Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains. Mycobacterium avium subsp. paratuberculosis (MAP) is known as the ethologic agent of Johne’s disease (JD), an incurable gastrointestinal inflammation in ruminants . Ac. Ac31]. g and 4 °C for 2 min, and then the whey phase (the liquid between the cream and pellet) was aspirated into a new Eppendorf tube (1.5 mL) and stored at −28 °C to be analyzed later . Ac. Ac31]. Cutoffs corresponding to H-MELISA L3P- and L5P-positivity were adjusted to optical densities of 0.8895 and 0.59925, respectively . Accordingly, 27.66% and 63.83% of 47 BTMs were identified as being H-MELISA-positive, producing various titers of antibodies directed against the L3P and L5P epitopes, respectively. Among the negative controls determined by the results of the milk qPCR and commercial milk ELISA , 11.76% r (45) = 0.5, p = 0.00039; Interestingly, the Pearson correlation test revealed a moderate positive association between antibody reactivity against two MAP-derived lipopeptides of L3P and L5P , 69.14% and 32.09% of cases were detected positive by H-MELISA L3P and H-MELISA L5P, respectively. Surprisingly, the majority of negative controls  we) we31]) r (79) = 0.37, p = 0.00059; Data generated by the two homemade milk ELISA (L3P/L5P) were further analyzed by Pearson correlation test. This analysis indicated that a low positive association existed between antibody reactivity against the two MAP-derived lipopeptides of L3P and L5P (900-positive colonies and milk samples using PCR IS1311-restriction enzyme (RE) analysis (hinf-1). In fact, the amplification of IS900 target was important prior strain typing giving us an overview about the presence/lack of MAP in samples, regardless of the type of MAP strains. However, insertion sequence IS1311 was selected in strain typing since this region has polymorphism within subspecies and this characteristic could be exploited in restriction analysis to discriminate various types of MAP strains (S/C/bison) from each other.To understand whether or not the positivity status of samples in H-MELISA L3P/L5P could predict the type of MAP strains (S/C) in BTMs or individual (MIF) level milk samples, strain typing was performed on qPCR IS900-positive. Among them, only one sample (82B) was detected positive by PCR IS1311 as well. Restriction enzyme analysis (REA) on the PCR product demonstrated that this colony (sample 82B) is a C-type MAP, as its band pattern matched the C-type MAP used as the reference strain. Furthermore, IS1311 was PCR amplified in 44.12% and 42.11% of IS900-positive BTMs and individual (MIF) milk samples respectively (23 out of 53 samples). Interestingly, IS1311 strain typing analysis showed that all three classes of MAP  existed among Sardinian sheep-milk samples -positive DNAs, extracted from colonies or milk samples, revealed that C (46.67%) and S (75%) classes were the predominant strain types at the level of BTM and individual sample (MIF) respectively. Although, the overall rates of H-MELISA-L3P/L5P positivity at both the herd and individual levels were in accordance with this strain-type orientation, there were some C- and S-type samples that did not follow the expected patterns in reactivity against L3P and L5P, in which, among the four BTMs recognized as being S-type, sample 33B showed higher antibody reactivity against L3P, whereas samples 20B and 31B had similar optical densities against both L3P and L5P, and sample 13B was more responsive against L5P  based on H-MELISA-L3P/L5P positivity was also noticed in the MIF category. Three of six S-type isolates were highly reactive against L3P  that contribute to the production of lipopeptides in various strains (S and C types) and are constituted by five and three modules in C-type and S-type MAP strains, respectively [The level of antibodies directed against MAP could comparably be measured in milk samples as well as sera  through ectively . This suectively .p < 0.0001) induced a significant specificity and sensitivity to milk-ELISA assessments [The present study, for the first time, used the MAP surface-exposed lipopeptide of L3P as a capture antigen in an in-house milk ELISA in order to screen antibodies directed against MAP in sheep-milk samples. Furthermore, the functionality of L3P was compared with the other lipopeptides (L5P) performing H-MELISA L5P on the same samples. To select the best cutoffs corresponding positivity, ROC-curve analysis was performed. Cutoffs representing positivity were selected carefully, since any miscalculation in this step could influence the specificity and sensitivity of the assay significantly . The impessments . Accordiessments . To the 900 has 16–22 copies [Our analysis depicted that the overall rates of H-MELISA-L3P/L5P positivity varied by the source of milk samples . More samples were reactive against L5P and L3P at the BTM and individual (MIF) levels, respectively. Surprisingly, the majority of negative controls  represented titers of antibodies directed against L5P and L3P epitopes in BTMs and individual milk samples, respectively. An issue that might be discussed, here, concerns why these negative controls should contain so much antibody against L3P and L5P epitopes to be characterized as being MAP-infected, while no evidence of MAP DNA was recognized in these samples. According to our analysis, MAP DNA could not be detected in a sample unless presenting at detectable concentrations. Although target IS2 copies  in wholeIn addition, the comparison between the results of H-MELISA L3P/L5P and other antibody-assessment methods depicted that H-MELISA L5P, at the BTM level, and H-MELISA L3P, at the individual level (MIF), had the lowest level of agreement with commercial milk ELISA and serum ELISA/commercial milk ELISA, respectively. This may demonstrate that H-MELISA L3P/L5P could function even better than commercial ELISA tests (milk or serum) and even detect minor titers of antibodies against MAP in milk samples. In fact, the types of capture-antigen molecules used in the structure of H-MELISA L3P/L5P and commercial ELISA tests could influence the functionality of the techniques significantly. Most commercial ELISA tests are designed to detect antibodies against crude extracts or specific epitopes of C-type MAP (as capture antigens) in milk or serum samples. Based on our interpretations in MAP-relevant infections, a noticeable proportion of antibodies against MAP are induced against its lipopeptides, since lipopeptides, as well as lipids, exist with higher frequency in the whole MAP cell structure compared with other epitopes. Therefore, we selected L3P and L5P as capture-antigen molecules in H-MELISA tests to target as many existing antibodies as possible against these epitopes in our milk samples.Based on the positivity patterns in H-MELISA L3P/L5P, it could be interpreted that C-type and S-type MAP are dominant strains at the BTM and individual (MIF) levels, respectively, since previous studies confirmed that L3P and L5P are specific lipopeptides native in S- and C-type MAP strains .1311-REA hinf-1 analysis, in which all three MAP strains  occurred among Sardinian sheep communities. In herd (BTM) group, C-type MAP was the dominant strain (46.67%) rather than S-type (26.67%), bison-type (20%), or an unknown strain . In contrast, S-type was more prevalent (75%) at the individual (MIF) level, as compared with C-type and bison-type, which had the lowest incidences, at 12.5% each. A question that might be raised, here, concerns why C-type MAP, a native strain in cow, should be a common strain among sheep animals at the BTM level. While the S and C types were initially defined as the indigenous strains isolated from, respectively, sheep and bovine, further studies revealed that this host orientation couldn’t inevitably predict the types of MAP strain (S or C) found in milk samples thereof [Recent assumptions have been proven by PCR IS thereof . In fact thereof ,16,17. P thereof ,47. This1311 sequence [Although, the distribution of MAP-type strains (S/C) at both the herd and individual levels was accordant with the overall rates of H-MELISA-L3P/L5P positivity, some S- or C-type MAP strains did not follow the predicted patterns in H-MELISA analysis; the majority (44.44%) of S- and C-type MAP strains were detected positive by both H-MELISA L3P and L5P, 22.22% were S type or C type as expected from their positivity patterns in H-MELISA L3P/L5P, and only 11.11% were positive but classified in unexpected categories. Surprisingly, the result of H-MELISA L3P/L5P on BTMs and individual milk samples (MIF) depicted that the majority of bison-type isolates were more reactive against L3P than L5P, at both the herd and individual levels. Previous molecular analysis unveiled that bison-type MAP is derived from a C-type MAP strain that underwent a single nucleotide polymorphism in the ISsequence ,19, but sequence ,25, demosequence ,26. To tMycobacterium avium complex (MAC) or other species, such as M. bovis, has been rejected [At first glance at this recent result, an ambiguity might be found in the stratification of MAP strain types (S or C), based on the result of H-MELISA L3P/L5P. We hypothesized that the antibody reactivity against L3P or L5P might be influenced by either a cross reactivity between the two lipopeptides or a possible co-infection of the studied animals with the two MAP strains. Previous studies straightforwardly showed that MAP strains (S and C types) have various lipopeptides (L3P and L5P) . While trejected , the therejected . Howeverrejected . This isrejected .1311 fragments confirmed that the three different MAP strains  circulated among animals at the herd and individual levels. Our assumption about the presence of a mixed infection, specifically, in the herd group (BTM), was fortified when a Pearson analysis explained a moderate and low positive correlation between antibody reactivity against L3P and L5P at the herd  = 0.5, p = 0.00039) and individual levels  = 0.37, p = 0.00059), respectively. This result indicated that the patterns of antibody reactivity against L3P and L5P are more similar at the BTM level rather than the MIF level.On the other hand, the possibility of a mixed infection with both MAP (S and C) strains should be examined carefully for the possibility that some samples  might contain enough antibodies directed against both L3P and L5P to be recognized positive by both H-MELISA L3P and H-MELISA L5P simultaneously. As the result of RE on amplified ISMAP-derived lipopeptides, L3P and L5P, could be considered potent capture antigens in serum/milk antibody-detection analyses. Our result suggest that these lipopeptides, in the format of an in-house milk ELISA and as a complementary MAP-screening test, could sensitively predict the infected animals (sheep) at the BTM and individual levels.; it was found that H-MELISA L3P/L5P could enhance the discovery of positive milk samples among the specimens that were potentially negative by other MAP-diagnostic assays. Accordingly, the majority of sheep-milk samples were detected positive by H-MELISA L5P and H-MELISA L3P at the levels of the herd (BTM) and individual sample (MIF) respectively. On the one hand, the overall rates of H-MELISA L3P/L5P confirmed the higher incidence of C-type and S-type classes of MAP in the herd (BTM) and individual (MIF) categories, respectively. Contrary to this overall estimation, some S- or C-type MAP strains did not show the expected reactivity against L3P and L5P, in which either they were positive by both H-MELISA L3P and L5P, or they had reverse reactivity against L3P/L5P. This might express the possibility of either cross-reactivity between L3P and L5P, due to the similarity in the overall structures of their two epitopes, or a within-herd mixed infection of both MAP strains. However, the presence of various MAP strain-types (S/C/Bovine), along with a moderate correlation between antibody reactivity against L3P and L5P that was estimated by Pearson correlation test, urged us to conclude that co-infection with both MAP strains (S and C) happens among the studied animals, at the herd level (BTM). These findings suggest that the L3P and L5P antigens could be useful for improving existing diagnostic tests, especially considering the MAP-strain diversity affecting these animals.


