Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow.

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with Nε -(5-carboxyfluorescein)-l-lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (Ka ) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.

Cancer cells as a new source of induced pluripotent stem cells.

Over the last 2 decades, induced pluripotent stem cells (iPSCs) have had various potential applications in various medical research areas, from personalized medicine to disease treatment. Different cellular resources are accessible for iPSC generation, such as keratinocytes, skin fibroblasts, and blood or urine cells. However, all these sources are somatic cells, and we must make several changes in a somatic cell's transcriptome and chromatin state to become a pluripotent cell. It has recently been revealed that cancer cells can be a new source of iPSCs production. Cancer cells show similarities with iPSCs in self-renewal capacity, reprogramming potency, and signaling pathways. Although genetic abnormalities and potential tumor formation in cancer cells pose a severe risk, reprogrammed cancer-induced pluripotent stem cells (cancer-iPSCs) indicate that pluripotency can transiently overcome the cancer phenotype. This review discusses whether cancer cells can be a preferable source to generate iPSCs.

Bone marrow mesenchymal stromal cells for diabetes therapy: touch, fuse, and fix?

Bone marrow mesenchymal stromal cells (BM-MSCs) have anti-inflammatory and pro-survival properties. Naturally, they do not express human leukocyte antigen class II surface antigens and have immunosuppressive capabilities. Together with their relatively easy accessibility and expansion, they are an attractive tool for organ support in transplantation and regenerative therapy. Autologous BM-MSC transplantation alone or together with transplanted islets improves ß-cell function, graft survival, and glycemic control in diabetes. Albeit MSCs' capacity to transdifferentiate into ß-cell is limited, their protective effects are mediated mainly by paracrine mechanisms through BM-MSCs circulating through the body. Direct cell-cell contact and spontaneous fusion of BM-MSCs with injured cells, although at a very low rate, are further mechanisms of their supportive effect and for tissue regeneration. Diabetes is a disease of long-term chronic inflammation and cell therapy requires stable, highly functional cells. Several tools and protocols have been developed by mimicking natural fusion events to induce and accelerate fusion in vitro to promote ß-cell-specific gene expression in fused cells. BM-MSC-islet fusion before transplantation may be a strategy for long-term islet survival and improved function. This review discusses the cell-protective and anti-inflammatory characteristics of BM-MSCs to boost highly functional insulin-producing cells in vitro and in vivo, and the efficacy of their fusion with ß-cells as a path to promote ß-cell regeneration.

Electrostatically induced pKa shifts in oligopeptides: the upshot of neighboring side chains.

pKa values of homorepeat hexapeptides with a 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) chromophore attached at the peptide C termini, through an asparagine derivative (Dbo), namely His6-Dbo (H6), Lys6-Dbo (K6), and Arg6-Dbo (R6), were determined by a novel fluorescence-based method. The fluorescence lifetime of Dbo in the peptides (τ) was measured as a function of pH. The side chains collide with Dbo intramolecularly and quench it efficiently only when they are deprotonated (i.e., pH ≥ side chain pKa). The pKa values of the H6, K6, and R6 peptides, attributable to side chain ionization, were found to be depressed compared to the pKa values of the His, Lys, and Arg residues in their free amino acid forms. We further looked into the structural changes of the peptides by molecular dynamics (MD) simulations; the peptides were structurally more expanded when their side chains are protonated. The structural expansion of the peptides reflects an electrostatic repulsion between the protonated side chain residues, which also accounts for the observed decrease in pKa values, which corresponds to a facilitated deprotonation, assisted by electrostatic repulsion.

Identification of lncRNAs associated with the progression of acute lymphoblastic leukemia using a competing endogenous RNAs network.

