corona extraction can vary between laboratories, and thus there is a need for robust and standardized methods 109, 126. Moreover, MS is laborious, expensive and requires a high level of expertise, and such high-throughput analytical experiments are subject to multiple errors and variabilities arising from laboratory-to-laboratory differences in sample preparation and analysis 123. AI and machine learning approaches can overcome these technical barriers and further elucidate the impact of the protein corona by predicting protein adsorption to NPs as well as their biological impacts (Box 1). Knapp A, Ripphahn M, Volkenborn K, Skoczinski P, Jaeger K-E. Activity-independent screening of secreted proteins using split GFP. J Biotechnol. 2017;258:110–6. Adli, M., Zhu, J. & Bernstein, B.E. Genome-wide chromatin maps derived from limited numbers of hematopoietic progenitors. Nat. Methods 7, 615–618 (2010). Organic - All of our ingredients are organically grown wherever possible, without the use of herbicides and pesticides which can be damaging to our environment. All of our products are made from 100% plant-based sources to minimise the impact made from animal agricultureWang K, Richards FM (1975) Reaction of dimethyl-3,3'-dithiobispropionimidate with intact human erythrocytes. Cross-linking of membrane proteins and hemoglobin. J Biol Chem 250:6622–6626 Yeom S-J, Kim M, Kwon KK, Fu Y, Rha E, Park S-H, Lee H, Kim H, Lee D-H, Kim D-M. A synthetic microbial biosensor for high-throughput screening of lactam biocatalysts. Nat Commun. 2018;9:1–12. Schallmey M, Singh A, Ward OP. Developments in the use of Bacillus species for industrial production. Can J Microbiol. 2004;50:1–17. As the final step towards a split GFP-based in vivo protein detection assay, target and detector proteins should be co-produced in B. subtilis. To analyze if the co-expression allows a comparative analysis of GUS11 accumulation inside live cells, a two-plasmid system was employed consisting of pBSMul1 [ 32] carrying for the gene of interest and pHT01 ([ 33], MoBiTec, Deutschland) encoding the detector protein. This strategy basically allows for an easy changing of the target protein without the need to change any other part of the split GFP assay system. The applicability of a two-plasmid system was initially evaluated with B. subtilis DB430 double transformants harboring plasmids pBS-Xnt-GUS11 for generating different amounts of intracellular GUS11 and pHT01-sfGFP to produce sfGFP instead of the non-fluorescent detector due to easier visualization . The production of GUS11 was again determined by hydrolytic activity, while the sfGFP amount was determined by the measurable fluorescence (see Additional file 1: Figure S1). GUS11 production remained tunable with different spacer lengths also in the presence of the second plasmid and, additionally, the sfGFP production was only slightly influenced by increasing GUS11 production, which confirmed the feasibility of an online monitoring system based on the expression of genes localized on two plasmids. Increased transcript stability enables sufficient detector production Yu C-H, Dang Y, Zhou Z, Wu C, Zhao F, Sachs MS, et al. Codon usage influences the local rate of translation elongation to regulate co-translational protein folding. Mol Cell. 2015;59(5):744–54.

Kluger R, Alagic A (2004) Chemical cross-linking and protein-protein interactions-a review with illustrative protocols. Bioorg Chem 32:451–472 Veganska mješavina WHOLE Ispunite svoj dan mješavinom pregrambenih dodataka WHOLE. Zašto je WHOLE izvrstan za svakodnevno konzumiranje? Jer sadrži čak 20g proteina po mjerici i samo 3,6 g ugljikohidrata. Mješavina je potpuno biljnog i prirodnog podrijetla. Sadrži čak 22 esencijalna vitamina i minerala. Uz to sadrži i ekstrakt iz korijena Ašvagande i kurkume. Ah da, jesmo li već rekli da svaka mjerica sadrži 5 milijardi dobrih bakterija? I najbolje od svega: Mješavinu možete pripremiti za manje od minute, što je idealno ako vam nedostaje vremena. Veganska mješavina prehrambenih dodataka je ALL-in-ONE napitak, jer sadrži kvalitetne proteine, čak 22 važna vitamina i minerala, ekstrakt kurkume, Ašvagandu i čak 5 milijardi dobrih bakterija po mjeri. Harbison, C.T. et al. Transcriptional regulatory code of a eukaryotic genome. Nature 431, 99–104 (2004).Jurischka S, Bida A, Dohmen-Olma D, Kleine B, Potzkei J, Binder S, Schaumann G, Bakkes PJ, Freudl R. A secretion biosensor for monitoring Sec-dependent protein export in Corynebacterium glutamicum. Microb Cell Fact. 2020;19:11. Kaleta C, Schäuble S, Rinas U, Schuster S. Metabolic costs of amino acid and protein production in Escherichia coli. Biotechnol J. 2013;8(9):1105–14. Once the aforementioned knowledge and understanding is achieved, researchers will be more capable of designing and developing new or adapted nanotherapeutics (such as nanoimmunotherapy by activating desired immune system pathways and mRNA and gene editing nanocarriers), by precisely designing and controlling the protein corona composition and decoration of the surface of NPs. The effects of the contributing factors (such as sex and age) on the safety and efficacy of NPs are strongly dependent on the type of NPs and their potential payload (such as mRNA, proteins and active biomolecules and pharmaceuticals). For monitoring the role of sex and age on the interactions of payload-free NPs with biosystems, for example, researchers can use ‘empty’ coronavirus-like particles and/or membranes. NPs that have targeting capabilities are obtained by functionalizing their surface with targeting species (such as antibodies, small molecules and nucleic acids), enabling their localization to desired locations in the body for payload release and/or imaging purposes. However, the formation of the protein corona can shield these targeting moieties and cause mistargeting and unfavourable biodistribution 20.