While some effector proteins specifically target bacterial or eukaryotic cells, others can target both types of cells (trans-kingdom effectors). In Salmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, which are differentially distributed among serotypes. Salmonella enterica serotype Dublin (S. Dublin) is a cattle-adapted pathogen that harbors both T6SSSPI-6 and T6SSSPI-19. Interestingly, while both systems have been linked to virulence and host colonization in S. Dublin, an antibacterial activity has not been detected for T6SSSPI-6 in this serotype. In addition, there is limited information regarding the repertoire of effector proteins encoded within T6SSSPI-6 and T6SSSPI-19 gene clusters in S. Dublin. In the present study, we demonstrate that T6SSSPI-6 and T6SSSPI-19 of S. Dublin CT_02021853 contribute to interbacterial competition. Bioinformatic and comparative genomic analyses allowed us to identify genes encoding three candidate antibacterial effectors located within SPI-6 and two candidate effectors located within SPI-19. Each antibacterial effector gene is located upstream of a gene encoding a hypothetic immunity protein, thus conforming an effector/immunity (E/I) module. Of note, the genes encoding these effectors and immunity proteins are widely distributed in Salmonella genomes, suggesting a relevant role in interbacterial competition and virulence. Finally, we demonstrate that E/I modules SED_RS01930/SED_RS01935 (encoded in SPI-6), SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both encoded in SPI-19) contribute to interbacterial competition in S. Dublin CT_02021853.The Type VI Secretion System (T6SS) is a multiprotein device that has emerged as an important fitness and virulence factor for many Gram-negative bacteria through the injection of effector proteins into prokaryotic or eukaryotic cells  Salmonella enterica  distributed between species S. enterica and S. bongori  represents a cattle-adapted pathogen that can lead to serious economic problems in bovine production, where it causes a severe systemic disease  has emerged as an important fitness and virulence factor for many Gram-negative bacteria . The T6SSalmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22 . Standard conditions for amplification were: 2 min at 95°C, followed by 30–35 cycles of 94°C for 45 s, 55°C for 30 s, and 72°C for a suitable time (1 min/kb) according to DNA polymerase processivity, and a final extension step at 72°C for 5 min.Plasmid DNA was isolated using the “QIAprep Spin Miniprep Kit” . PCR products were purified using the “QIAquick PCR Purification Kit” . DNA samples were analyzed by electrophoresis in 1% agarose gels and were visualized under UV light after RedGel  staining. Primers were designed using the “Vector NTI Advance 11.0” software  and are listed in S. Dublin CT_02021853 with deletions of single genes or gene clusters were constructed by the one-step inactivation procedure using the Lambda Red recombination system . Then, bacteria were made electrocompetent by serial washes with ice-cold, sterile 15% glycerol and transformed by electroporation with 500–600 ng of each PCR product. Transformants were selected on LB agar supplemented with the corresponding antibiotic at 37°C. Correct allelic replacement in each mutant was confirmed by PCR amplification using specific forward primers (Out5) together with reverse primer K1 (that hybridizes within the Kan resistance cassette) or reverse primer C3  in the chromosome of the attacker or the prey strain, respectively. To do this, phage P22 HT 105-1 int-201 was used to transduce mutant alleles ΔphoN::Kan and ΔphoN::Cam into the corresponding mutant strains harboring unmarked deletions of genes encoding effectors or E/I protein pairs.When required, the antibiotic resistance cassette was removed by transforming each mutant with the temperature-sensitive plasmid pCP20, which encodes the Flp recombinase . Transfo600nm of 0.5, and mixed at a 1:1 (attacker/prey) ratio. Then, 25 μl of the mixture was incubated at 37°C for 24 h in triplicate on MacConkey agar plates, a condition reported to induce the expression of T6SS gene clusters in Salmonella . An 80% identity and 80% sequence coverage threshold were used to select positive matches. Sequence conservation was analyzed by multiple sequence alignments using MAFFT  and T-CoSPI-6 and T6SSSPI-19 to interbacterial competition by S. Dublin CT_02021853, we performed a competition assay in MacConkey agar as it has been shown that bile salts upregulate the expression of T6SS-related genes in S. Typhimurium and S. Dublin strains  effector protein (S. Dublin CT_02021853 does not encode the Tae4/Tai4 and RHSorphan/RhsI E/I pairs described in S. Typhimurium 14028s (enterica : (i) Taeerotypes , (ii) th protein , and  analysis by the Bastion6 prediction pipeline ; (ii) bioinformatic analysis to identify the presence of putative immunity proteins through detection of signal peptide , transmembrane domains (TMHMM), and operon prediction  encodes a 210 amino acid protein with a predicted endonuclease RHS-HNHc protein domain ; Table 3orphan protein of S. Typhimurium 14028s  encodes a large 924 amino acid protein with both a RHS and an Ntox47 protein domain, involved in degrading target cell RNA . It shoum 14028s . Since Rchimeras , it is pmain protein (SED_RS01910). Taking into account that there are RHS proteins with C-terminal Tox-URI2 domains  encodes a small 113 amino acid protein with similarity to bacterial polymorphic toxins in the Tox-URI2 family, a DNAse protein domain that has been linked to RHS-CT proteins exported by the T6SS . SED_RS2 domains , it is pMany T6SS-associated RHS proteins have C-terminal endonuclease effector domains, which degrade DNA or RNA in the target cell . InteresSalmonella, despite its clear contribution to intestinal colonization and antibacterial activity . This ORF encodes a 290 amino acid protein with a predicted LysM domain (IPR018392) at its C-terminus. This domain is present in several T6SS effector proteins and has been linked to peptidoglycan hydrolase activity (Mycobacterium tuberculosis protein which functions as a lysozyme inhibitor (S. Enteritidis (SEN1001) has been linked to the ability of the bacteria to colonize the murine host and survive within infected macrophages . Of note, S. Enteritidis strains harbor an internal deletion within the SPI-6 T6SS gene cluster that is located upstream of the SED_RS01930/SED_RS01935 E/I module , SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both located in SPI-19) and generated non-polar deletion mutants of either the effector-encoding gene or the whole E/I module in S. Dublin CT_02021853. Next, we performed interbacterial competition assays using different combinations of attacker and prey strains. Noteworthy, in all three E/I modules analyzed we observed that the survival of the mutant lacking the effector-encoding gene (and keeping intact the gene encoding the cognate immunity protein) was not affected after coincubation with the wild-type strain (SPI-6 or T6SSSPI-19 in S. Dublin CT_02021853. Also, these results indicate that SED_RS01930, SED_RS06235, and SED_RS06335 encode functional effector proteins, while SED_RS01935, SED_RS06230, and SED_RS06340 encode their cognate immunity proteins. Further experimental evidence is required to fully confirm that each identified effector protein is secreted via T6SSSPI-6 and/or T6SSSPI-19.We aimed to evaluate if a selected group of E/I modules identified by our bioinformatic analysis contribute to interbacterial competition in e strain . These re strain . Finallye strain . These rSPI-6 and T6SSSPI-19 contribute to interbacterial competition by S. Dublin CT_02021853. We identified novel effector proteins and cognate immunity proteins encoded in SPI-6 and SPI-19 T6SS gene clusters and confirmed the contribution of three novel E/I pairs to interbacterial competition in S. Dublin CT_02021853. The biochemical characterization of the putative effector proteins identified is currently undergoing in our laboratory.Altogether, our results indicate that both T6SSS. Dublin acquired and maintained two distinct and functional T6SSs remains unanswered. We and others have proposed that the presence of multiple T6SSs could contribute to Salmonella fitness in different environments and/or hosts. Thus, the presence of putative antibacterial T6SS effector proteins suggests that S. Dublin may use them to differentially target bacterial cells within the cattle microbiota. Additional studies are required to confirm this hypothesis.The question of why The original contributions presented in the study are included in the article/FA, CB, CS, and DP: conceptualization, formal analysis, validation, writing-original draft preparation, and writing-review and editing. FA, CB, MB-I, DR, CS, and DP: methodology and investigation. CB, CS, AM-S, and DP: resources, project administration, and funding acquisition. CS and DP: supervision. FA, CB, and DP: visualization. All authors read and approved the final manuscript.This work was supported by FONDECYT grant 1181167, ANID Millennium Science Initiative/Millennium Initiative for Collaborative Research on Bacterial Resistance, MICROB-R, NCN17_081. CS was supported by FONDECYT grants 1171844, and 1212075. CB was supported by FONDECYT grant 1201805, ECOS-ANID ECOS200037, and HHMI-Gulbenkian International Research Scholar grant #55008749. FA was supported by CONICYT/ANID fellowship 21191925.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Moreover, the fluorescence quenching efficiency of Fe3+ ions displays an obvious linear relationship to Fe3+ concentrations in a wide range of concentrations (up to 200 µM) and with a detection limit of 1.89 µM. Therefore, the generated N-CQDs may be utilized as a robust fluorescence sensor for detecting pH and Fe3+ ions.Nitrogen-doped carbon quantum dots (N-CQDs) were synthesized in a one-step hydrothermal technique utilizing L-lactic acid as that of the source of carbon and ethylenediamine as that of the source of nitrogen, and were characterized using dynamic light scattering, X-ray photoelectron spectroscopy ultraviolet-visible spectrum, Fourier-transformed infrared spectrum, high-resolution transmission electron microscopy, and fluorescence spectrum. The generated N-CQDs have a spherical structure and overall diameters ranging from 1–4 nm, and their surface comprises specific functional groups such as amino, carboxyl, and hydroxyl, resulting in greater water solubility and fluorescence. The quantum yield of N-CQDs (being 46%) is significantly higher than that of the CQDs synthesized from other biomass in literatures. Its fluorescence intensity is dependent on the excitation wavelength, and N-CQDs release blue light at 365 nm under ultraviolet light. The pH values may impact the protonation of N-CQDs surface functional groups and lead to significant fluorescence quenching of N-CQDs. Therefore, the fluorescence intensity of N-CQDs is the highest at pH 7.0, but it decreases with pH as pH values being either more than or less than pH 7.0. The N-CQDs exhibit high sensitivity to Fe Carbon quantum dots (CQDs) are regarded as a novel class of fluorescence nanomaterials. Meanwhile CQDs exhibit many unique advantages, compared with traditional fluorescent nanomaterials, such as excellent biocompatibility , good so3+ ions. Therefore, it is essential to detect Fe3+ ions. Qi et al. [3+ ions and tetracycline antibiotics. Du et al. [2+ and Fe3+ ions in oral ferrous gluconate samples. Chen et al. [3+ ions.CQDs were originally