HK3 overexpression associated with epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a common cancer worldwide. The main cause of death in CRC includes tumor progression and metastasis. At molecular level, these processes may be triggered by epithelial-mesenchymal transition (EMT) and necessitates specific alterations in cell metabolism. Although several EMT-related metabolic changes have been described in CRC, the mechanism is still poorly understood. RESULTS: Using CrossHub software, we analyzed RNA-Seq expression profile data of CRC derived from The Cancer Genome Atlas (TCGA) project. Correlation analysis between the change in the expression of genes involved in glycolysis and EMT was performed. We obtained the set of genes with significant correlation coefficients, which included 21 EMT-related genes and a single glycolytic gene, HK3. The mRNA level of these genes was measured in 78 paired colorectal cancer samples by quantitative polymerase chain reaction (qPCR). Upregulation of HK3 and deregulation of 11 genes (COL1A1, TWIST1, NFATC1, GLIPR2, SFPR1, FLNA, GREM1, SFRP2, ZEB2, SPP1, and RARRES1) involved in EMT were found. The results of correlation study showed that the expression of HK3 demonstrated a strong correlation with 7 of the 21 examined genes (ZEB2, GREM1, TGFB3, TGFB1, SNAI2, TWIST1, and COL1A1) in CRC. CONCLUSIONS: Upregulation of HK3 is associated with EMT in CRC and may be a crucial metabolic adaptation for rapid proliferation, survival, and metastases of CRC cells.

Near-infrared fluorescent proteins.

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

Application of PCR-based approaches for evaluation of cell-free DNA fragmentation in colorectal cancer.

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination.

The Detection of Cancer Epigenetic Traces in Cell-Free DNA.

Nucleic acid fragments found in blood circulation originate mostly from dying cells and carry signs pointing to specific features of the parental cell types. Deciphering these clues may be transformative for numerous research and clinical applications but strongly depends on the development and implementation of robust analytical methods. Remarkable progress has been achieved in the reliable detection of sequence alterations in cell-free DNA while decoding epigenetic information from methylation and fragmentation patterns requires more sophisticated approaches. This review discusses the currently available strategies for detecting and analyzing the epigenetic marks in the liquid biopsies.

Far-red fluorescent tags for protein imaging in living tissues.

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Breast cancer organoid model allowed to reveal potentially beneficial combinations of 3,3'-diindolylmethane and chemotherapy drugs.

Epigenetic alterations represent promising therapeutic targets in cancer treatment. Recently it was revealed that small molecules have the potential to act as microRNA silencers. Capacity to bind the discrete stem-looped structure of pre-miR-21 and prevent its maturation opens opportunities to utilize such compounds for the prevention of initiation, progression, and chemoresistance of cancer. Molecular simulations performed earlier identified 3,3'-diindolylmethane (DIM) as a potent microRNA-21 antagonist. However, data on DIM and microRNA-21 interplay is controversial, which may be caused by the limitations of the cell lines.

Practical and reliable FRET/FLIM pair of fluorescent proteins.

BACKGROUND: In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. RESULTS: Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter). CONCLUSION: The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.

Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing.

Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends.

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs.

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

Exome analysis of carotid body tumor.

BACKGROUND: Carotid body tumor (CBT) is a form of head and neck paragangliomas (HNPGLs) arising at the bifurcation of carotid arteries. Paragangliomas are commonly associated with germline and somatic mutations involving at least one of more than thirty causative genes. However, the specific functionality of a number of these genes involved in the formation of paragangliomas has not yet been fully investigated. METHODS: Exome library preparation was carried out using Nextera® Rapid Capture Exome Kit (Illumina, USA). Sequencing was performed on NextSeq 500 System (Illumina). RESULTS: Exome analysis of 52 CBTs revealed potential driver mutations (PDMs) in 21 genes: ARNT, BAP1, BRAF, BRCA1, BRCA2, CDKN2A, CSDE1, FGFR3, IDH1, KIF1B, KMT2D, MEN1, RET, SDHA, SDHB, SDHC, SDHD, SETD2, TP53BP1, TP53BP2, and TP53I13. In many samples, more than one PDM was identified. There are also 41% of samples in which we did not identify any PDM; in these cases, the formation of CBT was probably caused by the cumulative effect of several not highly pathogenic mutations. Estimation of average mutation load demonstrated 6-8 mutations per megabase (Mb). Genes with the highest mutation rate were identified. CONCLUSIONS: Exome analysis of 52 CBTs for the first time revealed the average mutation load for these tumors and also identified potential driver mutations as well as their frequencies and co-occurrence with the other PDMs.

In vivo imaging of ligand receptor binding with Gaussia luciferase complementation.

Studies of ligand-receptor binding and the development of receptor antagonists would benefit greatly from imaging techniques that translate directly from cell-based assays to living animals. We used Gaussia luciferase protein fragment complementation to quantify the binding of chemokine (C-X-C motif) ligand 12 (CXCL12) to chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. Studies established that small-molecule inhibitors of CXCR4 or CXCR7 specifically blocked CXCL12 binding in cell-based assays and revealed differences in kinetics of inhibiting chemokine binding to each receptor. Bioluminescence imaging showed CXCL12-CXCR7 binding in primary and metastatic tumors in a mouse model of breast cancer. We used this imaging technique to quantify drug-mediated inhibition of CXCL12-CXCR4 binding in living mice. We expect this imaging technology to advance research in areas such as ligand-receptor interactions and the development of new therapeutic agents in cell-based assays and small animals.

A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore.

The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(em), 635 nm; chromophore-forming triad Met(63)-Tyr(64)-Gly(65)), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 A. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser(143), Leu(174), and Arg(197) residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val(93), Arg(122), Glu(155), Arg(157), Asp(159), His(169), Ile(171), Asn(173), Val(192), Tyr(194), and Val(216), are most likely responsible for the observed monomeric state of the protein in solution.

Bright monomeric red fluorescent protein with an extended fluorescence lifetime.

Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.

Bright far-red fluorescent protein for whole-body imaging.

For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.