Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

Conjugation of chemical handles and functional moieties to DNA during solid phase synthesis with sulfonyl azides

Labelling of oligonucleotides with dyes, targeting ligands, and other moieties has become ever more essential in life-sciences. Conventionally, modifications are introduced to oligonucleotides during solid phase synthesis by special phosphoramidites functionalised with a chemical handle or the desired functional group. In this work, we present a facile and inexpensive method to introduce modifications to oligonucleotides without the need for special phosphoramidites. Sulfonyl azides are applied to react with one or more selected phosphite intermediates during solid phase synthesis. We have prepared 11 sulfonyl azides with different chemical handles such as amine, azide, alkyne, and thiol, and we have further introduced functionalities such as pyrene, other dyes, photo-switchable azobenzenes, and a steroid. The method is compatible with current phosphoramidite-based automated oligonucleotide synthesis and serves as a simple alternative to the unstable and expensive special phosphoramidites currently used for conjugation to oligonucleotides.     

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.     

AN ANALYSIS OF LEAF ELONGATION IN XANTHIUM PENSYLVANICUM PRESENTED IN RELATIVE ELEMENTAL RATES

Maksymowych , Roman . (Villanova U., Villanova, Pa.) An analysis of leaf elongation in Xanthium pensylvanicum presented in relative elemental rates . Amer. Jour. Bot. 49(1): 7–13. Illus. 1962.—Xanthium plants were grown vegetatively, and leaves, whose developmental stages were specified by a previously described leaf plastochron index (L.P.I.), were marked with India ink along the midrib and photographed during 3 successive days. The relative elemental rates of elongation, d(dX/dpl)/dX were estimated during the whole course of development. The pattern of elongation was not constant but was changing with increasing plastochron age of the leaf. The elements of a young leaf of L.P.I. 0.75 elongated with a constant relative rate. In older leaves, the d(dX/dpl)/dX values were progressively declining toward the tip of the lamina. After L.P.I. 6.3 the only increment in length was due to the elongation of the elements of the petiole. The pattern of growth distribution is discussed in terms of relative elemental rates with respect to cell division and cell elongation in various portions of the lamina and is correlated with the basipetal trend of tissue differentiation in the developing Xanthium leaf.     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.     

ACE16k: a 128x128 focal plane analog processor with digital I/O

This paper presents a new generation 128x128 Focal-Plane Analog Programmable Array Processor -FPAPAP, from a system level perspective. It has been manufactured in a 0.35 microm standard digital 1P-5M CMOS technology. It has been designed to achieve the high-speed and moderate-accuracy -8b- requirements of most real time -early-vision applications. External data interchange and control are completely digital. The chip contains close to four million transistors, 90% of them working in analog mode. It achieves peak computing values of 0.33TeraOPS while keeping power consumption at reasonable limits -82.5GOPS/W. Preliminary experimental results are also provided in the paper.