founded by Xu et al.  in 2004,i et al.  utilizedu et al.  adopted n et al.  synthesi3+ ions, suggesting that they might be exploited as fluorescent probe of Fe3+ ions. The research of N-CQDs prepared from lactic acid would expand the scope of application of lactic acid.L-lactic acid is a widely used bio-based chemical containing hydroxyl and carboxyl groups, while ethylenediamine contains amino groups, which may be good candidates for preparing N-CQDs. In this work, we used a one-step hydrothermal technique to create N-CQDs utilizing L-lactic acid as carbon source and ethylenediamine as nitrogen source, respectively. The obtained N-CQDs featuring surface functional groups present excellent water solubility and fluorescence. The N-CQDs were investigated in terms of their morphology, surface structure, and optical properties. Those N-CQDs have been synthesized that were spherical particles with overall diameters ranging from 1–4 nm. When irradiated by ultraviolet light with the wavelength of 365 nm, N-CQDs would emit blue light, and their emission wavelength reaches to the maximum of 414 nm during the excitation wavelength of ultraviolet light being 310 nm, which indicates that the optical characteristics of N-CQDs depend on the excitation wavelength. Meanwhile, the fluorescence of N-CQDs also reveals a pH-dependent dependency. Furthermore, the obtained N-CQDs showed better specificity and sensitivity to Fe3 (A.R.), FeCl2·4H2O (A.R.), CuCl2·2H2O (A.R.), MgCl2·6H2O (A.R.), NiCl2·6H2O (A.R.), CaCl2 (A.R.), SnCl2 (A.R.), KCl (A.R.), CoCl2·6H2O (A.R.), and LiCl·2H2O (A.R.) were all bought from Nanjing Chemical Reagent Company . A dialysis bag with a molecular weight cut-off of 1000 Da was purchased from Shanghai Yuanye Bio-Technology Co., Ltd . The deionized water was purified using an Ulupure system  throughout all the experiments. All reagents used were available commercially and did not involve purification.L-lactic acid  was purchased from Musashino Chemical  Corporation. Ethylenediamine (A.R.), hydrochloric acid (A.R.), NaOH (A.R.), FeClWe synthesized N-CQDs through one-step hydrothermal method employing L-lactic acid as carbon source and ethylenediamine as nitrogen source ,33 Sche. 30.0 g In this section, the LG16-A high-speed centrifuge  was used to centrifuge the sample to eliminate big particles of contaminants, while the FD-1A-50 freeze drier  was used for freeze drying to create N-CQDs solids.−1, and the KBr tablet was being used as a carrier. The microstructures of N-CQDs were examined using JEM-2100  High-resolution transmission electron microscopy (HRTEM). To determine the elemental composition, X-ray photoelectron spectroscopy (XPS) using an Al Kα monochromatized source was studied on AXIS UltraDLD . Fluorescence spectra were recorded on Perkinelmer Fluorescent FL6500 . All fluorescence spectra were measured with the excitation slit being 5 nm, the emission slit being 10 nm, and the scanning speed being 1200 nm/min. Zetasizer Nano-ZS  was being used to evaluate the size and distribution of N-CQDs nanoparticles.The UV-vis spectrophotometer UV-2450  was applied to detect UV-vis spectra. 0.05 mg/mL N-CQDs aqueous solution prepared by dispersing the solid N-CQDs in deionized water was used for both UV-vis and fluorescence spectra below. Fourier-transformed infrared spectroscopy (FTIR) was measured with the FTIR-360 . The scanning range for the FTIR spectra was 450–4500 cmSQ) is 0.54. The quantum yield of N-CQDs (NQ) is calculated that used the following equation [Q stands for the quantum yield and I for the integrated area of fluorescence intensity, while A for the absorbance and φ for the refractive index of the solvent (being 1.33). The standard sample and N-CQDs are denoted by the subscripts S and N, respectively.Quinine sulfate dissolved in 0.1 M sulfuric acid is used as the standard sample, and its quantum yield QA certain amount of N-CQDs solid was dissolved in deionized water to yield 0.05 mg/mL N-CQDs solution. Aqueous solutions with pH values being from 1 to 14 were prepared with hydrochloric acid and sodium hydroxide, respectively. 1 mL of N-CQDs solution and 1 mL hydrochloric solution (or NaOH solution) with different pH values were added to a quartz cuvette and mixed for 1 min, and then the fluorescence test was performed at ambient temperature. All fluorescence spectra were recorded at 310 nm for excitation, and 414 nm for emission.+, Ca2+, Co2+, Cu2+, Fe3+, Mg2+, Fe2+, Ni2+, Sn2+ and K+) were, respectively, dissolved in deionized water to prepare various chloride solutions with the concentration of 1 mM. To investigate the selectivity of N-CQDs aqueous solution to various metal ions, 0.5 mL chloride solution containing different metal ions and N-CQDs aqueous solution with a volume of 2 mL were added to a quartz cuvette and mixed at ambient temperature for 1 min before even being detected. To evaluate the detection range of Fe3+ ions and the sensitivity of N-CQDs to Fe3+ ions, 0.5 mL of Fe3+ solutions with their concentrations varying from 0 μM to 200 μM were mixed with N-CQDs aqueous solution with a volume of 2 mL at ambient temperature for 2 min, and then the mixed solutions were detected. 0.5 mL FeCl3 solution and 0.5 mL other chloride solution were mixed with N-CQDs aqueous solution with a volume of 2 mL, and then the mixture solutions were detected to determine the fluorescence quenching of N-CQDs triggered by Fe3+ together with other metal ions.The above-mentioned aqueous solution of N-CQDs had a concentration of 0.05 mg/mL. The chlorides containing various metal ions . The XPS spectrum of N-CQD in The UV-Vis absorption spectrum was used to characterize the optical property of the generated N-CQDs. The UV–Vis absorption spectrum of N-CQDs aqueous solution in The surface of the obtained N-CQDs includes amino, hydroxyl and carboxyl functional groups, as can be seen by XPS and FTIR. We may utilize these functional groups to detect the effect of pH values on N-CQDs fluorescence intensity. The fluorescence intensities of N-CQDs with different pH values at the excitation wavelength of 310 nm were given in In short, the pH-dependent fluorescent behavior of N-CQDs might be ascribed to functional groups on the surface of N-CQDs. When the surface functional groups of N-CQDs are protonated, fluorescence quenching occurs mainly due to the agglomeration of N-CQDs particles . Therefo3+ ions for N-CQDs is obviously superior to that of other metal ions, as N-CQDs fluorescence intensity would decrease by about 99.6% due to Fe3+ ions, indicating that they have quite an excellent selectivity for Fe3+. To detect the fluorescence quenching of N-CQDs induced by Fe3+ in combination with other metal ions, a mixture of Fe3+ ions with other metal ions was added to an aqueous solution of N-CQDs, and then the mixed solutions were detected. 3+ ions on N-CQD fluorescence quenching is little affected by other metal ions. These findings suggest that they have a specific recognition effect on Fe3+ and a strong anti-interference ability.The hydroxyl, carboxyl and amino surface functional groups of N-CQDs can form complexes with metal ions . As the 3+ ions, the effect of Fe3+ ions concentrations on N-CQDs fluorescence intensity at 310 nm excitation wavelength was shown in 3+ concentration increasing from 0 μM to 200 μM. 0F − F)) and Fe3+ concentrations ranging from 25 to 175 μM with the correlation coefficient of 0.99602. Thus, the concentration of Fe3+ ions could be computed using the equation below.F and 0F denote the fluorescence intensities of N-CQDs in the presence and the absence of Fe3+, respectively. The concentration of Fe3+ ions is represented by C. According to the above equation, the limit of detection is calculated to be 1.89 μM based on three times the standard deviation rule , and it is far below the requirement that Fe3+ ion concentration in drinking water is less than 5.4 μM. 3+ to that of CQDs for Fe3+ reported in other literatures, in which the linear range of Fe3+ detection was ordered from small to large range. The obtained N-CQDs have quite a broad linear detection range and are far more sensitive to Fe3+ ions.In order to make sure the sensitivity of N-CQDs to Fe3+ with the carboxyl group, amino group and hydroxyl group on the surface of N-CQDs may be responsible for the fluorescence quenching caused by Fe3+ [3+ with N-CQDs surface functional groups [3+ ions and N-CQDs, and lead to the formation of a non-radiative electron/hole recombination [3+ ions significantly quench the fluorescence of N-CQDs. Furthermore, the particle size of N-CQDs significantly increases with the addition of Fe3+ ions, for the diameter distributing of N-CQDs increases from 2.6 nm to 62.3 nm  are significantly higher than that of the CQDs reported in literatures. Furthermore, the emission intensity of N-CQDs exhibits excitation wavelength dependence. Their fluorescence intensity steadily decreases as the excitation wavelength increases from 310 to 380 nm. Meanwhile, the fluorescence intensity of N-CQDs shows sensitivity to pH values. In comparison to other metal ions, N-CQDs have quite a high specificity and sensitivity for Fe


Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features. Coronaviruses (CoVs) include a remarkable diversity of animal and human-infecting species. There are seven human-infecting species, each causing disease of varying severity, ranging from mild or asymptomatic infections resembling the common cold to severe wheezing, bronchitis, or pneumonia ,2. TheseEnveloped CoV particles enclose a single positive-sense RNA genome. The limiting envelopes contain three major proteins, matrix (M), envelope (E), and spike (S). M glycoproteins, which have three envelope-spanning transmembrane domains, are the most abundant CoV membrane proteins. The transmembrane domains are involved in M oligomerization, which generates a lattice-like network that is considered fundamental in driving CoV particle budding into the lumen of intracellular membranous organelles, principally the ER-Golgi intermediate compartment ,10. The While several studies have documented that the membrane protein (M) and small envelope protein (E) are sufficient to form coronavirus VLPs, the question of whether these two proteins are all that is needed for efficient SARS-CoV2 assembly requires further investigation. Notably, the role of the cytoplasmic nucleocapsid protein (N) in virus assembly needs further investigations. N proteins bind virion genomic RNAs, forming helical ribonucleoprotein (RNP) complexes, which interact with M proteins and may in turn promote virus particle assembly . N proteWith the aim to further dissect CoV assembly processes, we developed a SARS-CoV2 VLP system that mimics the natural SARS-CoV2 assembly and cell entry processes. The VLP system utilizes an 11 amino acid Nluc fragment called “HiBiT”, which is appended to CoV structural proteins and serves as a marker for virus assembly, secretion from VLP-producing cells, and subsequent VLP entry into target cells. The HiBiT marker is identified by protein complementation with a larger Nluc fragment called “LgBiT”, with resulting Nluc detection by luminometry . Here, wv/v) fetal bovine serum  and 1% penicillin-streptomycin . All the cells were cultured at 37 °C in a 5% CO2 incubator and used for experiments after only short-term passaging. All cell cultures were confirmed to be negative for mycoplasma as determined by myco PCR testing.HeLa cells (ATCC) and human embryonic kidney cells  were grown in the complete Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% , CMV