Two specialized alternative rescue pathways, coordinated by ArfA and ArfB, have been recently discovered. The SmpB-tmRNA mediated trans-translation pathway, in addition to re-mobilizing stalled translation complexes, co-translationally appends a degradation tag to the associated nascent polypeptides, marking them for proteolysis by various cellular proteases. Another unique feature of trans-translation, not shared by the alternative rescue pathways, is the facility to recruit ribonuclease R (RNase R) for targeted degradation of non-stop mRNAs, thus preventing further futile cycles of translation. The distinct C-terminal lysine-rich (K-rich) domain of RNase R is essential for its recruitment to stalled ribosomes. To gain new insights into the structure and function of RNase R, we investigated its global architecture, the spatial arrangement of its distinct domains, and the identities of key functional residues in its unique K-rich domain. Small-angle X-ray scattering models of RNase R reveal a tri-lobed structure with flexible N- and C-terminal domains, and suggest intimate contacts between the K-rich domain and the catalytic core of the enzyme. Alanine-scanning mutagenesis of the K-rich domain, in the region spanning residues 735 and 750, has uncovered the precise amino acid determinants required for the productive engagement of RNase R on tmRNA-rescued ribosomes. Theses analyses demonstrate that alanine substitution of conserved residues E740 and K741result in profound defects, not only in the recruitment of RNase R to rescued ribosomes but also in the targeted decay of non-stop mRNAs. Additionally, an RNase R variant with alanine substitution at residues K749 and K750 exhibits extensive defects in ribosome enrichment and non-stop mRNA decay. In contrast, alanine substitution of additional conserved residues in this region has no effect on the known functions of RNase R. In vitro RNA degradation assays demonstrate that the consequential substitutions (RNase RE740A/K741A and RNase RK749A/K750A) do not affect the ability of the enzyme to degrade structured RNAs, indicating that the observed defect is specific to the trans-translation related activities of RNase R. Taken together, these findings shed new light on the global architecture of RNase R and provide new details of how this versatile RNase effectuates non-stop mRNA decay on tmRNA-rescued ribosomes.Decoding of aberrant mRNAs leads to unproductive ribosome stalling and sequestration of components of the translation machinery. Bacteria have evolved three seemingly independent pathways to resolve stalled translation complexes. The  RNase R process  and play process , the Smp process . Despite process .trans-translation related activities. In this study, we used small-angle X-ray scattering (SAXS) techniques to determine the global solution structure of RNase R. Our analysis reveals the overall architecture of RNase R, the high degree of similarity of its core domain to RNase II, and the relative spatial positioning of the flexible N- and C-terminal domains with respect to its catalytic core. Additionally, systematic mutational and biochemical analyses of the K-rich domain of RNase R highlight the importance of residues E740–K741 and K749–K750 in engaging tmRNA-rescued ribosomes and facilitating selective degradation of non-stop mRNAs.While the crystal structure of RNase II has revealed its structural organization at atomic resolution , informaEscherichica coli strain MG1655 rnr::kan was obtained by P1 transduction using the Keio rnr::kan disruption strain (cI-NS), which encodes for an N-terminal His6 epitope but lacks in-frame stop codons, served as the source of the non-stop reporter mRNA. A control reporter plasmid (pλ-cI-S), which encodes for an N-terminal His6 epitope and has two tandem in-frame stop codons, served as the source of the “normal” or stop codon containing reporter mRNA. A pACYCDuet-1 plasmid (prnr) harboring the E. colirnr gene under the control of the arabinose inducible PBAD promoter was used as a template to synthesize RNase R variants by site directed mutagenesis. For purification of RNase R and its variants, the coding region of E. colirnr genewas cloned into the MCS of pET15b under the control of isopropyl β-D-thiogalactoside (IPTG)-inducible T7 promoter. This plasmid was used as a template to synthesize RNase R variants by site directed mutagenesis. The plasmids were transformed into W3110 (DE3) rnrrnb strain for over-expression and purification of the corresponding RNase R variants, as previously described  using a Pilatus 300K located 3.4 m from the sample for small-angle X-ray data. Wide-angle X-ray scattering data were collected simultaneously with SAXS data using a Photonic Science CCD camera located 0.47 m from the sample. Twenty microliters of sample were continuously flowed through a 1 mm diameter capillary and exposed to a 400 μm × 200 μm X-ray beam with a wavelength of 0.9184 Å for 30 s of total measurement time. Scattering data were collected at concentrations of 2.2, 3.7, 4.2, 5.0, and 10 mg/mL for RNase RWT and at concentrations of 0.7, 2.8, 3.0, 3.5, and 6.0 mg/mL for RNase R723. Each concentration was measured in triplicate. Normalization for beam intensity, buffer subtraction, and merging of data from both detectors were carried out using PRIMUS (Rg) was calculated using a Guinier approximation, I(q)=I(0)exp(-q2 Rg2 /3), where a plot of I(q) and q2 is linear for q <1.3/Rg (P(r), and maximum particle dimension, Dmax (I(0), is the theoretical scattering at a q value of 0 and is proportional to the molecular weight of the sample (I(0)/c, where c is sample concentration, was identical for all RNase R measurements. For RNase R723, ten independent ab initio beads models that describe the experimental data were calculated using DAMMIN  of 0.71 Å, were subsequently aligned, averaged, and filtered by occupancy using the DAMAVER suite of programs , 250 mM KCl, and 1 mM DTT within 24 h of data acquisition and stored at 4g PRIMUS . The rad <1.3/Rg . Three ion, Dmax . The line sample . I(0)/c,g DAMMIN . The resprograms . An elecprograms . For RNang BUNCH .Escherichica coli strain MG1655 rnr::kan harboring the reporter plasmid pλ-cI-NS and the various prnr variants were grown at 37°C in Luria-Bertani (LB) broth containing 0.01% arabinose, 100 μg/ml of Ampicillin, and 30 μg/ml of Chloramphenicol. At an optical density at 600 nm (OD600) of ~0.6, expression of the λ-cI-NS reporter mRNA was induced with a final concentration of 1 mM IPTG for 1 h. Equal number of cells were harvested and total RNA was isolated using TRI Reagent (MRC Inc.). To test the activity of RNase R variants, in vitro RNA degradation assays were performed as previously described (trpAt-A10: 5′-GCAGCCCGCCUAAUGAGCGGGCAAAAAAAAAA-3′). Reaction mixtures containing the 32P-labeled trpAt-A10 substrate and the desired RNase R variants were assembled and incubated at 37°C. Aliquots of 5 μl were taken at indicated time points and quenched by the addition of 5 μl of the formaldehyde gel-loading dye. Full-descriptions of the in vivo reporter mRNA stability and ribosome enrichment assays have been described elsewhere  and lysed by French Press. Ribosomes were pelleted from cleared cell lysates by centrifugation at 29,000 rpm for 16–18 h through a 32% sucrose cushion in Buffer E. Ribosomes translating the reporter mRNAs were purified from tight-coupled ribosomes by Ni2+-NTA affinity chromatography. The ribosome-associated RNase R was detected by Western blot analysis using polyclonal anti-RNase R antibody. Protein bands were quantified using ImageJ.escribed . The olilsewhere . A briefWT). Our results provide secondary structure predictions for the N- and C-terminal domains