Acute lymphoblastic leukemia (ALL) is a malignancy of bone marrow lymphoid precursors. Despite effective treatments, the causes of its progression or recurrence are still unknown. Finding prognostic biomarkers is needed for early diagnosis and more effective treatment. This study was performed to identify long non-coding RNAs (lncRNAs) involved in ALL progression by constructing a competitive endogenous RNA (ceRNA) network. These lncRNAs may serve as potential new biomarkers in the development of ALL. The GSE67684 dataset identified changes in lncRNAs and mRNAs involved in ALL progression. Data from this study were re-analyzed, and probes related to lncRNAs were retrieved. Targetscan, miRTarBase, and miRcode databases were used to identify microRNAs (miRNAs) related to the discovered genes and lncRNAs. The ceRNA network was constructed, and the candidate lncRNAs were selected. Finally, the results were validated with reverse transcription quantitative real-time PCR (RT-qPCR). The ceRNA network outcomes demonstrated that the top lncRNAs associated with altered mRNAs in ALL are IRF1-AS1, MCM3AP-AS1, TRAF3IP2-AS1, HOTAIRM1, CRNDE, and TUG1. Investigations of the subnets linked to MCM3AP-AS1, TRAF3IP2-AS1, and IRF1-AS1 indicated that these lncRNAs were considerably related to pathways associated with inflammation, metastasis, and proliferation. Higher expression levels of IRF1-AS1, MCM3AP-AS1, TRAF3IP2-AS1, CRNDE, and TUG1 were found in ALL samples compared to controls. The expression of MCM3AP-AS1, TRAF3IP2-AS1, and IRF1-AS1 is significantly elevated during the progression of ALL, playing an oncogenic role. Due to their role in the main cancer pathways, lncRNAs could be suitable therapeutic and diagnostic targets in ALL.

A Newly Characterized Potentially Probiotic Strain, Lactobacillus brevis MK05, and the Toxicity Effects of its Secretory Proteins Against MCF-7 Breast Cancer Cells.

Among seven strains of lactic acid bacteria (LAB) isolated from traditional dairy products, a Lactobacillus strain was identified through 16S rRNA gene sequencing and tentatively designated as Lactobacillus brevis MK05. This strain demonstrated the highest probiotic potential through biochemical analysis, including acid and bile salt resistance, as well as antibacterial activity. The collected cell-free supernatant (CFC) of L. brevis MK05 culture, compared with MRS broth with pH equal to the pH for CFC, revealed antimicrobial activity against Escherichia coli (ATCC 25922) and Staphylococcus aureus subsp. aureus (ATCC 25923), possibly due to the presence of antibacterial metabolites other than organic acids. This strain was, therefore, selected to assess the biological activity of its partially purified secretory proteins against MCF-7 cancer cells and normal fibroblast cells via the MTT assay. The partially purified cell-secreted proteins of this strain (hereafter referred to as Lb-PPSPs) showed a time and dose-dependent anti-cancer and apoptosis induction function. There was a remarkable decline in the survival rate of MCF-7 cells at doses equal to and higher than 0.5 mg/mL after 48 h. The changes in expression of the three genes involved in the apoptosis pathway (BAX, BCL-2, and BCL2L11) in MCF-7 cells treated with the Lb-PPSPs confirm its cytotoxic activity and apoptosis induction.

Development of a novel method for the purification of C-phycocyanin pigment from a local cyanobacterial strain Limnothrix sp. NS01 and evaluation of its anticancer properties.

