Identification of novel dysregulated circular RNAs in early-stage breast cancer

Breast cancer is a major cause of cancer-related death in women worldwide. Non-coding RNAs are a potential resource to be used as an early diagnostic biomarker for breast cancer. Circular RNAs are a recently identified group of non-coding RNA with a significant role in disease development with potential utility in diagnosis/prognosis in cancer. In this study, we identified 26 differentially expressed circular RNAs associated with early-stage breast cancer. RNA sequencing and two circRNA detection tools (find_circ and DCC) were used to understand the circRNA expression signature in breast cancer. We identified hsa_circ_0006743 (circJMJD1C) and hsa_circ_0002496 (circAPPBP1) to be significantly up-regulated in early-stage breast cancer tissues. Co-expression analysis identified four pairs of circRNA-miRNA (hsa_circ_0023990 : hsa-miR-548b-3p, hsa_circ_0016601 : hsa_miR-1246, hsa_circ_0001946 : hsa-miR-1299 and hsa_circ_0000117:hsa-miR-502-5p) having potential interaction. The miRNA target prediction and network analysis revealed mRNA possibly regulated by circRNAs. We have thus identified circRNAs of diagnostic implications in breast cancer and also observed circRNA-miRNA interaction which could be involved in breast cancer development.     

Hagenia from the early Miocene of Ethiopia: Evidence for possible niche evolution?

Fossil pollen believed to be related to extant Hagenia abyssinica were discovered in the early Miocene (21.73 Ma) Mush Valley paleoflora, Ethiopia, Africa. Both the fossil and extant pollen grains of H. abyssinica were examined with combined light microscopy, scanning electron microscopy, and transmission electron microscopy to compare the pollen and establish their relationships. Based on this, the fossil pollen grains were attributed to Hagenia. The presence of Hagenia in the fossil assemblage raises the questions if its habitat has changed over time, and if the plants are/were wind pollinated. To shed light on these questions, the morphology of extant anthers was also studied, revealing specialized hairs inside the anthers, believed to aid in insect pollination. Pollen and anther morphology are discussed in relation to the age and origin of the genus within a molecular dated phylogenetic framework, the establishment of complex topography in East Africa, other evidence regarding pollination modes, and the palynological record. The evidence presented herein, and compiled from the literature, suggests that Hagenia was an insect‐pollinated lowland rainforest element during the early Miocene of the Mush Valley. The current Afromontane habitat and ambophilous (insect and wind) pollination must have evolved in post‐mid‐Miocene times.     

Cell Squeezing as a Robust,Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.     

The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks

Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.     

ARF1 controls Rac1 signaling to regulate migration of MDA-MB-231 invasive breast cancer cells

ADP-ribosylation factors (ARFs) are monomeric G proteins that regulate many cellular processes such as reorganization of the actin cytoskeleton. We have previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced migration. In this study, we propose to define the molecular mechanism by which ARF1 regulates this complex cellular response by investigating the role of this ARF GTPase on the activation process of Rac1, a Rho GTPase, associated with lamellipodia formation during cell migration. Here, we first show that inhibition of ARF1 or Rac1 expression markedly impacts the ability of MDA-MB-231 cells to migrate upon EGF stimulation. However, the effect of ARF1 depletion can be reversed by overexpression of the Rac1 active mutant, Rac1 Q⁶¹L. Depletion of ARF1 also impairs the ability of EGF stimulation to promote GTP-loading of Rac1. To further investigate the possible cross-talk between ARF1 and Rac1, we next examined whether they could form a complex. We observed that the two GTPases could directly interact independently of the nature of the nucleotide bound to them. EGF treatment however resulted in the association of Rac1 with its effector IRSp53, which was completely abrogated in ARF1 depleted cells. We present evidences that this ARF isoform is responsible for the plasma membrane targeting of both Rac1 and IRSp53, a step essential for lamellipodia formation. In conclusion, this study provides a new mechanism by which ARF1 regulates cell migration and identifies this GTPase as a promising pharmacological target to reduce metastasis formation in breast cancer patients.     

Dynamic stability of a human standing on a balance board

The neuromuscular system used to stabilize upright posture in humans is a nonlinear dynamical system with time delays. The analysis of this system is important for improving balance and for early diagnosis of neuromuscular disease. In this work, we study the dynamic coupling between the neuromuscular system and a balance board—an unstable platform often used to improve balance in young athletes, and older or neurologically impaired patients. Using a simple inverted pendulum model of human posture on a balance board, we describe a surprisingly broad range of divergent and oscillatory CoP/CoM responses associated with instabilities of the upright equilibrium. The analysis predicts that a variety of sudden changes in the stability of upright postural equilibrium occurs with slow continuous deterioration in balance board stiffness, neuromuscular gain, and time delay associated with the changes in proprioceptive/vestibular/visual-neuromuscular feedback. The analysis also provides deeper insight into changes in the control of posture that enable stable upright posture on otherwise unstable platforms.     

Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.     

Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin I-converting enzyme

The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.