of RNase R  that are in good agreement with previous studies  support the notion that RNase R has a structurally conserved core domain that is highly similar to the known crystal structure of RNase II . Additional density is apparent near the N-terminus that most likely corresponds to residues 1–65 of RNase R. Having confirmed that the solution structure of RNase R displays a conserved molecular shape with RNase II, we collected SAXS data on RNase RWT, and carried out rigid-body modeling with the conserved core domain of RNase R. We combined the core domain modeling with ab initio C-α bead modeling for the missing N- and C-terminal domains using BUNCH . The resulting SAXS model of RNase RWT reveals a tri-lobed structure that is somewhat elongated, and is consistent with the presence of extensive flexible loops, as predicted by homology modeling. The scattering patterns from RNase R723 and RNase RWT  were quite similar, reflecting their largely analogous topologies, and the SAXS model of the N-terminal and core domains of RNase RWT correlated well with the overall shape of the RNase R723 envelope. Both models demonstrate an excellent fit to the raw scattering data , with discrepancies (χ2) of 0.68 for RNase R723 and 1.43 for RNase RWT. Visual comparison of the two models reveals the relative positioning of the K-rich C-terminal lobe with respect to the core and N-terminal domains. Both the I-TASSER and SAXS models imply intimately close links between the catalytic core and the K-rich domains of RNase R, suggesting a potential regulatory role for the K-rich domain. However, the extent of such contacts and the identity of functionally important amino acid residues in the K-rich domain that participate in any potential intra- or inter-molecular interactions have not been determined.To gain insight into the overall 3D architecture and relative domain positioning of RNase R, we determined its solution structure at low resolution using SAXS techniques. As a preliminary step, and as a complement to the low resolution SAXS data, we used the I-TASSER platform , employi studies . The thrRNase II . The I-T750) had no effect on the trans-translation related activity of RNase R (735), resulted in a discernible defect in the ability of the enzyme to degrade non-stop mRNA. RNase R truncation variant RNase R723, which lacks the entire K-rich region, exhibited a similar pronounced defect in degradation of non-stop mRNA . To ensure evaluation of a non-stop mRNA specific process, we utilized two related versions of this reporter , a non-stop transcript lacking in-frame stop codons (λ-cI-NS), and a “normal” control transcript possessing an in-frame stop codon (λ-cI-S). The λ-cI-NS non-stop transcript has been well characterized and is known to promote ribosome stalling at its 3′ end, whereas the stop codon containing λ-cI-S variant is readily translated and promotes efficient recycling of the translation machinery . Similarly, single and double alanine substitutions at residues K743 and K744 had no effect on the steady state level of the non-stop reporter mRNA . Alanine substitution at E740 or K741, either individually or in combination with other neighboring residues (RNase RR739A/E740A and RNase RK741A/K742A), resulted in measurable increases in the accumulation of non-stop mRNA , indicating a functional role for these adjoining residues in the mRNA decay activity of RNase R. To further investigate their functional contributions, we generated RNase RE740A/K741A, a double alanine substitution variant at residues E740 and K741. Assessment of RNase RE740A/K741A revealed profound defects in non-stop mRNA degradation . Alanine substitution of two additional conserved residues (K749 and K750) in the region between 735 and 750 (RNase RK749A/K750A) resulted in extensive accumulation of non-stop mRNA, suggesting that these residues also played an important role in the selective non-stop mRNA decay activity of RNase R . Collectively, these data implied that conserved residues E740–K741 and K749–K750 play a crucial role in the trans-translation mediated decay of non-stop mRNA.We expressed each RNase R alanine substitution variant alongside the λ-cI-NS reporter mRNA, and measured the relative steady state abundance of the non-stop transcript. For all mRNA stability assessments, we used RNase R variant  as a negT737A/R739A, RNase RE740A/K741A, and RNase RK749A/K750A) for further evaluation. First, we examined the relative differences in the steady state abundance of non-stop mRNA among these variants, using RNase RWT and RNase R723 as controls . This analysis showed no significant difference in the ability of RNase RWT and RNase RT737A/R739A in the selective degradation of the λ-cI-NS non-stop mRNA. In contrast, alanine substitution variants RNase RE740A/K741A and RNase RK749A/K750A consistently exhibited a 2.5 to 3-fold defect in degradation of the λ-cI-NS reporter; a defect that was statistically significant and similar to the defect exhibited by RNase R723 . To eliminate the possibility that the observed non-stop mRNA stability defects of these RNase R variants were due to difference in their relative abundance, we compared their steady state levels to RNase RWT and RNase R723 under identical experimental conditions. This analysis revealed no significant differences in the steady state levels of these RNase R variants , suggesting expression or stability difference could account for the observed non-stop mRNA decay defects exhibited by these RNase R variants.To obtain a more quantitative measure of the non-stop mRNA degradation defect of the alanine substitution mutants, we selected three double-alanine substitution variants  of the λ-cI-NS non-stop mRNA in the presence of RNase RWT, RNase RE740A/K741A, and RNase RK749A/K750A . Consistent with previous studies, RNase RWT degraded the λ-cI-NS report transcript very efficiently, with a t1/2 of 0.8 ± 0.3 min . RNase RE740A/K741A had a strong defect in the non-stop mRNA degradation, increasing t1/2 by roughly 4-fold to 3.2 ± 0.4 min . We found this deficiency in non-stop mRNA decay reminiscent of the defect observed with the RNase R723 truncation variant. Similarly, in the presence of RNase RK749A/K750A, we detected a substantial increase in the stability of the non-stop reporter mRNA with a t1/2 of 2.0 ± 0.1 min . Based on these findings, we conclude that residues E740–K741 and K749–K750 in the K-rich domain of RNase R play important but distinct roles in the selective degradation of non-stop mRNA.Next, we measured the E740A/K741A and RNase RK749A/K750A could be that these residues (K740–K741 and K749–K750) interact directly with the catalytic core of RNase R to modulate its ribonuclease activity. To assess this possibility, we examined the exoribonuclease activity of each alanine substitution variant in an in vitro RNA degradation assay. To this end, we overexpressed RNase RWT, RNase RE740A/K741A, and RNase RK749A/K750A, in an rnr- rnb- strain, and purified each protein to near homogeneity . The ribonuclease activity of each purified enzyme was assessed by its ability to degrade the trpAt-A10 substrate, a 32-nucleotide long structured RNA containing the trpA-terminator stem-loop followed by a stretch of 10 adenines at its 3′ end. Degradation reactions were initiated by the addition of the respective RNase R variant and terminated at the indicated time points by the addition of denaturing sample buffer. The reaction products were resolved by electrophoresis on denaturing polyacrylamide gels , with the activity of each RNase R variant indicated by the disappearance of the full-length substrates and appearance of lower-molecular-weight 2–5 nucleotide products. Using this assay, we observed