LgBiT expression plasmid , restriction enzymes , PCR and Gibson fragment assembly reagents (NEB), and size-exclusion columns . Rabbit polyclonal antibodies to detect the S (#JH5052), E (#JH5046), and M (#JH5054) of SARS-CoV2 were kind gift from Carolyn Machamer .GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGCTCGACCGGTGGCTCGAGCGGT, encoding the HiBiT tag (VSGWRLFKKIS) and sequence encoding a linker (GSTGSSG) to the 5′ end of the coding sequences for N gene . Similarly, the SARS-CoV2 E-HiBiT and SARS-CoV2 M-HiBiT genes were cloned in same parent vector with the HiBiT tag at the 3′ end of E and M genes, respectively. MERS-CoV HiBiT-N, MHV HiBiT-N, as well as HiBiT-tagged SARS-CoV2 N-NTD (1–175) and N-CTD (247–419) were cloned into pcDNA3.1+ expression vectors. Chimeric-HiBiT-N constructs were cloned using Gibson assembly. Briefly, the various gene fragments (encoding different domains of N protein) of MERS-CoV (GenBank: JX869059.2) and SARS-CoV2 were selectively obtained by performing PCR, followed by cloning into pcDNA3.1+ expression vector. The Nluc-N was cloned by tagging the NanoLuc gene to the 5′ end of the coding sequences for N gene. All clones were validated by sequencing, and expression was measured using the Promega’s Binary NanoLuc Technology (NanoBiT) as per manufacturer’s instructions.Human codon-optimized SARS-CoV2-S cDNAs were obtained from genscript (MC_0101081). Human codon optimized SARS-CoV2 E, M, N (GenBank: MN908947.3), and HiBit gene blocks were commercially synthesized . All DNAs were cloned using Gibson assembly. The SARS-CoV2 E and M genes were cloned in pcDNA3.1+ mammalian expression vector. The SARS-CoV2 N protein was tagged with an 11 amino acid peptide tag (HiBiT). The HiBit-N gene was also cloned in a pcDNA3.1+ expression vector by ligating the HiBiT-linker sequence g, 4 °C, 10 min, then at 3000× g, 4 °C, 10 min. VLPs in media were then concentrated 20-fold by ultrafiltration using 100 K Amicon ultra filters , and 0.5-mL volumes were then applied to size-exclusion chromatography (SEC) columns. Fractions were collected according to manufacturer’s instructions , and VLPs were identified in eluted fractions using Nano-Glo luciferase assay reagents . Briefly, aliqouts of each fraction were mixed with LgBiT protein in the presence of Nano-Glo luciferase assay substrate (Promega), and Nluc enzyme levels were measured in terms of relative light units (RLU) using a Veritas microplate luminometer. Fractions containing HiBiT-VLPs were aliquoted and stored at −80 °C.Adherent HEK293T cells at 80% confluence were co-transfected with equimolar amounts of plasmids encoding the CoV-S, E, M, and HiBiT-N proteins using LipoD transfection reagent . 1:3 DNA:LipoD mixtures were mixed in serum free-DMEM for 10 min at room temperature followed by dropwise addition onto cells. Four h later, media were replenished with DMEM-1% FBS. Media containing VLPs were collected at 24 h post-transfection, clarified by sequential centrifugation, first at 300× 2, 1 mM MnCl2, 2 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture ), lysates mixed 5:1 with 6× sample solubilizer (0.0625 M Tris·HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, 2% (w/v) SDS, 2% 2-mercaptoethanol), and then heated at 95 °C for 5 min. Proteins were electrophoresed on discontinuous Laemmli polyacrylamide gels, transferred to nitrocellulose membranes , and probed with primary antibodies and secondary HRP-conjugated antibodies and antibodies visualized by chemiluminescence (Thermo Fisher Scientific) as per manufacturer’s instructions. HiBiT-tagged proteins were identified by probing nitrocellulose membranes for 1 min with LgBiT protein in the presence of Nano-Glo luciferase assay substrate (Promega), followed by chemiluminescent visualization. Image processing was performed using FlourChem E (Protein Simple).Cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer  and SEMN (S+E+M+N) containing VLPs for 1.5 h at 4 °C. The cells were washed and lysed to quantify the associated Nluc activity using the Promega’s Nano-Glo luciferase assay system. The EMN VLPs were used to calculate the background.For the VLP-cell entry assay, HeLa cells were transfected with pcDNA-ACE2-LgBiT. Twenty-four h later, cells were inoculated with EMN and SEMN, containing VLPs for 1 h at 4 °C. Thereafter, cells were incubated with or without trypsin (20 ng/µL) in the presence of Nluc live cell substrate vivazine and transferred to 37 °C to initiate VLP-cell entry. Nluc activity was measured at various time points to assess kinetics of VLP entry. After 2 h, cells were dissolved in Nano Glo lysis buffer  to allow for maximal HiBiT:LgBiT complementation. The proportions of VLPs that entered cells in the 2 h time period were estimated relative to the maximal complementation after detergent cell lysis.t-test and ANOVA with Dunnette post hoc test were applied to statistical differences amongst the groups. In all the experiments, p < 0.05 was considered statistically significant.Results are expressed as means ± SE of the mean of at least three independent experiments. Unpaired CoV VLPs can be produced upon co-expression of the E, M, and N structural proteins in a mammalian expression system, and the S glycoprotein can efficiently incorporate onto these secreted VLPs . Recent To delineate the role of individual structural proteins in VLP assembly, we performed a series of transfections with E, M, and N, alone or together, purified secreted VLPs using SEC, and analyzed VLP proteins by Western blotting . Unlike The central role for N proteins in VLP production  promptedThese differences between SARS-2 and MERS N proteins provided opportunities to delineate the roles of individual domains of N protein. We made a series of MERS-SARS-2 chimeric N constructs A. The chMS3 and NSMS5, both of which are ~80% as effective as complete SARS-2 N and both of which retain the SARS-2 CTDs  was constructed in which a portion of the CTD was eliminated . This ΔNCoV VLPs are tools to evaluate particle assembly  and can We considered whether HiBiT tags could be placed on CoV structural proteins other than nucleocapsid, so that the membrane proteins might also be monitored using Nanobit approaches. To this end, we produced two more types of SARS-CoV2 VLPs having the HiBiT tags on C terminus of M and the E proteins. Production and SEC purification of the E- and M-HiBiT VLPs revealed that they could be obtained in yields similar to HiBiT-N VLPs -rich containing linker regions, and CTDs . Our resA remaining question is whether N protein: RNA binding has a role in particle morphogenesis. Both NTDs and CTDs were associated with RNA binding in SARS-CoV ,52, and The VLPs can, however, provide assay platforms for particle assembly and its inhibition. SARS-CoV2 assembly is a target for pharmacologic inhibition, and in silico approaches have identified several phytocompounds ,54 and aThe VLP systems described here also provide platforms for evaluating virus-cell entry. Here, we found that the 11-amino acid HiBiT tags used to monitor VLP production and entry can be appended to different structural proteins of SARS-CoV2, as demonstrated by the VLP-cell entry assays . This adOverall, we report a HiBiT-VLP system that can be utilized in BSL-1 laboratory settings to quantify features of CoV assembly and entry. The VLPs were used here to illuminate roles for N protein domains in SARS-CoV-2 assembly and to demonstrate utility in virus-cell entry assays. We anticipate the VLP systems will be adaptable to screens for inhibitors targeting SARS-CoV2 entry and assembly, thereby accelerating antiviral drug discovery for COVID19 and other CoV diseases.


C-MYC (MYC)-amplificated N-type SCLC (SCLC-N) with less immune infiltrate. Our data showed that c-Myc was the suppressor of NKG2DL in SCLC-N. Further, c-Myc suppressed the transcription of NKG2DL by recruiting HDAC3 to deacetylate H3K9ac at the promoter of MICA and MICB in SCLC-N and inhibited the cytotoxicity of NK cells. The above findings revealed the role of c-Myc/HDAC3 axis in the regulation of NKG2DL expression, supplying a new perception for comprehending the mechanism of SCLC-N immune escape, which was poorly understood and providing the therapeutic targets that SCLC-N may benefit from.Natural killer group 2, member D ligand (NKG2DL) is the most relevant ligand of NK cells to perform immune surveillance and is rarely expressed in most small cell lung cancer (SCLC) with the unclear mechanism. This study aimed to investigate the mechanisms underlying the NKG2DL deficiency in MYC in the regulation of NKG2DL, the most relevant NK-activating ligand in SCLC-N. Western Blotting, Immunofluorescence, flow cytometry, quantitative real-time PCR (qRT-PCR), Co-Immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and Cytotoxicity assay were used on H2227 cells, H446 cells, and other SCLC cell lines, and we found that c-Myc negatively regulated NKG2DL expression in SCLC-N cells. Mechanistically, c-Myc recruited HDAC3 to deacetylate H3K9ac at the promoter regions of MICA and MICB, suppressing the MICA/B expression of SCLC-N cells and the cytotoxicity of NK cells. Treatment with selective HDAC3 inhibitor up-regulated the expression of NKG2DL on SCLC-N cells and increased the cytotoxicity of NK cells. Furthermore, analysis of the CCLE and Kaplan-Meier plotter data performed the negative correlation between MYC and NKG2DL in SCLC-N cells and the correlation with the prognosis of lung cancer patients. Collectively, the results provided the new insight into the role and mechanism of c-Myc/HDAC3 axis in NKG2DL expression and innate immune escape of SCLC-N, suggesting the potential target for SCLC-N immunotherapy.SCLC is an aggressive malignancy with a very poor prognosis and limited effective therapeutic options. Despite the high tumor mutational burden, responses to immunotherapy are rare in SCLC patients, which may be due to the lack of immune surveillance. Here, we aimed to examine the role and mechanism of oncogene  Small cell lung cancer (SCLC), an extremely malignant subtype of lung cancer, is featured by rapid growth and tendency to metastasize, with a dismal prognosis and high relapse rate. Among all patients with SCLC, about two-thirds are diagnosed with advanced-stage disease that is generally accompanied by distant metastasis and has a five-year survival rate of less than 5% . SCLC isRAET1 genes, including Rae1α/β/γ/δ/ε, MULT1, and H60a/b/c [In SCLC, responses to immune checkpoint blockade (ICB) are rare, which may be related with the low infiltration by immune cells, especially cytotoxic lymphocytes ,8. SCLC H60a/b/c . NKG2D iH60a/b/c . Our preH60a/b/c . HoweverH60a/b/c , suggestRB1 and TP53, along with mutually exclusive expression of MYC paralog  [−NeuroD1−POU2F3− tumor tissues as subtype-I (SCLC-I) with high expression of inflammatory genes and increased number of immune cells including T cells, NK cells, and macrophages [MYC drove the SCLC-N and SCLC-Y subtype of SCLC in a temporal evolution by reprograming neuroendocrine fate [MYC may contribute to the construction of SCLC-N immune microenvironment and the effect of immunotherapy through modulating NKG2DL.SCLC has been treated as a single disease without patient stratification and exhibits genetic loss of both tumor suppressors N, MYCL) ,21,22,23N, MYCL) . The resrophages . Survivaine fate , SCLC-N ine fate , and SCLine fate , we specNKG2DL promoter and inhibited the transcription of NKG2DL in SCLC-N cells. The study might suggest the inhibition of HDAC3 to