C-phycocyanin (C-PC) pigment, as a natural blue dye, has particular applications in various fields. It is a water-soluble protein which has anticancer, antioxidant and anti-inflammatory properties. Here, we introduce an efficient procedure for the purification of C-PC pigment, followed by conducting a comprehensive investigation of its cytotoxic effects on human breast cancer (MCF-7) cells and the underlying mechanisms. A novel four-step purification procedure including the adsorption of impurities with chitosan, activated charcoal, ammonium sulfate precipitation, and ion exchange chromatography was employed, achieving a high purity form of C-PC with purity index (PI) of 5.26. SDS-PAGE analysis showed the purified C-PC with two discrete bands, subunit α (17 kD) and ß (20 kD), as confirmed its identity by Native-PAGE. A highly purified C-PC was employed to evaluate its anticancer activity and underlying molecular mechanisms of action. The inhibitory effects of highly purified C-PC on the proliferation of human breast cancer cells (MCF-7) have detected by MTT assay. The IC50 values for 24, 48, and 72 hours of exposure to C-PC were determined to be 5.92, 5.66, and 4.52 µg/µl, respectively. Flow cytometric analysis of cells treated with C-PC, by Annexin V/PI double staining, demonstrated to induce MCF-7 cells apoptosis. Also, the results obtained from propidium iodide (PI) staining showed that MCF-7 cells treated with 5.92 µg/µl C-PC for 24 h would arrest at the G2 phase and 5.66 and 4.52 µg/µl C-PC for 48 and 72 h could induce cell cycle arrest at both G2 and S phases. The oxidative damage and mitochondrial dysfunction were evaluated to determine the possible pathways involved in C-PC-induced apoptosis in MCF-7 cells. Our findings clearly indicated that the treatment of MCF-7 cells with C-PC (IC50 for 24 h) increased the production of reactive oxygen species (ROS). Consequently, an increase in the lipid peroxidation (LPO) level and a reduction in the ATP level, mitochondrial membrane potential (MMP), glutathione (GSH) and its oxidized form (GSSG), occurred over time. The reduced expression levels of anti-apoptotic proteins, Bcl2 and Stat3, plus cell cycle regulator protein, Cyclin D1, using Real-Time PCR confirm that the C-PC-induced death of MCF-7 human breast cancer cells occurred through the mitochondrial pathway of apoptosis. Collectively, the analyses presented here suggest that C-PC has the potential so that to develop it as a chemotherapeutic anticancer drug.

Cucurbiturils as supramolecular inhibitors of DNA restriction by type II endonucleases.

Cucurbiturils (CB6 and CB7) were shown to inhibit the enzymatically catalyzed restriction of plasmids and linear DNA. This effect can be inverted by supramolecular masking of the macrocycles through competitive complexation with polyamines. These experiments provide supramolecular control of biocatalytic processes.

Indicator displacement assays inside live cells.

The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells.

Coulomb repulsion in short polypeptides.

Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge-charge repulsion, side chain functional group solvation, N-terminal and side chain charge-charge repulsion, and side chain-side chain as well as side chain-backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each other or the main chain.

Corynebacterium jeikeium jk0268 constitutes for the 40 amino acid long PorACj, which forms a homooligomeric and anion-selective cell wall channel.

Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed.

Diffusion-enhanced Förster resonance energy transfer and the effects of external quenchers and the donor quantum yield.

The structural and dynamic properties of a flexible peptidic chain codetermine its biological activity. These properties are imprinted in intrachain site-to-site distances as well as in diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two chain sites labeled with a FRET donor and acceptor. Both could be obtained from time-resolved FRET measurements if their individual contributions to the FRET efficiency could be systematically varied. Because the FRET diffusion enhancement (FDE) depends on the donor-fluorescence lifetime, it has been proposed that the FDE can be reduced by shortening the donor lifetime through an external quencher. Benefiting from the high diffusion sensitivity of short-distance FRET, we tested this concept experimentally on a (Gly-Ser)(6) segment labeled with the donor/acceptor pair naphthylalanine/2,3-diazabicyclo[2.2.2]oct-2-ene (NAla/Dbo). Surprisingly, the very effective quencher potassium iodide (KI) had no effect at all on the average donor-acceptor distance, although the donor lifetime was shortened from ca. 36 ns in the absence of KI to ca. 3 ns in the presence of 30 mM KI. We show that the proposed approach had to fail because it is not the experimentally observed but the radiative donor lifetime that controls the FDE. Because of that, any FRET ensemble measurement can easily underestimate diffusion and might be misleading even if it employs the Haas-Steinberg diffusion equation (HSE). An extension of traditional FRET analysis allowed us to evaluate HSE simulations and to corroborate as well as generalize the experimental results. We demonstrate that diffusion-enhanced FRET depends on the radiative donor lifetime as it depends on the diffusion coefficient, a useful symmetry that can directly be applied to distinguish dynamic and structural effects of viscous cosolvents on the polymer chain. We demonstrate that the effective FRET rate and the recovered donor-acceptor distance depend on the quantum yield, most strongly in the absence of diffusion, which has to be accounted for in the interpretation of distance trends monitored by FRET.