that RNase RE740A/K741A and RNase RK749A/K750A were as proficient in degradation of this structured RNA substrate as RNase RWT . This result indicated that alanine substitutions at amino acid pairs E740–K741 and K749–K750 did not compromise the ability of RNase R to unwind and degrade this RNA substrate, implying that these defined alterations did not impact intramolecular interactions of the K-rich domain with the catalytic core but rather had a specific effect on the trans-translation dependent activity of RNase R.One possible mechanistic explanation for the observed non-stop mRNA decay defect of RNase R723 to degrade non-stop mRNA was due to a defect in ribosome enrichment . Quantification of ribosome enrichment data, from several independent experiments, showed greater than 3-fold RNase RWT enrichment on ribosome translating the λ-cI-NS non-stop reporter as compared to the λ-cI-S control transcript . In contrast, ribosome enrichment analysis of the RNase RE740A/K741A variant in rnr- cells revealed only background level of ribosome association in cells expressing either the λ-cI-NS or λ-cI-S reporter transcript . Although we consistently detected a small but reproducible increase in enrichment of the RNase RK749A/K750A variant on stalled ribosomes , this level of enrichment did not fully reflect the observed increase in non-stop mRNA half-life. We attribute this difference to the fact that the ribosome enrichment assay, which involves several in vitro processing steps, is not sensitive enough to detect intermediate level enrichment of RNase R variants, whereas the rifampicin-chase assay is more sensitive in detecting subtle differences in non-stop mRNA degradation rates. Based on these findings, we conclude that the non-stop mRNA decay defect of RNase RE740A/K741A and RNase RK749A/K750A is due to the failure of these variants to productively engage tmRNA-rescued ribosomes.The SmpB-tmRNA system rescues ribosomes stalled at the 3′ end of non-stop mRNAs and recruits RNase R to initiate non-stop mRNA decay . Our prerichment . This rent assay  to analytrans-translation related activity of RNase R. Given the nature of these amino acids, it was conceivable that they interacted with each other, perhaps by forming a salt bridge required for maintaining intramolecular contacts. The presence of these contacts might be important for the non-stop mRNA degradation activity of RNase R. We reasoned that swapping the positions of these residues could preserve these interactions, and hence the trans-translation activity of RNase R on stalled ribosomes. To assess this possibility, we constructed the RNase RE740K/K741E switch variant and performed ribosome enrichment assays. This analysis showed that the E–K switch variant (RNase RE740K/K741E) had a strong defect in association with tmRNA-rescued ribosomes . This finding indicated that the position and orientation of residues E740 and K741 are important for productive engagement of RNase R with the trans-translation machinery.Alanine substitution at residues E740 and K741 appears to have a profound effect on the E. coli RNaseR have yielded the first glimpses of its overall shape and domain arrangement, indicating a tri-lobed structure with a core catalytic domain that is highly similar to the known crystal structure of RNase II. Our data show that the unique N- and C-terminal domains of RNase R flank the catalytic core of the protein. Consistent with bioinformatics analysis  in the K-rich domain that are involved in the selective and SmpB-tmRNA dependent elimination of non-stop mRNA. Alanine substitution of residues E740 and K741 significantly weaken the recruitment of RNase R to tmRNA-rescued ribosomes and as a consequence diminish its ability to selectively degrade the associated non-stop mRNA. In addition, simultaneous mutation of residues K749 and K750 renders RNase R markedly defective in non-stop mRNA decay. It is interesting to note that the effect of these alanine substitutions is specific to the trans-translation related activity of RNase R, as they do not compromise its catalytic activity. These four conserved residues, particularly E740 and K741, might exert their influence on the activity of RNase R by participating in decisive intra- or intermolecular interactions. Our data suggest that these amino acids most likely participate in intermolecular interactions with components of the rescued ribosome. We proposed that ribosomal RNA or ribosomal proteins might interact directly with these residues, thereby enabling RNase R to productively engage stalled ribosomes. Taken together, these investigations have shed new light on the global architecture and the relative spatial arrangement of the N- and C-terminal domains of RNase R and highlight the functional significance of key conserved residues in the trans-translation related activities of this versatile enzyme.Previous work from our lab established that the unique K-rich domain of RNase R is required for the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties.Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells +/CD24-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44+/CD24low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments.We describe a sub-population expressing the stem-like marker CD44in vivo.Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity A.In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4 Results are shown in Additional file Luciferase via unpaired, two-sided, unequal variance t-test statistics. Calculated mean fold changes from each set of shRNA expression constructs and corresponding P-values for every gene present in the library are shown in Additional file Two separate barcode read-count ratios were calculated. In order to identify shRNAs, which are toxic to adherent cells or mammospheres, the ratios [tFor target validation, shRNA template sequences identified in the screen were synthesized individually (Sigma-Aldrich) and cloned into the pRSI9 vector backbone. Cloning and virus production were performed following the protocol provided by the manufacturer (Cellecta). Sequences were as follows: shATG4A-1 5′-ACCGGCAGATACAGATGAGTTGGTATGTTAATATTCATAGCATACCAGCTCATCTGTATCTGTTTT-3′; shATG4A-2 5′-ACCGGCCCGGAAAGAAATAGAATAATGTTAATATTCATAGCATTGTTCTATTTCTTTCCGGGTTTT-3′; shATG4A-3 5′- ACCGGGCTGTTGTAATGAGGAAATGGGTTAATATTCATAGCCCATTTCCTCATTGCAGCAGCTTTT-3′ and shCTRL 5′-ACCGGATCACAGAATCGTCGTATGTAGTTAATATTCATAGCTGCATACGACGATTCTGTGATTTTT-3′. For virus production, cloned shRNA plasmids were co-transfected with the packaging plasmids psPAX2 and pMD2.G into HEK293T cells. Viral supernatant was harvested 48 h post transfection and cleared from debris via centrifugation. Cells were transduced with lentivirus for 24 h in cell culture medium containing 8 μg/ml polybrene (Sigma-Aldrich) and selected with 2.5 μg/ml puromycin (Sigma-Aldrich) for 48 h. Following selection cells were recovered for 48 h in antibiotic-free culture medium.Total RNA was isolated from cells or mammospheres using RNeasy Mini Kit (Qiagen). Reverse transcription and PCR were performed using the One Step Quantifast SYBR Green RT-PCR Kit (Qiagen) with a LightCycler 480 system . For target gene amplification, the QuantiTect Primer Assay (Qiagen) was used. Target gene expression was normalised to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).3VO4, 1 mM NaF, 4 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitor mixture (Roche). the protein concentration was measured with the bicinchoninic acid (BCA) Protein Assay . Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane . The membrane was probed with antibodies; peroxidase-conjugated secondary antibodies detected the bands by ECL Plus (ThermoFisher Scientific). Antibodies were: anti-ATG4A  and anti-Tubulin .To determine the protein concentration, cells were lysed in TBS containing 1% Triton X-100, 10 mM NavSingle cell suspension of adherent cells or spheres was stained with CD24-FITC, CD44-PE/Cy7 and EpCAM-APC (Becton Dickinson), E-cadherin-PE  and Vimentin-Alex488 . The cells were measured using a FACS-Canto-II (Becton Dickinson) and data were analysed using FlowJo software .2 in 0.3% agarose with MammoCult medium (StemCell Technologies) on a 0.8% agar base layer. The culture was covered with 0.5 ml MammoCult medium and cultured for 14 days. For quantification, the wells were imaged using a microscope, and the colonies were analysed using ImageJ software.We suspended 2,500 cells/cmP <10-10) were used for pathway enrichment analysis using DAVID Functional Annotation Tool [SUM149 cells were cultured adherently and under mammosphere formation conditions in biological triplicates for two weeks. Spheres were filtered using a 40-μm cell strainer (Becton Dickinson) and RNA was isolated from spheres and adherent cells using RNeasy Mini Kit (Qiagen). RNA was analysed on HumanHT-12 v4 Expression BeadChip  according to manufacturer instructions. Raw data were normalised and grouped using Chipster. Genes with significant gene expression changes (ion Tool . Data weion Tool  under th+/CD24low/EpCAM-/low cells was confirmed via fluorescent-activated cell sorting (FACS).The described sub-population of SUM149 cell was enriched by depletion of EpCAM-expressing cells using EpCAM MicroBead Kit (Miltenyi Biotec). The depletion was performed according to the manufacturer’s protocol. Enrichment of CD444 cells in 15 μl PBS were mixed 1:1 (v/v) with Matrigel  prior to injection into the second left thoracic mammary fat pad of 8- to 9-week-old NOD SCID gamma (NSG) female mice. Tumour growth was monitored over a period of 15 weeks and tumour size was determined twice a week using a caliper. Significance values from Kaplan-Meier plots were calculated using the Wilcoxon test and GraphPad Prism software. For tissue staining, tumours were collected and embedded into paraffin according to routine procedures. H&E staining was done on 5-μm paraffin sections. Studies were approved by the local ethics committee at Regierungspräsidium Karlsruhe .Cells were transduced with plasmids expressing shATG4A-1 and -2 (shATG4A), the ATG4A open reading frame (ATG4A-OE), or a non-silencing control (shCTRL). This was followed by a selection of transduced cells with puromycin. For each injection, 4 × 10+/CD24low  and mammospheres larger 40 μm (tsphere), barcode sequences were recovered using PCR and quantified using next-generation sequencing. Each barcode sequence stands for a particular shRNA expression construct. Figure P <0.01). Pathway enrichment analysis using the DAVID Functional Annotation Tool [Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to proteasomal and ribosomal function (Table P <0.01) but had no impact on adherent proliferation (P >0.1) were used for pathway enrichment analysis. Pathway analysis showed the highest enrichment of candidate genes in Janus kinase (Jak)-signal transducers and activators of transcription (STAT) and cytokine signalling followed by mTOR and several cancer-related signalling pathways  as well as DNA replication (P = 2 × 10-14) were found to be down-regulated in mammospheres, which is in accordance with the reduced growth rate that cells exhibit under serum-free suspension conditions. Interestingly, strongest enrichment of up-regulated genes was seen for lysosome related genes (P = 2 × 10-18) and genes involved in oxidative phosphorylation (P = 3 × 10-14) indicating a requirement of lysosomal activity and energy generation under sphere forming conditions.In order to further investigate molecular differences between mammospheres and adherently cultured cells, gene expression profiles were compared; the results of a pathway enrichment analysis are summarized in Additional file A and ATG4B. It was further found that mRNA and protein levels of ATG4A, but not ATG4B, were elevated in spheres when compared to adherently cultured cells  seeded under serum-free suspension conditions. Inhibition of ATG4A reduced this figure to 18 and 15 spheres, respectively, and overexpression increased the number of spheres formed to 40  and luminal MCF-7 cells (CD44-/CD24+) [A inhibition reduced sphere formation in MDA-MB-231 cells, whereas its overexpression led to a dramatic increase  . It was A expression on the sub-population of cells with CSC properties described above, CD24/EpCAM levels were compared between control cells (CTRL) and cells with reduced (shATG4A) or increased ATG4A expression (ATG4A-OE). Under adherent culture conditions, inhibition of ATG4A was found to reduce the sub-population whereas its overexpression increased it , cells overexpressing ATG4A (ATG4A-OE) and control cells (CTRL) were injected into the mammary fat pad of NSG mice. Tumour formation was monitored over a period of 15 weeks. As shown in Figure P <0.01) whereas knockdown caused a reduced tumour burden (P <0.05) when compared to control cells. Furthermore, at 15 weeks post injection, 100% of animals from the control group, but only 50% from the ATG4A knockdown group, had developed a tumour . A schematic presentation of some of the identified candidate genes acting in the Jak-STAT pathway is shown in Figure +/CD24-/low stem-like breast cancer cells [Here, we exploited this enrichment to establish a high-throughput pooled RNAi screening system suitable to identify genes that are specifically involved in the maintenance of those rare cells with CSC properties. The system is based on the comparison of two separate RNAi screens performed under (i) adherent and (ii) mammosphere culture conditions Figure A. With ter cells  and was er cells . Last, Ser cells . Taken tDespite the advantages of a functional enrichment, culturing of cells as mammospheres also has some drawbacks when performing a high-throughput screen. For the analysis of the screen, we pooled all spheres bigger than 40 μm. Therefore, we could not distinguish between sphere size and number of spheres. Large spheres are believed to consist of more differentiated cells or early progenitors than smaller spheres. Another concern might be the formation of spheres because of cell aggregation instead of clonal growth. To overcome this hurdle, we chose an appropriate cell density to avoid any sphere fusion. Moreover, we validated our candidates in semi-solid soft agar colony formation assays that guarantee clonal sphere growth.A. Autophagy is a lysosome-dependant degradation pathway allowing cells to remove macromolecules and damaged organelles in order to survive stress conditions [+/CD24-/low phenotype in breast cancer cells [A (Figure A led to reduction of a sub-population with CSC properties (Figure A inhibition (Figure Besides Jak-STAT signalling, a number of previously uncharacterized candidate genes were identified in this screen, one of which was the positive regulator of autophagy, ATG4nditions ,38. Inteer cells  and furter cells -43. ATG4er cells . ATG4A cer cells . In a seer cells ,45. Thiser cells ,47. The n Figure A as welln Figure B. These A, are involved in the maintenance of cells with CSC properties. Analysis of additional cell lines using the described approach should greatly accelerate the search for novel molecular targets that could be used to tackle the cancer stem cell.In order to develop targeted CSC therapies, it is essential to understand the underlying molecular mechanisms of CSC maintenance. To study those mechanisms, we developed a high-throughput negative selection RNAi screening system and provide evidence that it is suitable to