be the selectively beneficial therapeutic approaches for MYC-amplificated SCLC-N.Here, the study aimed to identify the c-Myc-dependent mechanism of suppressing NKG2DL expression and innate immune response in SCLC-N and found that c-Myc targeted HDAC3 to deacetylate H3K9ac at v/v) FBS and antibiotics (100 IU/mL of penicillin and streptomycin). Human malignant non-Hodgkins Lymphoma cell line NK-92MI were maintained at 37 °C in NK-92MI special complete medium . All the cell lines were cultured in a 5% CO2 in air humidified incubator.Human SCLC cell lines NCI-H2227 (H2227), NCI-H446 (H446), NCI-H69 (H69), NCI-H524 (H524), and NCI-H196 (H196)  were maintained at 37 °C in RPMI-1640 medium or Dulbecco’s Modified Eagle Media supplemented with 10% (MYC encoding sequence (CDS) (Gene ID: 4609) was subcloned into CMV-MCS-IRES-EGFP-SV40-Neomycin vector . The empty vector was used as a negative control. All plasmids were isolated using TIANprep Mini Plasmid Kit  and transfected using Lipofectamine 3000 Reagent  according to the manufacture’s protocol. After 48 or 72 h, the cells were collected for the following studies.For the overexpression of c-Myc, full-length MYC siRNAs  or 40 nM of Negative Control siRNA  . The transfection was performed for 4 h using a dilution of 1:50 in Lipofectamine 2000  in serum-free DMEM followed by a 48 or 72 h incubation in FBS containing media.Endogenous c-Myc expression in H446 cells and H2227 cells was reduced using transient siRNA transfection. Around 24 h post-seeding when reaching 50% cell density, the cells were transfected with either 40 nM of H2227, H446, and H196 cells were co-cultured with Entinostat  or 10058F4  for 48 h and collected for the following studies. The H2227 and H446 cells transfected with MYC-OE plasmid for 48 h were incubated with RGFP966 . After 48 h, the cells were collected for flow cytometry analysis.3) were seeded into 96-well plates treated with different concentrations of Entinostat . After 48 h, the numbers of live cells were detected by Cell Counting Kit-8 (CCK8) . Absorbance at 450 nm was measured. Cell viability = (ODsample − ODspontaneous)/(ODcontrol − ODspontaneous) × 100%.SCLC cells (5 × 104) were seeded into the chamber of BD BioCoat Matrigel coated plates . After 24 h, migrated cells were recorded after staining with crystal violet.SCLC cells . Equal amounts of protein were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride membranes , blocked with skimmed milk and then incubated overnight at 4 °C with different primary antibodies in buffer containing 5% skimmed milk. Membranes were washed with TBS containing 0.05% Tween-20 three times, incubated with a secondary antibody for 1 h at room temperature, and then washed again three times. The blotting was examined using chemiluminescence  and analyzed with Image J software (version 1.8.0). Antibodies against GAPDH , c-Myc , NeuroD1 , YAP1 , ASCL1 , HDAC1 , HDAC2 , HDAC3 , and horseradish peroxidase-conjugated goat anti-rabbit IgG  were used.Western blotting was conducted as described previously . The parThe cells were seeded in 24-well plates. After overnight incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After being washed in PBS, the cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% bovine serum albumin. After 1 h incubation the cells were stained with rabbit anti-human c-Myc  at 4 °C overnight, followed by being incubated with fluorescein (FITC)-conjugated goat anti-rabbit IgG  for 1 h at room temperature. After being washed, the cells were counterstained with antifade mounting medium with DAPI . The images of the cells were taken under the fluorescence microscope.The cultured cells were harvested, washed twice with PBS containing 2% FBS, and stained with PE-labelled mouse anti-human MICA/MICB (320906) or PE-labelled mouse IgG2a κ isotype ctrl antibody  for 30 min on ice in the dark followed by being washed twice with PBS containing 2% FBS. All stained cells were analyzed by FACSCanto II . Live cells were carefully gated by forward and side scatter. Data were analyzed using FlowJo software (version 10).ACTB was used as an internal control. Relative mRNA expression levels were analyzed using the 2−ΔΔCt method.Total RNA was extracted using TRIzol Reagent  following the manufacturer’s instructions and reverse transcribed using First Strand cDNA Synthesis Kit . Analyses of qRT-PCR were performed using SYBR qPCR Mix  on a system . The primers used for analysis were shown in The lysates of the cultured H446 and H2227 cells were harvested and subjected to c-Myc immunoprecipitation using anti-c-Myc antibody . Antibody-protein complexes were captured using 20 μL protein A + G sepharose beads . Immunoprecipitates were then analyzed by Western Blotting. The HRP conjugated light-chain specific mouse anti-rabbit IgG antibody  was used as secondary antibody, and rabbit IgG  was used as a negative control.MICA and MICB promoters were searched by UCSC Genome Browser  and Cistrome Data Browser . ChIP assay kit  was used for ChIP assay following the manufacturer’s instructions. Briefly, cells were fixed with 1% formaldehyde and quenched with 0.125 M glycine. Next, the cells were sonicated using an ultrasonic cell disruptor in the lysis buffer. DNA was immunoprecipitated with either control IgG , H3K9ac , H3K14ac , H3K27ac , HDAC3 , or c-Myc  primary antibody. RNA and protein were digested using RNase A  and Protein K , respectively. DNA were purified using DNA Purification Kit  followed by qPCR analysis. The qPCR primers are listed in The predicted promoter sequence of 3 cells per well) and cultured overnight. Next, the SCLC cell lines were co-cultured with NK-92MI cells (E) at different E/T ratio . After 4 h, the lysate of SCLC cells was tested using LDH Cytotoxicity Assay Kit . Absorbance at 490 nm was measured using a microplate reader . Percent cell death was calculated as (ODsample − ODspontaneous)/(ODMax − ODspontaneous) × 100%.H2227, H196, or H446 cells (T) were seeded into 96-well plates . The correlation between MYC expression and NKG2DL expression was evaluated by Linear Regression test.Gene expression data of the SCLC cell lines were downloaded from CCLE .To investigate the association between t tests. Correlations between MYC and NKG2DL in SCLC cell lines were analyzed by Spearman’s rank test. p value < 0.05 was considered statistically significant.All experiments were performed in triplicate. Data were analyzed using GraphPad Prism software (version 8.0), and the results are presented as the mean ± SD. Comparisons between groups were conducted using analysis of unpaired MYC-amplificated SCLC-N cells [MYC was higher in H2227 cells than in H446 cells, while the expression level of MICA was lower in H2227 cells than in H446 cells   and majo-N cells , both of46 cells C. Furthe46 cells D,E. The MYC gene and named it MYC-OE. H2227 and H446 cells were transfected with MYC-OE and the overexpression of c-Myc in H2227 cells was determined by western blotting (+ cells were lower in MYC-OE-transfected H2227 cells than in empty vector (EV)-transfected cells  into H2227 cells to silence c-Myc. As a result, siMYC transfection silenced the expression of c-Myc  , and the patients with high MICA mRNA expression had better OS   , but notctively) , indicat= 0.019) B which a+ T cells and was the substantial mechanism of immune surveillance to initiate the immune response to find and eliminate NKG2DL+ tumor cells. In this study, we found that compared to the SCLC-Y cells, which was reported to express high level of inflammation gene and be infiltrated by more immune cells [MYC (MYC in the immune characteristics of SCLC. In addition, previous studies reported that H2227 and H446 cells were SCLC-N cells [MYC, which is consistent with the increased level of ULBP3 in both MYC-overexpressed and -silenced cells (MYC in H2227 cells (MYC in H446 cells (NKG2DL has been considered as an activating signal on the surface of cancer cells to trigger the cytotoxicity cells including NK cells and CD8ne cells , SCLC-N ne cells . Furtherlls [MYC , whose e-N cells  while H1-N cells , which w-N cells ,37,38,39ed cells . Notably27 cells C, while 46 cells , indicatNKG2DL (NKG2DL in SCLC-N cells and inhibiting the cytotoxic effect of NK cells (MYC (MYC (MICA and MICB could be regulated by HDAC3 (MICB mRNA was regulated by MYC (MICA might be regulated by the unknown specific mechanism which needed further study. Moreover, SCLC-I tumors, which obtained the highest total immune infiltrate and cytolytic activity score [MYC-amplificated SCLC-N and could solve the problem that c-Myc is largely unstructured, falling in the category of ontrinsically disordered proteins [MYC on the cytotoxicity of NK cells is worthy of further study.Additionally, the study on the mechanism revealed that c-Myc recruited HDAC3 to deacetylate histone H3K9ac, which was bound to the promoter region of NKG2DL , therebyNK cells . ConsistNK cells . Supportlls (MYC A. The spMYC (MYC , which wMYC (MYC E, indicaby HDAC3 B,F, onlyd by MYC C,G, indity score , expressproteins  and is dMYC and regulated the transcription of MYC [NKG2DL by deacetylating histone H3K9ac and the treatment with HDAC3 specific inhibitors increased the acetylation level of H3K9 (MICA/B and the level of H3K9ac were prevented by the silencing of MYC (Here, it was found that c-Myc in SCLC-N cells could combined with HDAC3, but not HDAC1 or HDAC2 A, indican of MYC ,47. The n of MYC . Due to  of H3K9 F and up- of H3K9 C. Since g of MYC B,F, it iMYC-amplificated SCLC cells, c-Myc was involved in regulating the expression of NKG2DL, the activating ligand for cytotoxic cells such as NK cells. The main mechanism might be that c-Myc recruited HDAC3 to deacetylate the histone H3K9ac, which bind to the promoter region of NKG2DL, thereby inhibiting the transcription of NKG2DL. Our findings suggested that the role of c-Myc in SCLC-N cells as a key modulator for immune characteristics and immunotherapy should be noted; compared with c-Myc, HDAC3 might be a better target for SCLC immunotherapy.To date, evidence for c-Myc to participate in the regulation and mechanism of SCLC immune microenvironment and immune characteristics is still lacking. Based on our study, we confirmed that in


