A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 Helicase to interact with RAD51 Recombinase for genome stabilization

The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.

Tunable “Nano-Shearing” : A physical mechanism to displace nonspecific cell adhesion during rare cell detection

We report a tunable alternating current electrohydrodynamic (ac-EHD) force which drives lateran fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over onspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific dsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.

PRMT2 and RORγ expression are associated with breast cancer survival outcomes

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Advanced mass spectrometry-based multi-omics technologies for exploring the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the primary hepatic malignancies and is the third most common cause of cancer related death worldwide. Although a wealth of knowledge has been gained concerning the initiation and progression of HCC over the last half century, efforts to improve our understanding of its pathogenesis at a molecular level are still greatly needed, to enable clinicians to enhance the standards of the current diagnosis and treatment of HCC. In the post-genome era, advanced mass spectrometry driven multi-omics technologies (e.g., profiling of DNA damage adducts, RNA modification profiling, proteomics, and metabolomics) stand at the interface between chemistry and biology, and have yielded valuable outcomes from the study of a diversity of complicated diseases. Particularly, these technologies are being broadly used to dissect various biological aspects of HCC with the purpose of biomarker discovery, interrogating pathogenesis as well as for therapeutic discovery. This proof of knowledge-based critical review aims at exploring the selected applications of those defined omics technologies in the HCC niche with an emphasis on translational applications driven by advanced mass spectrometry, toward the specific clinical use for HCC patients. This approach will enable the biomedical community, through both basic research and the clinical sciences, to enhance the applicability of mass spectrometry-based omics technologies in dissecting the pathogenesis of HCC and could lead to novel therapeutic discoveries for HCC.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

Development of reverse phase protein microarrays for clinical applications and patient-tailored therapy

While genomics provide important information about the somatic genetic changes, and RNA transcript profiling can reveal important expression changes that correlate with outcome and response to therapy, it is the proteins that do the work in the cell. At a functional level, derangements within the proteome, driven by post-translational and epigenetic modifications, such as phosphorylation, is the cause of a vast majority of human diseases. Cancer, for instance, is a manifestation of deranged cellular protein molecular networks and cell signaling pathways that are based on genetic changes at the DNA level. Importantly, the protein pathways contain the drug targets in signaling networks that govern overall cellular survival, proliferation, invasion and cell death. Consequently, the promise of proteomics resides in the ability to extend analysis beyond correlation to causality. A critical gap in the information knowledge base of molecular profiling is an understanding of the ongoing activity of protein signaling in human tissue: what is activated and “in use” within the human body at any given point in time. To address this gap, we have invented a new technology, called reverse phase protein microarrays, that can generate a functional read-out of cell signaling networks or pathways for an individual patient obtained directly from a biopsy specimen. This “wiring diagram” can serve as the basis for both, selection of a therapy and patient stratification.

Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

Drosophila 3′ UTRs are more complex than protein-coding sequences

The 3′ UTRs of eukaryotic genes participate in a variety of post-transcriptional (and some transcriptional) regulatory interactions. Some of these interactions are well characterised, but an undetermined number remain to be discovered. While some regulatory sequences in 3′ UTRs may be conserved over long evolutionary time scales, others may have only ephemeral functional significance as regulatory profiles respond to changing selective pressures. Here we propose a sensitive segmentation methodology for investigating patterns of composition and conservation in 3′ UTRs based on comparison of closely related species. We describe encodings of pairwise and three-way alignments integrating information about conservation, GC content and transition/transversion ratios and apply the method to three closely related Drosophila species: D. melanogaster, D. simulans and D. yakuba. Incorporating multiple data types greatly increased the number of segment classes identified compared to similar methods based on conservation or GC content alone. We propose that the number of segments and number of types of segment identified by the method can be used as proxies for functional complexity. Our main finding is that the number of segments and segment classes identified in 3′ UTRs is greater than in the same length of protein-coding sequence, suggesting greater functional complexity in 3′ UTRs. There is thus a need for sustained and extensive efforts by bioinformaticians to delineate functional elements in this important genomic fraction. C code, data and results are available upon request.

Failure of amino acid homeostasis causes cell death following proteasome inhibition

The ubiquitin-proteasome system targets many cellular proteins for degradation and thereby controls most cellular processes. Although it is well established that proteasome inhibition is lethal, the underlying mechanism is unknown. Here, we show that proteasome inhibition results in a lethal amino acid shortage. In yeast, mammalian cells, and flies, the deleterious consequences of proteasome inhibition are rescued by amino acid supplementation. In all three systems, this rescuing effect occurs without noticeable changes in the levels of proteasome substrates. In mammalian cells, the amino acid scarcity resulting from proteasome inhibition is the signal that causes induction of both the integrated stress response and autophagy, in an unsuccessful attempt to replenish the pool of intracellular amino acids. These results reveal that cells can tolerate protein waste, but not the amino acid scarcity resulting from proteasome inhibition.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Determination of strain-rate-dependent mechanical behavior of living and fixed osteocytes and chondrocytes using atomic force microscopy and inverse finite element analysis

The aim of this paper is to determine the strain-rate-dependent mechanical behavior of living and fixed osteocytes and chondrocytes, in vitro. Firstly, Atomic Force Microscopy (AFM) was used to obtain the force-indentation curves of these single cells at four different strain-rates. These results were then employed in inverse finite element analysis (FEA) using Modified Standard neo-Hookean Solid (MSnHS) idealization of these cells to determine their mechanical properties. In addition, a FEA model with a newly developed spring element was employed to accurately simulate AFM evaluation in this study. We report that both cytoskeleton (CSK) and intracellular fluid govern the strain-rate-dependent mechanical property of living cells whereas intracellular fluid plays a predominant role on fixed cells’ behavior. In addition, through the comparisons, it can be concluded that osteocytes are stiffer than chondrocytes at all strain-rates tested indicating that the cells could be the biomarker of their tissue origin. Finally, we report that MSnHS is able to capture the strain-rate-dependent mechanical behavior of osteocyte and chondrocyte for both living and fixed cells. Therefore, we concluded that the MSnHS is a good model for exploration of mechanical deformation responses of single osteocytes and chondrocytes. This study could open a new avenue for analysis of mechanical behavior of osteocytes and chondrocytes as well as other similar types of cells.

Delivery of nitric oxide for analysis of the function of cytochrome c'

On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.

The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event

In this paper, the complete mitochondrial genome of Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini) is reported; a circular molecule of 15,245 bp in size. For A. issoria, genes are arranged in the same order and orientation as the complete sequenced mitochondrial genomes of the other lepidopteran species, except for the presence of an extra copy of tRNAIle(AUR)b in the control region. All protein-coding genes of A. issoria mitogenome start with a typical ATN codon and terminate in the common stop codon TAA, except that COI gene uses TTG as its initial codon and terminates in a single T residue. All tRNA genes possess the typical clover leaf secondary structure except for tRNASer(AGN), which has a simple loop with the absence of the DHU stem. The sequence, organization and other features including nucleotide composition and codon usage of this mitochondrial genome were also reported and compared with those of other sequenced lepidopterans mitochondrial genomes. There are some short microsatellite-like repeat regions (e.g., (TA)9, polyA and polyT) scattered in the control region, however, the conspicuous macro-repeats units commonly found in other insect species are absent.