identify genes which, like ATG4Kyoto encyclopedia of genes and genomes; MOI: Multiplicity of infection; NSG: NOD SCID gamma; PBS: Phosphate-buffered saline; RT-PCR: Reverse transcription polymerase chain reaction; shRNAi: Short hairpin RNA interference; S-P: Sub-population; STAT: Signal transducers and activators of transcription.BCA: Bicinchoninic acid; CSC: Cancer stem cell; DMEM: Dulbecco’s modified eagle’s medium; EDTA: Ethylenediaminetetraacetic acid; EMT: Epithelial-mesenchymal transition; FACS: Fluorescence activated cell sorting; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H&E: Hematoxylin and eosin; IL: Interleukin; JAK: Janus kinase; KEGG: The authors declare that they have no competing interests.JW optimised RNAi screening conditions, conducted RNAi screens, performed or participated in all experiments and their analysis, and drafted the manuscript. DLD participated in and performed the validation experiments of ATG4A. JF assisted in the data analysis. KMD performed the xenotransplantation experiments and completed the tissue staining. CF evaluated the immunohistochemical staining. JDH participated in the design of the study and in writing the manuscript. MB designed the study, supervised the experiments and drafted the manuscript. All authors read and approved the final manuscript.Read-count ratios. Read-count ratios from 27,500 shRNA expression constructs.Click here for fileP-values for all genes.Average and significance for each gene. Average impact on sphere formation with corresponding Click here for fileSignalling pathway enrichment RNAi screen. Pathway enrichment analysis of genes identified via mammosphere formation RNAi screen.Click here for fileSignalling pathway enrichment expression profile. Pathway enrichment analysis of genes differentially expressed between adherently cultured cells versus mammospheres.Click here for file


This results in a burst of virus replication in the T cells that is much greater than that resulting from direct, cis infection of either APC or T cells, or trans infection between T cells. Such APC-to-T cell trans infection first involves a complex set of virus subtype, attachment, entry, and replication patterns that have many similarities among APC, as well as distinct differences related to virus receptors, intracellular trafficking, and productive and nonproductive replication pathways. The end result is that HIV-1 can sequester within the APC for several days and be transmitted via membrane extensions intracellularly and extracellularly to T cells across the virologic synapse. Virus replication requires activated T cells that can develop concurrently with the events of virus transmission. Further research is essential to fill the many gaps in our understanding of these trans infection processes and their role in natural HIV-1 infection. Since the 1990s we have known of the fascinating ability of a complex set of professional antigen presenting cells  to mediate HIV-1  This could be a major pathogenic process in HIV-1 infection and development of acquired immunodeficiency syndrome (AIDS). This process of trans  infection of virus going across from the APC to the T cell is in contrast to direct, cis  infection of T cells by HIV-1. Note that this is a narrow definition of trans infection, as direct, cell-to-cell spread of virus is a classic phenomenon in viral infections , and chemokine receptor blockers (RANTES) [The virologic synapse is where critical virus transmission events occur between the DC and T cell involving conventional HIV-1 CD4 and chemokine receptors on the T cells. Thus, 1/ICAM-1  which ar1/ICAM-1   , 247.trans infection. Activation of Rho guanine nucleotide-exchange factor LARG in DC after HIV-1 infection is one such factor involved in formation of virus-T cell synapses [Various intracellular processes are operative in forming the DC-T cell virologic synapse that is required for efficient synapses . During synapses . Althougsynapses , 249, bo+ effector-memory T cell (Tem) subset is most susceptible to trans infection by R5 HIV-1, which is related to expression of the CCR5 coreceptor [+ naïve T cells (Tn) are more susceptible to trans infection with X4 virus in relation to expression of CXCR4.Importantly, the CD4receptor . In conttrans infection is further dependent on expression of HIV-1 nef [cis infection of immature DC and recruitment of T cells to DC [trans infection by upregulating DC-SIGN expression on DC and promoting DC-T cell clustering [+ T cells [cis infection of DC that need to be clarified.The efficiency of IV-1 nef , 251. Nels to DC . Nef heiustering , while e T cells . However T cells . Thus, ttrans infection could be blocked by neutralizing antibodies [trans infection was protected from such effects [ trans infect T cells with X4 virus [in vivo HIV-1 infection. These seemingly conflicting results could be related to the viral target of the antibody. Thus, neutralizing antibody directed against gp41 is much more effective at blocking trans infection than that directed to gp120 [In contrast to early studies suggesting that tibodies , later i effects . In factX4 virus , suggestto gp120 . This istrans infection. Indeed, NK cells drive DC maturation [trans infection of T cells. Some NK cells express CD4 and are susceptible to HIV-1 cis infection. Infection of NK cells by HIV-1 has been controversial, but accumulating data support that a CD4-expressing NK cell subset is permissive for HIV-1 replication [trans infection mediated by DC. Additional studies are needed to better define NK cell interactions with DC and T cells in relation to HIV-1 trans infection.NK cells have bidirectional interactions with DC where they promote maturation of DC and production of cytokines that activate T cells , 259. Thturation . As disclication . Moreovelication . This cocis and trans infection, it is logical that coinfections could affect these processes. This has been studied in the context of an enhancing role of genital coinfections on HIV-1 trans infection in sexual transmission. Bacterial vaginosis is a mildly symptomatic condition, for example, discharge, odor, and pain, occurring when the predominant commensal lactobacilli in the vaginal tract are largely replaced by Gram negative or positive bacteria [trans infection [trans infection [trans infection [Given the importance of DC maturation and T cell activation in both bacteria . It is abacteria . Mucosalbacteria , althougnfection . Neverthnfection , which anfection , 267. Thnfection . TLR2 trnfection . Finallynfection . This sutrans infection is through translocation of bacterial products across the gastrointestinal (GI) tract to the blood, which leads to systemic immune activation. Indeed, there is extensive evidence for early, enhanced HIV-1 replication in resident CD4+ T cells of the GI tract, with lysis of the infected cells and associated disruption of the epithelial-blood barrier [+ T cells are under a steady state of activation and express the primary CD4 receptor and CCR5 and α4β7 coreceptors, making them fertile ground for either cis or trans infection. Moreover, recent studies have revealed that there is selective destruction of the Th17 cell subset of CD4+ T cells in the GI tract [+ DC are also constitutively activated in relation to chronic TLR signaling by microbial products [E. coli [A second context of bacterial coinfection enhancing HIV-1  barrier . Lamina GI tract . These cproducts . These D[E. coli . This isMycobacterium tuberculosis (MTB), infection of DC is also notable [trans infection by the DC. Microscopy studies indicate that MTB-HIV-1 coinfection of DC decreases degradation of the virus, resulting in greater sequestration of HIV-1 in the intracellular trans infection pathway [Tuberculosis is the leading cause of death in HIV-1 infected persons . Althoug notable . MTB bin notable . Soluble notable . This ma pathway . trans infection. Thus, infection of MDDC with Candida albicans augments HIV trans infection of T cells in relation to increases in expression of CD80 and CD86 on the DC [Cryptococcus neoformans and Penicillium marneffei, both of which can be lethal to HIV-1 infected persons, enhance DC-mediated HIV-1 trans infection in association with increases in DC-T cell conjugates and CD4+ T cell activation [Several diverse types of fungi induce maturation of DC and coincidental enhancement of HIV-1 n the DC . Cryptoctivation , 282.  