B7 homolog 3 (B7-H3) is a recently found superfamily B7 molecule and therefore has significant involvement in immunological regulation. However, the relationships of B7-H3 expression with the tumor microenvironment (TME), response to immunotherapy, and prognosis in head and neck squamous cell carcinoma (HNSCC) are still unknown. In the present analysis, we determined B7-H3 as a novel biomarker that predicts the prognosis and response to immunotherapy in HNSCC. B7-H3 expression is enhanced in HNSCC compared to normal sample and is stably expressed in HNSCC cell line. Besides, high B7-H3 expression is correlated with a dismal prognosis and resistance to immunotherapy and contributes to an immunosuppressive microenvironment. Moreover, single-cell RNA sequencing (scRNA-seq) analysis shows that B7-H3 is mainly expressed in the stromal as well as malignant cells. In conclusion, the study provides insight in understanding the prognostic value of B7-H3 in HNSCC and highlights its involvement in promoting the immunosuppressive microenvironment, which presents an attractive strategy for antibody-based immunotherapy. HNSCC is the sixth most common malignancy globally, with a highly aggressive and heterogeneous phenotype , 2. EvenB7 superfamily molecules play essential roles in regulating anti-tumor immunity. For instance, B7-H1 termed as programmed death-ligand 1 (PD-L1) is a critical immune checkpoint molecule that has a predominantly inhibitory role in adaptive immunity and suppresses proliferation as well as activation of T cells –10. The This study aimed to (i) identify the relationship of TME and B7-H3 expression in HNSCC; (ii) evaluate its prognostic value; (iii) exploit B7-H3 as a biomarker that predicts the response of immune checkpoint therapy; (iv) elucidate the potential signaling pathways of HNSCC in the development and progression of HNSCC.The immortalized NP69 nasopharyngeal epithelial cell line has been bought from MeisenCTCC  and was cultured in Keratinocyte medium . CAL-27 and Tca-8113 cell lines were cultured in RPMI-1640 medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin .Briefly, cells were harvested and stained with fluorescein isothiocyanate (FITC)-anti-human B7-H3  at 4°C for 0.5 h. After incubation, the mean fluorescence intensity (MFI) of B7-H3 was determined by a flow cytometer .https://cancergenome.nih.gov/). The proteomics and DNA methylation were collected from the clinical proteomic tumor analysis consortium  (http://research-pub.gene.com/IMvigor210CoreBiologies), a fully documented R statistical computing environment software and data package. The single-cell RNA-seq data were obtained from the GEO database   (s/cptac) . IMvigors/cptac) . RNA-seqE139324) , 19.https://cibersort.stanford.edu/) including plasma cells, memory B cells, naive B cells, T cells CD4 naïve, T cells CD8, activated CD4 memory T cells, resting CD4 memory T cells, T cells regulatory (Tregs), T cells follicular helper, monocytes, T cells gamma delta, NK cells activated, NK cells resting, macrophages: M0; M1; M2, activated dendritic cells, resting dendritic cells, resting mast cells, activated mast cells, neutrophils, & eosinophils. The relationship between immune cells infiltration and B7-H3 expression was determined using the Pearson correlation coefficient.CIBERSORT was performed to quantify the 22 types of immune cells infiltration among each HNSCC sample . A leukoGSEA algorithm was performed to reveal the altered signaling pathways in the B7-H3 low- and high group, as recognized by their enrichment in the MSigDB Collection . For each analysis, gene set permutations were carried out 1000 times.http://tide.dfci.harvard.edu/) which enables a prediction of ICB response using two major tumor immune evasion mechanisms: T cell dysfunction induced in tumors with high cytotoxic T lymphocyte (CTL) infiltration and T cell infiltration inhibited in tumors with low CTL level (Tumor immune dysfunction and exclusion (TIDE) analysis has been conducted to determine ICB response. Jiang et al. designed an analytic technique known as TIDE  was used to evaluate the response to chemotherapy by performing the R package “pRRophetic” . GDSC ishttp://tisch.comp-genomics.org/home/”) is a scRNA-seq data source that focuses on TME and gives specified annotation of cell-type at a single-cell level, which allows TME exploration across multiple cancers (The tumor immune single-cell hub (TISCH) is used to analyze single-cell RNA-seq data derived from the GSE103322 and GSE139324. TISCH (“The Wilcoxon signed-rank test has been applied for comparing B7-H3 expression. Receiver operating characteristic (ROC) curve was used to determine the diagnostic value of B7-H3 using R software. To assess the prognostic value, Kaplan-Meier analysis was carried out with the Survival and Survminer R package. The statistically significant P < 0.05 was applied.Data from the TCGA database signified that B7-H3 is extremely elevated in tumor samples compared to normal samples or adjacent samples  analysis in HNSCC cell lines (CAL-27 and Tca8113) and immortalized NP69 nasopharyngeal epithelial cell line . The result of t-distributed stochastic neighbor embedding (t-SNE) presented that 11 clusters were identified in HNSCC-GSE103322 and HNSCC-GSE139324. For HNSCC-GSE103322 data, B7-H3 was mainly expressed in the stromal cells  and malignant cells, while B7-H3 is almost undetectable in the immune cells .The results published here are in part based upon data generated bythe TCGA Research Network .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Galanin has a vast diversity of biological functions, including modulation of feeding behavior. However, the clinical application of natural galanin is not practicable due to its rapid in vivo breakdown by peptidases and lack of receptor subtype specificity. Much effort has been put into the development of receptor-selective agonists and antagonists, and while receptor selectivity has been attained to some degree, most ligands show overlapping affinity. Therefore, we aimed to develop a novel ligand with specificity to a single galanin receptor subtype and increased stability. To achieve this, a lanthionine amino acid was enzymatically introduced into a galanin-related peptide. The residue’s subsequent cyclization created a conformational constraint which increased the peptide’s receptor specificity and proteolytic resistance. Further exchange of certain other amino acids resulted in a novel methyllanthionine-stabilized galanin receptor agonist, a G1pE-T3N-S6A-G12A-methyllanthionine[13–16]-galanin-(1–17) variant, termed M89b. M89b has exclusive specificity for GAL2R and a prolonged half-life in serum. Intranasal application of M89b to unfasted rats significantly reduced acute 24 h food intake inducing a drop in body weight. Combined administration of M89b and M871, a selective GAL2R antagonist, abolished the anorexigenic effect of M89b, indicating that the effect of M89b on food intake is indeed mediated by GAL2R. This is the first demonstration of in vivo activity of an intranasally administered lanthipeptide. Consequently, M89b is a promising candidate for clinical application as a galanin-related peptide-based therapeutic.The regulatory (neuro)peptide galanin is widely distributed in the central and peripheral nervous systems, where it mediates its effects via three G protein-coupled receptors (GALThe online version contains supplementary material available at 10.1007/s13311-021-01155-x. Galanin is a 29 amino acid peptide that was first isolated from porcine intestine in 1983 . The test also indicated an interaction between time and treatment (p < 0.001). Tukey’s post hoc test showed significant differences between M89b- vs. all other groups . Remarkably, intranasal M89b also resulted in a significant reduction of body weight , while other treatment groups exhibited no body weight change.In accordance with the literature , intrana38) Fig. a. The te47) Fig. b. A more47) Fig. c. The reist M871 . Adminis2R specificity and highly increased stability. M89b has no activity on GAL1R or GAL3R, not even at concentrations as high as 10 µM, and its affinity via GAL2R is slightly lower compared to wild-type galanin or GAL-(1–15) in most assays.The functions of galanin-related peptides, such as galanin, GALP, and spexin, are mediated by GALRs. Over the last two decades, a wide range of GALR agonists and antagonists have been developed. Most of them displayed limited and/or unknown specificity for the three receptors , 55, 56.1R. This observation is in accordance with the fact that GAL-(2–11) is a non-GAL1R selective GALR agonist [2R affinity. Furthermore, in silico analyses indicated that the binding cavity of GAL2R is about twice the size of that of GAL1R and about three times the size of that of GAL3R [2R specificity of M89b.Since the N-terminal region of galanin is essential for activity, our developmental approach started with a C-terminally truncated galanin analog. Accordingly, the introduction of a methyllanthionine before position 13 strongly affected activity on GALRs. Importantly, the first N-terminal amino acid of wild-type galanin seems to have a role in receptor specificity. Its absence appears to abolish the ability of galanin to stimulate GAL agonist , 58. Theof GAL3R . The rel2R-specific agonist [As expected, introduction of the pyroglutamate and the methyllanthionine clearly protected against peptidase-mediated breakdown and resulted in greatly increased stability of M89b. In an in vitro stability test using 5% serum, which contains many different peptidases , 61, M89 agonist . The inc2R and GAL3R [Additionally, Reyes-Alcaraz et al. observed that Thr3 of wild-type galanin is critically important for activation of GALnd GAL3R . However2R agonist, the M1160 peptide is the only other ligand developed so far as having strict GAL2R specificity, as all others still retain activity on GAL1R and/or GAL3R at high concentrations [2R. Importantly, no data on the stability of M1160 are available [2R-specific variants exerted anxiolytic effects in mice [2R over GAL1R have been discovered and showed analgesic properties in rodent models of inflammation, neuropathic pain, acute pain, and epilepsy [3R has not been reported.Besides the spexin-based GALtrations , 63. Comvailable . Neverthvailable . Spexin- in mice  and ther in mice  after icepilepsy , 64, 65.2R specificity and the high in vitro stability together with the enhanced in vivo stability strongly support the clinical usefulness of M89b. Indeed, in the present study, we provide evidence that M89b is functionally active in vivo.Taken together, the strict GAL1R- nor GAL2R-KO mice displayed any marked phenotype related to feeding behavior [1R agonist, M617, induced consumption of high-fat milk in rats, whereas a selective GAL2R agonist, M1153, did not [1R- and GAL2R-KO mice [2R-specific antagonist M871. To our knowledge, this is the first study showing in vivo activity of a lanthionine peptide upon intranasal application. Furthermore, our data support recent findings on a GAL2R-selective spexin analog [2R signaling. However, there are no studies available which tried to block the effects of GALP on food intake with GALR-specific antagonists to prove the involvement of GALRs in GALP-mediated effects. As chronic administration might lead to resistance to treatment, it will have to be elucidated whether chronic administration of M89b reduces the body weight of obese animals. A first hint that this could be the case comes from the observation that intranasal administration of the spexin-based GAL2R agonist SG2A for 14 days decreased food intake and body weight [The galanin system plays a role in feeding, as GALRs are expressed in brain regions important for appetite control . Galaninbehavior , 67, an  did not . However-KO mice , indicat-KO mice . Thus, wn analog , which iy weight . Taken tIntranasal delivery of peptides to the brain has been reported for a limited number of peptides , includi2R-selective agonist offers therapeutic opportunities for treating a variety of brain diseases. GAL2R has, for instance, been reported to be neuroprotective in experimental autoimmune encephalomyelitis and a cuprizone model mimicking aspects of multiple sclerosis [2R in a multiple sclerosis patient has been reported [2R in this disease. The current study raises the possibility of an anti-obesity effect of M89b that could be confirmed via chronic or repeated administration in future studies.In conclusion, intranasal delivery of a GALclerosis , 71. A nreported , indicatSupplementary file1 (PDF 616 KB)Supplementary file2 (PDF 3506 KB)Supplementary file3 (PDF 1358 KB)Supplementary file4 (PDF 462 KB)Supplementary file5 (PDF 468 KB)Supplementary file6 (PDF 628 KB)Supplementary file7 (PDF 507 KB)Supplementary file8 (PDF 230 KB)Below is the link to the electronic supplementary material.


