Joint Resource Allocation for Multiuser Opportunistic Beamforming Systems with OFDM-NOMA.

Opportunistic beamforming (OBF) is an effective technique to improve the spectrum efficiencies (SEs) of multiple-input-multiple-output (MIMO) systems, which can obtain multiuser diversity gains with both low computation complexity and feedback information. To serve multiple users simultaneously, many multiple-access schemes have been researched in OBF. However, for most of the multiple-access schemes, the SEs are not satisfactory. To further improve the SE, this paper proposes a downlink multiuser OBF system, where both orthogonal frequency division multiplexing (OFDM) and non-orthogonal multiple-access (NOMA) methods are applied. The closed-form expressions of the equivalent channels and SE are derived in frequency selective fading channels. Then, an optimization problem is formulated to maximize the SE, although the optimization problem is non-convex and hard to solve. To obtain the solution, we divide the optimization problem into two suboptimal issues, and then a joint iterative algorithm is applied. In the proposed optimization scheme, the subcarrier mapping ϑ, user pairing knc and allocated power Pknc are determined to maximize spectrum efficiency (SE) and reduce bit error ratio (BER). According to numerical results, the proposed method achieves approximately 5 dB gain on both SE and BER, compared to the existing beamforming methods with low feedback information. Moreover, the SE of the proposed method is approximately 2 (bps/Hz) higher than sparse code multiple-access (SCMA), when the number of waiting users and the ratio of transmit power to noise variance are respectively 10 and 20 dB. It is indicated that the proposed scheme can achieve high and low BER with the limited feedback and computation complexity, regardless of the transmit power and the number of waiting users.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation.

The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1ß or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pretreated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1ß or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1ß or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.

Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway.

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

Patching and single-strand ligation in nonhomologous DNA end joining despite persistence of a closely opposed 3'-phosphoglycolate-terminated strand break.

Previous work showed that in human nuclear extracts, double-strand break substrates bearing partially complementary (-ACG) 3'-phosphoglycolate (PG)-terminated 3' overhangs are joined by a mechanism involving annealing of the terminal CG dinucleotides, PG removal, single-base gap filling and ligation. However, in these extracts only a minority of the breaks are rejoined, and most of the 3'-PG termini remain intact even after several hours. To determine whether the presence of a persistent 3'-PG prevents patching and ligation of the opposite strand, a substrate was constructed with two -ACG overhangs, one PG-terminated and one hydroxyl-terminated. after incubation in HeLa cell nuclear extracts, two major repair products of similar yield were formed: a fully repaired duplex and a nicked duplex in which the initial 3'-PG terminus remained intact. These results indicate that patching and ligation can proceed to completion in the unmodified strand despite persistence of the 3'-PG-terminated break in the opposite strand. The break in the PG-containing strand could then presumably be rejoined by a single-strand break repair pathway.

Loop 1 modulates the fidelity of DNA polymerase lambda.

Differences in the substrate specificity of mammalian family X DNA polymerases are proposed to partly depend on a loop (loop 1) upstream of the polymerase active site. To examine if this is the case in DNA polymerase λ (pol λ), here we characterize a variant of the human polymerase in which nine residues of loop 1 are replaced with four residues from the equivalent position in pol ß. Crystal structures of the mutant enzyme bound to gapped DNA with and without a correct dNTP reveal that the change in loop 1 does not affect the overall structure of the protein. Consistent with these structural data, the mutant enzyme has relatively normal catalytic efficiency for correct incorporation, and it efficiently participates in non-homologous end joining of double-strand DNA breaks. However, DNA junctions recovered from end-joining reactions are more diverse than normal, and the mutant enzyme is substantially less accurate than wild-type pol λ in three different biochemical assays. Comparisons of the binary and ternary complex crystal structures of mutant and wild-type pol λ suggest that loop 1 modulates pol λ's fidelity by controlling dNTP-induced movements of the template strand and the primer-terminal 3'-OH as the enzyme transitions from an inactive to an active conformation.

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase lambda in human nuclear extracts.

Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3' overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase lambda. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or -GTA) 3' overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote error-prone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase lambda was replaced with that of polymerase beta, could not substitute for polymerase lambda in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.

Coordinate 5' and 3' endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase.

Previous work showed that, in the presence of DNA-dependent protein kinase (DNA-PK), Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5'-->3' exonucleolytic resection of double-stranded DNA. This resection required a 5'-phosphate, but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent and did not require a 5'-phosphate. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nt from the 5'-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase-mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.

Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts.

Previous work suggested that phosphorylation of DNA-PKcs at several serine/threonine (S/T) residues at positions 2609-2647 promotes DNA-PK-dependent end joining. In an attempt to clarify the role of such phosphorylation, end joining was examined in extracts of DNA-PKcs-deficient M059J cells. Joining of ends requiring gap filling prior to ligation was completely dependent on complementation of these extracts with exogenous DNA-PKcs. DNA-PKcs with either S/T --> A or S/T --> D substitutions at all six sites in the 2609-2647 cluster also supported end joining, but with markedly lower efficiency than wild-type protein. The residual end joining was greater with the S/T --> D-substituted than with the S/T --> A-substituted protein. A specific inhibitor of the kinase activity of DNA-PK, KU57788, completely blocked end joining promoted by wild type as well as both mutant forms of DNA-PK, while inhibition of ATM kinase did not. The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors. Overall, the results support a role for autophosphorylation of the 2609-2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609-2647 cluster in regulating end joining.

Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease.

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.

The growth arrest function of the human oncoprotein mouse double minute-2 is disabled by downstream mutation in cancer cells.

We have reported earlier that ectopic expression of mouse double minute-2 (MDM2) induces G1 arrest in normal cells. To explain occasional overexpression of MDM2 in cancer cells, we searched for deletion or substitution mutation in the growth suppressor domains of MDM2 in several breast cancer cell lines that overexpress the oncoprotein. Our results suggest the absence of alteration (deletion or substitution) in the open reading frame of MDM2 transcripts in such cells. Because the breast cancer cell line MCF-7 overexpresses MDM2, we isolated the full-length MDM2 transcript from this cell line. The MDM2 cDNA synthesized from transcripts isolated from MCF-7 cells induced inhibition of G1 to S phase transition in normal human diploid cells such as WI38, suggesting that the genetic alterations in breast cancer cells that overexpress MDM2 disable the growth arrest function of the oncoprotein. Consistently, overexpression of full-length MDM2 in MCF-7 cells over its high endogenous level did not inhibit G1-S transition efficiently. Although MDM2 overexpression was accompanied by CDK4 overexpression or absence of cdk4 inhibitor p16 in most breast cancer cells, we found remarkably high levels of cyclin A rather than cyclin E in these cells. Ectopic expression of cyclin A released MDM2-mediated inhibition of G1-S transition in normal human diploid WI38 cells. We propose that cancer cells expressing high levels of cyclin A escape MDM2-mediated G1 arrest, which may account for a selective growth advantage over normal cells.