Plasmodium falciparum increases HIV-1 viral load and enhances progression to AIDS [trans infection could be due to hemozoin, a pigment that is released in excess after destruction of the infected erythrocytes. The hemozoin-loaded DC are partially activated, leading to increased activation and trans infection of CD4+ T cells [Coinfection of malarial parasites with HIV-1 is common in coendemic areas . Infecti to AIDS . The enh T cells , 286.trans infection by malarial parasites, leishmania parasites inhibit trans infection by DC [In contrast to enhancement of HIV-1 on by DC . The mec+ T cells and not DC are the first, primary targets in HIV-1 and SIV sexual transmission models [Although much of this review is premised on the hypothesis that DC serve as the major, initial sites of HIV-1 infection in the mucosa, it needs to be acknowledged that this is a controversial concept. Indeed, there is extensive evidence that CD4n models . Other sn models  as well n models . cis infect MDDC in vitro [cis infection in vitro [trans infection, as it can inhibit this process in vitro, possibly by blocking HIV-1 binding to CLR on the DC by high-mannose N-linked carbohydrates [cis and trans infection. Thus, uterine epithelial cells secrete transforming growth factor β that decreases DC-SIGN expression on immature DC, consequently inhibiting trans infection by the DC [trans infection of activated CD4+ T cells. HIV-1 captured on spermatozoa via heparan sulfate can efficiently in vitro . Vaginalin vitro . Howeverhydrates . Also, ty the DC . In conty the DC . This retrans infection of regional CD4+ T cells. However, systemic infection with HIV-1 could require trafficking of HIV-1 infected DC to draining lymph nodes. Recent evidence in a humanized mouse model indicates that HIV-1-infected CD4+ T cells are very motile, circulating to lymph nodes where they tether to other T cells to form virologic synapses that facilitate cell-to-cell transmission [DC infected at these mucosal transmission sites likely spread HIV-1 through smission . Similartrans infection by microbicides and antiviral drugs. Trans infection by both R5 and X4 viruses can be at least partially blocked by many different nonnucleoside reverse transcriptase inhibitors (NNRTIs) [trans infection [cis infection of DC and DC-mediated trans infection of T cells can be inhibited by protease inhibitor saquinavir [cis infection of DC [Based on the premise that DC are early targets during sexual transmission of HIV-1, there has been a good deal of interest in assessing inhibition of HIV-1 (NNRTIs) . Others nfection . Both ciquinavir . Severalon of DC . cis and trans infection, some of whose mechanisms of action are still unclear. These include amphibian-derived antimicrobial peptides [Various experimental microbicides act through different inhibitory pathways to decrease HIV-1 peptides , HIV-1 ipeptides , Miltefopeptides , SAMMA, peptides , HIV-1 fpeptides , the nempeptides , sulfonapeptides , a solubpeptides , Dendronpeptides , Inmunofpeptides , carbohypeptides , 311, olpeptides , trimannpeptides , 314, anpeptides .α in response to HIV-1 and other viral infections. The pDC were distinct from myeloid DC in morphology and phenotype, clarifying previous work indicating that a natural IFN-producing cell with a dendritic cell phenotype was the major producer of IFN-α [In 1999, Siegal et al.  defined of IFN-α , 318. Thof IFN-α . cis infection with HIV-1 than are myeloid DC, even though they both express CD4 and HIV-1 coreceptors [in vitro [in vivo [It appears that pDC are less permissive for productive, eceptors , 320. Neeceptors , 321 andeceptors , 323 caneceptors . Moreovein vitro , there i[in vivo .cis infection of pDC, it is not surprising that pDC can trans infect T cells with R5 and X4 HIV-1 [+ T cells [cis infection of the pDC is not required for trans infection of T cells with HIV-1 [Given these data on expression of HIV-1 receptors and productive X4 HIV-1 , 325. Thtrans infection with HIV-1 has been challenged by work showing that, regardless of their state of activation, pDC derived from the same donors as myeloid DC did not trans infect their T cells [α produced by the pDC. Clearly more information is needed to define whether pDC are able to trans infect T cells with HIV-1.The ability of pDC to mediate  T cells . This istrans infection of CD4+ T cells similar to DC [+ T cells. Two basic principles of HIV-1 trans infection were established with this pioneering study, that is, that antigen-specific activation of the T cells results in greater trans infection by the MDM and that anti-CD4 and anti-gp120 antibodies block trans infection. It took years for this phenomenon to be extended, as in 1999 Carr and colleagues [+ T cells much more efficiently than cis infection of T cells. This process required cell contact, occurred in both allogeneic and autologous systems, and was enhanced by preactivation of the T cells. Overall, trans infection mediated by MDM appears as efficient as that mediated by myeloid DC.Circulating monocytes and their tissue-transformed macrophages are APC that can mediate HIV-1 ar to DC . This waar to DC , where blleagues  reportedtrans infection mediated by monocytes and macrophages is largely dependent on the pathways and proficiency of preceding cis infection in these APCs. Given that blood-derived monocytes/macrophages express CD4 and CCR5, but not CXCR4, they are highly susceptible to R5 HIV-1, albeit with much lower production of infectious virus than cis infection of CD4+ T cells [in vivo [cis infection with either R5 or X4 strains of HIV-1 [+ T cells. As with DC, the efficiency of HIV-1  T cells . However T cells . It is a[in vivo , 332. Tiof HIV-1 , 44, 292of HIV-1 . These icis replication. For example, prostatic acidic phosphatase (PAP) in seminal fluid forms amyloid fibrils that enhance HIV-1 infection of macrophages [Interestingly, semen contains enhancers of HIV-1 rophages .cis replication efficiency, that is, production of infectious virus, varies greatly among both R5 and X4 HIV-1 strains but is relatively poor in monocytes/macrophages compared to T cells [+ T cells. In contrast to CD4+ T cells, HIV-1 resides and accumulates in specialized compartments within monocytes/macrophages, where new virions form and are eventually released to the extracellular environment [The overall outcome is that  T cells . This isironment . cis infection. Thus, the TLR2 ligand PAM3CSK4, a synthetic triacylated lipopeptide mimic of the acylated amino terminus of bacterial lipopeptide, activates HIV-1 transcription in macrophages [Monocytes and macrophages express a variety of TLRs depending on their blood and tissue distribution . These crophages . This efrophages .