Artificial proteincages have potential as programmable, protectivecarriers of fragile macromolecules to cells. While natural cages andVLPs have been extensively exploited, the use of artificial cagesto deliver active proteins to cells has not yet been shown. TRAP-cageis an artificial protein cage with an unusual geometry and extremelyhigh stability, which can be triggered to break apart in the presenceof cellular reducing agents. Here, we demonstrate that TRAP-cage canbe filled with a protein cargo and decorated with a cell-penetratingpeptide, allowing it to enter cells. Tracking of both the TRAP-cageand the cargo shows that the protein of interest can be successfullydelivered intracellularly in the active form. These results providea valuable proof of concept for the further development of TRAP-cageas a delivery platform. Adenoviruses,for example, are highly efficient and bind quite specifically to theCAR receptor.7Transport of molecularcargos to cells is desirable for a rangeof applications including delivery of drugs, genetic material, orenzymes. A number of nanoparticles have been employed to achieve thisincluding liposomes,9 which allows substituent proteins to be expressed as relativelysmall individual subunits with/without cargos, thus circumventingpossible production problems associated with large complex formationin the cell. Artificial cages also allow other features such as geometriesnot seen in natural cages,8 which widensthe possible library of building blocks, and replacement of enhancementof protein–protein interactions with other interactions, leadingto control of disassembly.10 To date, a number ofartificial protein cages have been produced including tandem fusionsof proteins with two- and threefold rotational symmetries able toform a 12-subunit tetrahedral cage,13 a nanocube structureof 24 subunits with an octahedral symmetry,14 a 60-subunit icosahedral cage structure that self-assembles fromtrimeric protein building blocks,15 andco-assembling, two-component 120-subunit icosahedral protein complexescomparable to those of small viral capsids9 and designed peptides able to form networks that close to form cages.16 Several examples exist where artificial proteincages have been filled with various cargos including siRNA,18 mRNA,19 and fluorescent dyes.20 However, only a handful of cases have demonstrated delivery of cargosto cells by artificial cages.21 To the best of our knowledge, delivery of protein cargos to cellsmediated by artificial protein cages has not previously been demonstrated. In this work, we show for thefirst time that an artificial protein cage is capable of deliveringa functional protein cargo to the cell interior.Artificial proteincages are constructed by proteins which do notnaturally form cage structures and in which interactions between constituentproteins are modified to promote their assembly. The advantage ofusing such an approach is that the resulting cages can be given propertiesand capabilities that may not be available or feasible in naturallyoccurring forms. This includes triggerable assembly,23 In nature, TRAP is involved incontrol of tryptophan synthesis and has been well characterized structurallyand biochemically.27 It has also been used as a versatile building block in bionanoscience.30 TRAP-cage consists of 24 TRAP rings, forming an approximately 22nm-diameter, 2.2 MDa hollow sphere with a lumen roughly 16 nm in diameter.Each TRAP ring in the cage is bound to five TRAP ring neighbors, andthe structure contains six square holes approximately 4 nm in diameter.Unusually, compared to other natural and most artificial cages, thering subunits in the cage are held together not by a network of protein–proteininteractions. Instead, single gold(I) ions bridge opposing thiolsof the cysteine residues between rings in proteins where the naturallyoccurring lysine at position 35 is replaced with cysteine. The cysteinesof 10 of the 11 monomers of each ring in the cage are bridged in thisway with those of the eleventh remaining unbridged and available toreact, for example, with maleimide-labeled dyes. As the constituentTRAP ring is made from 11 monomers, it approximates a hendecagon,which would not be expected to assemble to form a regular-faced convexpolyhedron. However, by assembling into an approximation of an Archimedeansolid (a snub cube) and allowing for small amounts of deviation/flexibility,an “almost regular” TRAP-cage can form.8We previouslyproduced an artificial protein cage using a buildingblock consisting of the naturally occurring ring-shaped protein, TRAP(trp RNA-binding attenuation protein), referred to as TRAP-cage 1a.8,22,28 Despite this high stability,TRAP-cage breaks apart readily in the presence of low concentrationsof reducing agents including the cellular reducing agent glutathione.8 This feature raises the prospect that the TRAP-cagemay have utility as a system for delivering cargos to cells, as itcan be expected to retain its structure, protecting the cargo untilentering cells where intracellular reducing agents will result indisassembly and subsequent cargo release.TRAP-cage is extremely stable and able to survivetemperaturesof 95 °C for at least 3 h and high levels of denaturing agentssuch as 7 M urea.Here, we show thatTRAP-cage can be deliberately filled with aprotein cargo, and we use a negatively supercharged variant of greenfluorescent protein, GFP(-21), as an exemplar molecule. Furthermore,we show that TRAP-cage can be used to deliver such cargos to the interiorsof human cells. This cell penetration is itself controllable, as itonly occurs if the surface of TRAP-cage is modified, for example,by cell-penetrating peptide. The results are a first step toward developmentof TRAP-cage as a potentially useful tool for delivering medicallyrelevant cargos to cells and more generally demonstrate the potentialfor artificial protein cage systems as therapeutic agents.8 Supercharged (-21) His-tagged GFP protein (MGHHHHHHGSACELMVSKGEELFTGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK31) was expressed from pET28a encoding the GFPgene and produced in BL21(DE3) cells. The protein was purified usingNi-NTA. Briefly, cells were lysed by sonication at 4 °C in 50mM Tris-HCl, pH 7.9, 150 mM NaCl, 5 mM MgCl2, and 5 mMCaCl2, in the presence of protease inhibitors (Thermo FisherScientific), and lysates were centrifuged at 20 000 g for 0.5 h at 4 °C. The supernatant was incubated with agarosebeads coupled with Ni2+-bound nitrilotriacetic acid  pre-equilibrated in 50 mM Tris,pH 7.9, 150 mM NaCl, and 20 mM imidazole (buffer A). After three washesof the resin (with buffer A), the protein was eluted with 50 mM Tris,pH 7.9, 150 mM NaCl, and 300 mM imidazole (buffer B). Fractions containingHis-tagged GFP(-21) were pooled and subjected to size exclusion chromatographyon a HiLoad 26/600 Superdex 200 pg column  in 50 mMTris-HCl, pH 7.9, and 150 mM NaCl at room temperature. Protein concentrationswere measured using a Nanodrop spectrophotometer using a wavelengthof 280 nm.TRAP-cage (TRAP monomersequence: MYTNSDFVVIKALEDGVNVIGLTRGADTRFHH-SEKLDKGEVLIAQFTEHTSAIKVRGKAYIQTRHGVIESEGKK)production and purification were performed as described previously.GFP encapsulation was conducted by mixing equal volumesof 100 μM negatively supercharged (-21) His-tagged GFP with1 μM pre-formed TRAP-cage incubated overnight in 50 mM Trisand 150 mM NaCl (pH 7.9). Purification of TRAP loaded with GFP(-21)was carried out by size exclusion chromatography using a Superose6 Increase 10/300 column  in 50 mM HEPES, pH 7.5, and150 mM NaCl. Fractions containing TRAP-cage were collected and analyzedby native PAGE using 3–12% native Bis-Tris gels (Life Technologies)followed by fluorescence detection using a Chemidoc detector (BioRad)with excitation at 488 nm.Samples of purifiedTRAP-cage filled with His-tagged GFP(-21) protein were divided intotwo portions and incubated under reducing (1 mM TCEP) or nonreducing(no TCEP) conditions. Next, samples were passed through a Ni-NTA resin(Thermo Fisher Scientific) under gravitational flow, in which 100μL of each sample was introduced onto 50 μL of the resinequilibrated with buffer A. Three samples were collected: (i) flowthrough, (ii) wash with buffer A, and (iii) elution with buffer B.Samples were analyzed by native PAGE, followed by fluorescence detection and western blot. For theSDS-PAGE and western blot, samples collected from the Ni-NTA pull-downassay were denatured by addition of TCEP  and boiling for 15 min followed by separation via Tris/Glycinegel electrophoresis. Gels were then subjected to electrotransfer  in 25 mM Tris, 192 mM glycine, and 20% methanol buffer ontoan activated PVDF membrane. The membrane was blocked with 5% skimmedmilk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T),followed by 1.5 h of incubation with the mouse monoclonal anti-GFPantibody  and anti-mouse secondary antibody conjugated withhorse radish peroxidase. The signal was developed using a Pierce ECLwestern blotting substrate (Thermo Fisher Scientific) and visualizedin a BioRad Chemidoc detector.Alexa Fluor-647 C2 maleimide fluorescent dye  and cell-penetrating PTD4 peptide were conjugatedto the TRAP-cage filled with GFP(-21) via crosslinking reactions withcysteines and lysines present in the TRAP protein.Figure S3a). Briefly, aliquots of TRAP-cage loaded with GFP(-21) (11.36 nM)were mixed with maleimide-conjugated Alexa-647 at concentrations rangingfrom 0.1 to 100 μM. Samples were then separated by native gelelectrophoresis and visualized by fluorescence detection in a Chemidocsystem, with excitation at 647 nm. Reactions where no free Alexa-647is present in the sample and no GFP(-21) interference with the Alexa-647signal is observed were considered to possess optimal decoration conditionsand used in further experiments. The yield of TRAP-cage labeling withAlexa-647 was quantified using fluorescence detection of the labeledTRAP protein.To achievefluorescent labeling, TRAP-cage carrying GFP(-21)  was mixed with a Alexa-647 C2 maleimide dye ;the reaction was conducted in 50 mM HEPES with 150 mM NaCl, pH 7.5,for 2.5 h at room temperature with continuous stirring at 450 rpm.The optimal interaction ratio of maleimide-conjugated Alexa-647 toTRAP-cage was assessed by titration  labelingby Alexa-647, TRAP-cage loaded with GFP(-21) with and without Alexa-647labeling was subjected to denaturing gel separation and western blottingfollowed by detection with the anti-GFP antibody.N-hydroxysuccinimide, all reagents dissolved in ddH2O. Subsequently, an excess of activated PTD4 peptides was added toTRAP-cage filled with GFP(-21) and labeled with Alexa-647 and incubatedfor 2.5 h at room temperature, with continuous stirring at 450 rpm.The reaction was stopped by addition of 5 μL of 200 mM Tris-HCl,pH 7.5. The conjugation efficiency was verified by native PAGE, fluorescentgel imaging, and HPLC analysis.For the cell-penetratingpeptide decoration, PTD4 peptide  was mixed with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimidehydrochloride  and g for 5 min at room temperature, and the supernatant wasapplied onto hydrophilized carbon-coated copper grids (STEM Co.) Sampleswere then negatively stained with 3% phosphotungstic acid, pH 8, andvisualized using a JEOL JEM-2100 instrument operated at 80 kV.TRAP-cage filled with GFP(-21),TRAP-cage filled with GFP(-21) and labeled with Alexa-647, and TRAP-cagefilled with GFP(-21) and fully decorated were imaged using transmissionelectron microscopy (TEM). Samples were typically diluted to a finalprotein concentration of 0.025 mg/mL and centrifuged at 10,0005 per well and cultured