α activated deamination of viral DNA [cis replication of HIV-1 in MDM [Monocytes are more resistant to productive HIV-1 infection than macrophages, which has been associated with presence of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) . This cyiral DNA . Also, r1 in MDM . γ, TNF-α, and LPS, and M2 being activated by anti-inflammatory agents including IL-4, IL-10, and IL-13 [γ and TNF-α induce activation of the M1 subset of MDM, with downregulation of CD4 and consequent resistance to HIV-1 infection [γ-activated M1 cells can, however, maintain low levels of both R5 and X4 HIV-1 DNA and low virus replication [cis infection of monocyte/macrophages that are beyond the scope of this review [Macrophages have been divided into M1 and M2 subsets distinguished by their polarized function, with M1 being activated by proinflammatory factors such as IFN-nd IL-13 . Polariznfection . IFN-γ-alication . Regardis review . An exciting, recently discovered mechanism by which macrophages restrict HIV-1 replication is that they express the dideoxynucleotide hydrolase SamHD1 , 347. Thcis infection of monocytes/macrophages and therefore likely to affect trans infection of T cells. Autophagy is a catabolic process where cellular components are degraded by lysosomal machinery and thereby has a critical role in antigen processing by APC [+ T cells [+ T cells by X4 or R5 Env, leading to apoptosis, uninfected macrophages are resistant to this process [Finally, the phenomenon of autophagy has in recent years been shown to be central to HIV-1 g by APC . Althoug T cells . Whereas process . In HIV- process , 353.in vitro [cis and trans infection. However, MDM treated in vitro with IL-13 [trans infection. M2a MDM derived by IL-4 treatment express DC-SIGN that facilitates cis infection with both R5 and X4 HIV-1 and subsequent efficient trans infection of T cells [α and IFN-γ treatment express little DC-SIGN and do not efficiently trans infect T cells. Monocytes and MDM normally do not express DC-SIGN in vitro , thus neth IL-13 , 355 or th IL-13 , 357 exp T cells . In contMDM also express the C-type lectin mannose-binding receptor (macrophage mannose receptor (MMR); CD206) which binds mannosylated carbohydrates on gp120 with high efficiency . Virus btrans infection is that certain resident tissue macrophages [trans infection. It has been suggested that placental CD163+ macrophages (Hofbauer cells) are infected by HIV-1 via their DC-SIGN receptors and that this could represent a pathway for mother-to-child transmission of HIV-1 [trans infection of T cells by IL-13-treated MDM that express both [cis replication of HIV-1 in MDM [trans infection mediated by MDM involves other nonreplicative pathways of HIV-1 in these APC. This concept fits with the extensive data that HIV-1 replicates relatively poorly in MDM and tissue resident macrophages [in vivo and MDM in vitro, is associated with HIV-1 cis infection of MDM and their capacity to mediate trans infection [Of particular pertinence to HIV-1 rophages , 361 expof HIV-1 . Howeveress both . Interes1 in MDM . This surophages . In thisnfection .trans infection of T cells [trans infection mediated by MDM can be blocked by inhibitors of MMR binding. This process differs, however, from trans infection mediated by DC in that virus does not survive beyond 1 day after binding to MDM. Notably, this latter finding contrasts with evidence that HIV-1 survives for weeks within macrophages and can still trans infect T cells [trans infection mediated by monocytes/macrophages is based largely on inefficient cis infection of these APC with HIV-1 virions assembling in late endosomes or multivesicular bodies. This involves tetraspanins including CD63 and CD81 that are constitutively expressed in these microvesicles. The virion-containing vesicles are important components in the trans infection T cell pathway, where they accumulate at the point of the virologic synapse. Efficient trans infection may also involve multiple points of cell contact, termed polysynapses, which are rosette-like structures of an infected macrophage surrounded by many T cells [α, and is related to enhanced trans infection [Given the ability of MMR to bind HIV-1 with high efficiency, this pathway has been linked to  T cells . Thus, H T cells . Regardl T cells . Finallynfection .trans infects T cells within 6 hours of cell-to-cell contact [trans infection of T cells mediated by macrophages [Using the HIV-1-infected MDM model, it is evident that movement of the vesicular body to the virologic synapse is dependent on Gag . HIV-1 f contact . Interesrophages . Hence, trans infection of T cells by macrophages is through filopodial bridges or tunneling nanotubes [trans infection.Another intriguing physical pathway for HIV-1 anotubes . Murine anotubes , suggestanotubes . HIV-1 canotubes , 376. Imanotubes . These atrans infection of CD4+ T cells that are also present [trans infection mediated by DC [trans infection through functional natural IgA and IgG antibodies specific for the carbohydrate recognition domain of DC-SIGN [trans infection in breast milk include stimulation of TLR3 expressed on breast milk macrophages with its double-stranded RNA ligand poly (I:C) [α and IFN-β that are induced by TLR3 activation.Neonatal infection with HIV-1 has largely been prevented by ART prophylaxis during pregnancy . Neverth present . Interesed by DC . This oced by DC . Human m DC-SIGN . Strategly (I:C) . This prcis infection [trans infection of T cells [+ T cells trafficking through the lymphatics are infected by the FDC-bound virus, with virus replication being driven by TNF-α produced by the FDC [Although B lymphocytes are highly activated and modified beginning in the earliest phases of HIV-1 infection, they are not considered a significant, major target for HIV-1 nfection . Indeed,nfection , 385. Mo T cells –390. The T cells . Virus i T cells . CD4+ T  the FDC .+ T cells, that is, CD40L and IL-4 [cis infection is non-productive, yet virus can retain its infectivity for at least 2 days within the B cells. Interestingly, most of the captured virions are destroyed in B cell lines engineered to express DC-SIGN [trans infect T cells [In 2006, two laboratories discovered that DC-SIGN is expressed on peripheral blood and lymphatic tissue B cells , 395, in DC-SIGN , 216. Ho T cells , 396. trans infection of CD4+ T cells [trans infection mediated by B cells can be blocked with anti-DC-SIGN mAb as well as mannan. Although B cells express high levels of CXCR4, treatment with anti-CXCR4 mAb prior to addition of HIV-1 to B cells does not inhibit trans infection of the T cells. Thus, although cis infection of B cells does not involve productive replication of infectious virions like that of DC and monocytes/macrophages, B cells are as efficient as these other professional APC in mediating HIV-1 trans infection. This DC-SIGN dependent process of B cell mediated trans infection is not unique to HIV-1, as a similar pathway has been reported for hepatitis C virus trans infection of hepatoma cells [Comparable to DC-SIGN-expressing B cell lines, activated blood and tissue-derived B cells are highly efficient in mediating HIV-1  T cells . As withma cells .