for further 16 h prior to the experiments.Cells were then incubated with 50 μg (6 nM) of TRAP-cage filledwith the cargo, labeled with Alexa-647 only, or decorated with Alexa-647and PTD4 peptide in 50 mM HEPES with 150 mM NaCl, pH 7.5, supplementedwith 10% FBS for 15 min, 2, and 4 h at 37 °C, 5% CO2. After the incubation, cells were washed three times for 5 min withphosphate-buffered saline (PBS) (EURx), harvested with trypsin (1mg/mL), and centrifuged at 150g for 5 min. Subsequently,cells were washed thrice in PBS by centrifugation (150g for 3 min) and re-suspended in 0.5 mL of PBS. Cells were run ina Navios flow cytometer (Beckman Coulter), and the fluorescence of12,000 cells was collected for each sample. Untreated cells and cellstreated with TRAP-cage filled with cargo and labeled with Alexa-647only were used as negative controls. Obtained data for three independentexperiments were analyzed with Kaluza software (Beckman Coulter).The percentage of Alexa-647/GFP(-21) positive cells and median fluorescenceintensity was determined for each sample.For TRAP-cage internalization experiments,MCF-7 and HeLa cells were seeded into 12-well plates (VWR) in 800μL of DMEM medium with 10% FBS at a density of 2.5 × 105 per well in 800 μL of DMEM medium with 10% FBS)and further stimulated with 50 μg (6 nM) of TRAP-cage filledwith the cargo, labeled with Alexa-647 only, or decorated with Alexa-647and PTD4 peptide in 50 mM HEPES with 150 mM NaCl, pH 7.5, supplementedwith 10% FBS for 4 h at 37 °C and 5% CO2. Next, cellswere washed with PBS (three times for 5 min), fixed with 4% paraformaldehydesolution , and permeabilized with 0.5%Triton-X100 in PBS . Actin filaments werestained with phalloidin conjugated to Alexa-568 in PBS . Cover slips were thenmounted on slides using Prolong Diamond medium with DAPI (Thermo FisherScientific). Fluorescent images were acquired under an Axio Observer.Z1inverted microscope , equipped with theLSM 880 confocal module with a 63× oil immersion objective. Imageswere processed using ImageJ 1.47v .For fluorescentlaser scanning confocal microscopy observations, cells were grownon 15 mm glass cover slips plated into 12-well plates (2.5 ×1036 The fact that the constituent TRAP rings do not assemble into TRAP-cageuntil the addition of gold(I)8 means thatprotein cargos below approximately 4 nm (the size of the fourfoldholes in TRAP-cage8) have two possibleroutes for encapsulation—they may bind to TRAP rings priorto assembly or they may be added after TRAP-cage formation and allowedto diffuse into the cage through the fourfold holes. We chose negativelysupercharged GFP(-21) as a model cargo , similar to but lower than the rangefound in a number of other filled protein cages,38 although somehave shown considerably greater numbers of cargos.39 Where the magnitude of charge complementarity is lower,a low cargo loading is expected, and while we have used a superchargedGFP, the cage interior bears only small patches of positive chargesassociated with the wild-type protein, likely accounting for the relativelylow loading efficiency, which could, in future work, be addressedby modifying TRAP-cage further, such that it carries a higher densityof positive charges within the cage interior. Alternatively, differentmethods of cargo capture  could be explored,as described for other protein cages.40To fill TRAP-cage,we took advantageof the fact that the only significant patch of positive charge onthe surface of the TRAP ring lies on the face lining the interiorof the cage 1b,c. In el cargo 1d. This el cargo 1e. His-tel cargo 2a. To veel cargo 2b. TheseFigure S3a). The yield of TRAP-cage labeling with Alexa-647was quantified using fluorescence detection of labeled TRAP-cage ofknown concentration. The average number of Alexa-647 molecules boundto TRAP-cage was estimated to be 2 . Although the cargo GFP(-21) contains three cysteine residues,control reactions showed no detectable labeling of GFP(-21) with Alexa-647.In order to be able to track TRAP-cage independentlyfrom its cargo, we labeled it with the Alexa-647 fluorescent dye.For this, we crosslinked the maleimide group on the dye with the 24available cysteines lining the six 4 nm holes of TRAP-cage that arenot involved in ring–ring interactions. By titration, we establishedthe optimal amount of Alexa-647 to be added, this being the concentrationat which the TRAP-cage is readily labeled and no free dye is presentin the sample. It was assessed by native PAGE combined with fluorescentmeasurements to detect both GFP(-21) and Alexa-647 .We aimed to modifythe TRAP-cage in order to promote its cell entry. For this, we chosePTD4 (YARAAARQARA)—an optimized TAT-based cell-penetratingpeptide that shows a significantly improved ability to penetrate cellmembranes, being more amphipathic with a reduced number of argininesand increased α-helical content.In reactions optimized for Alexa-647 labeling,we observed an increasein the apparent molecular weight of TRAP-cage after reaction withPTD4 2c, asvi2 atmosphere in Dulbecco’s modified eaglemedium (DMEM) without or with fetal bovine serum (FBS) at variousconcentrations. The results showed that the TRAP-cage structure isstable in DMEM culture medium within 18 h of incubation at 37 °Cand 5% CO2 .Before embarking on cell delivery tests, we first assessed whetherTRAP-cage was structurally stable, that is, did not disassemble undercell culture conditions. Stability was checked at 37 °C and ina 5% CO44 Human cancercell lines MCF-7 and HeLa were incubated in the presence of TRAP-cageand TRAP-cage filled with GFP(-21) and decorated with Alexa-647 andPTD4 peptide. The number of cells, TRAP-cage dose, and stimulationtime used in cell viability tests correspond to the conditions underwhich the internalization of the TRAP-cage experiments was performed.Untreated cells were used as a control. The data showed that bothunmodified TRAP-cage and TRAP-cage filled with GFP(-21) and decoratedwith Alexa-647 and PTD4 do not significantly affect the viabilityof MCF-7 and HeLa cells for at least 4 h of incubation .In order to determine the effect of TRAP-cage on cell viability,alamarBlue assays were carried out. This test is based on the naturalability of viable cells to convert resazurin, a blue and nonfluorescentcompound, into resofurin, a red and fluorescent molecule by mitochondrialand other reducing enzymes.Figure S6a,b).Delivery ofTRAP-cage to cells was studied using human cancer cell lines MCF-7and HeLa. Cells were incubated for different time periods with thepurified TRAP-cages containing encapsulated GFP(-21) and labeled withAlexa-647 only or with Alexa-647 and PTD4 and analyzed by flow cytometry.The fluorescent signal due to both Alexa-647 and GFP(-21) increasedwith prolonged incubation time in both cell lines treated with TRAP-cagewith GFP(-21) labeled with Alexa-647 and PTD4 peptide 3a,b. TheS6c).In order to discriminatebetween fluorescent signals from TRAP-cageswhich were internalized in the cells and those which were adsorbedexternally on the cell membrane, confocal microscopy was used. TRAP-cagecontaining GFP(-21) and labeled with Alexa-647 but lacking PTD4 wasnot observed in the cells. In contrast, TRAP-cage containing GFP(-21)and decorated with PTD4 showed a clear signal in the cell interiorat 4 h after stimulation , where the lipid envelope was derived fromthe host cell membrane. The EPNs were able to deliver the cages, meaningthat entry to cells was achieved by the enveloping, host-derived membrane,not the protein cage.The demonstrationof delivery of the active protein cargo to cellinteriors via an artificial protein cage is of interest given thesmall number of previous studies on artificial protein cage-mediateddelivery of cargos to cells demonstrated for nonprotein cargos. Notably,Edwardson and co-workers showed that an artificial protein cage loadedwith siRNA can be taken up by different mammalian cells and can releaseits cargo to induce RNAi and knockdown of target gene expression.S7). While differentiating between intracellularGFP(-21) in the cytoplasm and the endosome is challenging, evidencesupporting cytoplasmic localization comes from the lag observed inthe appearance of the GFP(-21) signal. IF GFP(-21) was localized inthe endosome, we would expect degradation to result in a decreaserather than an increase in the signal over time.The high stabilityof TRAP-cage coupled with its ability to break apart in the presenceof modest concentrations of cellular reducing agents suggests thatTRAP-cage in the cytoplasm should readily disassemble, releasing theGFP(-21) cargo. As TRAP-cage and GFP(-21) possess discrete and trackablesignals, we hypothesized that cage disassembly and release of GFP(-21)may be strongly inferred if the Alexa-647 and GFP(-21) signals becamenon-colocalized after cell entry. To assess this possibility, we trackedboth signals over time after addition to MCF-7 and HeLa cancer cells.Notably, in both cell lines tested, during the first 90 min of incubation,TRAP-cage was mainly present at the cell boundaries, as indicatedby the strong localization of the Alexa-647 signal there, and theGFP(-21) signal was barely detectable. However, after 3 h of incubation,the TRAP-cage signal (Alexa-647) became weaker and appeared to bedistributed more evenly in the cell, whereas the GFP(-21) signal wasclearly detectable, likely due to its release from the TRAP-cages4 and S7.Figure S11).To further confirm that TRAP-cage with GFP(-21)is successfullydelivered to the cell interior and the cargo is released, we performedin-cell ELISA, where we detect GFP(-21) with a specific antibody,at several time points. These results demonstrated that the longerthe cells were incubated with TRAP-cage filled with GFP(-21), themore detectable the GFP(-21) signal, suggesting that the cargo proteinbecomes more accessible for the antibody, that is, released or exposedby cage opening . Given the relatively low occupancyof GFP(-21) inside TRAP-cage and the fact that on average, each 24-ringcage bears only two Alexa-647 dye molecules, cage disassembly could,in principle, lead to separation of rings with electrostatically attachedGFP(-21) from Alexa-647 simply by virtue of the fact that the ringto which GFP(-21) attached is not labeled with Alexa-647. For futurepotential use, this is not necessarily problematic, depending on theidentity of the protein cargo, which will be active as long as anybinding/active site is accessible.Thechange in relative signal strengths of TRAP-cage-associatedAlexa-647 versus GFP(-21) once in the cell is suggestive of intracellularbreak-up of the cage and release of the cargo. A possible explanationis that when Alexa-647 and GFP(-21) are in close proximity to eachother due to association with TRAP-cage, the GFP(-21) fluorescencemay be decreased due to a (non-FRET) quenching effect from the dye.Once GFP(-21) is released by TRAP-cage disassembly, average GFP(-21)to Alexa-647 distances become larger, resulting in an increase inthe detected GFP(-21) fluorescence. This possibility is supportedby the observation that the signal from intracellular GFP(-21) isvisibly brighter when it is delivered using TRAP-cage lacking Alexa-647. An engineeredcage, by contrast, is an existing cage  thathas been engineered, that is, altered to give it certain properties.We demonstrated a relatively low cargo-filling efficiency, and inthe future study, this could be addressed by modifying TRAP-cage furthersuch that it carries a higher density of positive charge within thecage interior. Alternatively, different methods of cargo capture  could be explored, as described for otherprotein cages.