+ T cells, which are abundant throughout chronic immune activation that is prevalent in HIV-1 infection. CD40 is a normal constituent on B cell surfaces. Thus, the continuous interaction of CD40L on activated T cells with CD40 on B cells is one pathway of chronic B cell stimulation during HIV-1 infection [trans infection of autologous CD4+ T cells compared to virions without CD40L [CD40L is expressed on activated CD4nfection . Intrigunfection . Such vinfection . Importaut CD40L . cis and trans infection is the role of cross-talk among DC, B cells and other cell types. Thus, HIV-1 cis infection of DC is enhanced by coculture with CD4+ T or B lymphocytes but not with CD8+ T cells [cis and, hence, trans infection is the role of the tumor necrosis factor receptor superfamily [cis and trans infection mediated by B cells. Another area of potential interest regarding HIV-1 trans infection mediated by B cells is a possible role of TLR both in vitro and in vivo [cis and trans infection. An emerging concept in HIV-1  T cells . In addierfamily . Specifierfamily , which i in vivo . For exa in vivo . TLRs ha in vivo . These f+ T cells by macrophages [+ T cells via CD45, CD2, and CD28, respectively. The final result is enhanced HIV-1 replication in the T cells.B cells have also been implicated as intermediaries in enhanced HIV-1 infection of CD4rophages . This prtrans infections, there are still major gaps in our understanding of this phenomenon. In particular, there is a paucity of information on the importance HIV-1 trans infection in natural HIV-1 infection and disease. There are inherent difficulties and highly challenging complexities in proving that this process is involved in virus transmission and disease pathogenesis. Nevertheless, studies of trans infection would be of interest in persons who control HIV-1 infection in the absence of ART, who range in definition and terminology as long-term nonprogressors, viral controllers, and elite controllers [cis and trans infection. However, APC from these individuals have not been studied for their ability to trans infect T cells. We can speculate that the efficiency of this process will depend in part on the integrity of functional activity of the APC and CD4+ T cells, that is, their expression of receptors and intracellular and cell-cell pathways known to be involved in both HIV-1 cis and trans infections. Despite years of extensive studies on HIV-1 trollers –408. Myetrollers , which c


DRAM) as one of the genes that were more upregulated by S121F than wild-type p53. Although knockdown of DRAM was not sufficient for reducing the ability of S121F to induce apoptosis, DRAM overexpression enhanced the ability in a wild-type p53-dependent manner. Here, we show that DRAM is an important gene for the enhancement of p53-dependent apoptosis. Additional analysis of the mechanism of super p53-dependent apoptosis may lead to the identification of novel drug targets for cancer therapy.Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive mechanisms in human tumorigenesis. Till date, “super p53” mutants exhibiting more potent ability to induce apoptosis than wild-type p53 have been reported. These super p53s may provide a clue for development of novel therapeutic targets. However, the major mechanism underlying the super p53-dependent apoptosis remains unclear. To identify critical gene(s) in this mechanism, we performed a comprehensive and comparative expression analysis in p53-null Saos-2 cells with conditional expression of wild-type p53 and S121F, which was previously reported as a super p53 mutant. We identified damage-regulated autophagy modulator ( Tumor suppressor p53 protein is activated by various cellular stresses, including DNA damage and hypoxia, and is posttranslationally phosphorylated and acetylated. Activated p53 binds to specific DNA sequences in the regulatory regions of downstream genes, resulting in cellular events such as cell cycle arrest and apoptosis –4. Thus p53-dependent apoptosis is one of the most critical pathways of cell death in tumor-suppressive functions. Apoptosis is an evolutionarily conserved programmed cell death process. So far, numerous studies have reported results from protein investigations on p53-dependent apoptotic mechanisms. A number of p53-target gene products, such as BAX, PUMA, NOXA, p53AIP1, and PTEN, elicit apoptosis. Apart from the known transactivation-dependent mechanism, a transactivation-independent mechanism via mitochondria has also been reported . EventuaPrevious studies of p53 mutants have revealed the presence of “super p53” mutants that exhibit increased ability to induce apoptosis than wild-type p53 ,29. BecaDRAM) is one of the genes that are more strongly induced by S121F than wild-type p53. DRAM has been reported as a direct target of p53 and a critical factor for p53-dependent apoptosis and autophagy in specific contexts [We first established Saos-2 cell lines that contained doxycycline (Dox)-inducible wild-type p53, S121F (a known super p53 mutant), or R175H (a known common mutant), and performed microarray analysis to identify an important candidate gene for the strong ability of S121F to induce apoptosis. In this study, we show that damage-regulated autophagy modulator (contexts . FurtherTP53−/−) and SF126  were cultured in RPMI 1640 containing 10% fetal bovine serum at 37°C. p53-inducible cell lines were cultured in a medium containing 200 μg/mL hygromycin B .Human cell lines Saos-2 . The polymerase chain reaction (PCR) primers for the DRAM cDNA were 5′-GTAGAATTCATGCTGTGCTTCCTGAGGGGAAT-3′ and 5′-GCCGGATCCAATATACCATTGATTTCTGTGG-3′. The DRAM cDNA was sequenced using an automated CEQ2000XL DNA analysis system . These expression vectors were transfected using Effectene transfection reagent .The wild-type p53 expression vector, pCR259-p53-WT, and the S121F mutant p53 expression vector, pCR259-p53-S121F, were constructed as described previously . A FLAG-p53-inducible cell lines were established as described previously . Wild-tySaos-2-Mock, Saos-2Tet-p53-WT, -S121F, and -R175H cells were grown to 70–80% confluence in 6-cm dishes and further incubated with or without Dox for 24 h. Total RNA was extracted using the RNeasy Mini kit (Qiagen). Total RNA (800 ng) from each sample was used for the microarray analysis. The quality of total RNA was assessed using the 2100 Bioanalyzer . Comprehensive and comparative mRNA expression analysis was performed using the Whole Human Genome microarray (Agilent Technologies) according to the manufacturer's protocol. The gene expression data were analyzed using Gene Spring v9.2 (Agilent Technologies) and TIGR open access software TMEV v4.0. The results are shown using TMEV v4.0.DRAM mRNA levels were normalized to ACTB mRNA levels. Relative DRAM mRNA levels were expressed as ratios of normalized DRAM mRNA levels with Dox to those without Dox.Total RNA was extracted using the RNeasy Mini kit 24 h after Dox treatment. cDNA was generated from 2 μg of total RNA using the High Capacity cDNA Reverse Transcription kit (Life Technologies). Real-time quantitative RT-PCR (qRT-PCR) was performed in duplicate using TaqMan Gene Expression Assays (Life Technologies), and reactions were analyzed using the CFX96 thermal cycler . Cells were harvested and resuspended in lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 5 mmol/L EDTA, and 1% protease inhibitor cocktail (Sigma-Aldrich). The lysate was analyzed by Western blotting as described previously  using anDRAM mRNA. The sequence of nonsilencing control (NSC) was 5′-CTTACTCTCGCCCAAGCGAGAG-3′. p53-inducible cell lines  were transduced with lentivirus targeting DRAM mRNA or targeting no mRNA (NSC). Cells were selected for stable integration of the lentivirus by incubation with 1 μg/mL puromycin . All cells were examined under a fluorescence microscope BZ9000  to detect green fluorescent protein (GFP) expression, which is an indicator of transduction efficiency.DRAM expression was knocked down using the shRNA-mir GIPZ lentiviral vector  targeting the sequence, 5′-CACAACACTATAAGAAATA-3′ in the 3′-UTR of Cells were treated as described previously  and analt-test was performed and P < 0.05 was considered statistically significant.All experiments were repeated at least three times. The results are expressed as mean ± standard deviation (SD). The Student's P < 0.01 for all). Furthermore, the subG1 fraction of S121F was significantly higher than that of wild-type p53 at 24, 48, and 72 h . Apoptosis was not induced in R175H. These results indicate that S121F has an increased potent ability to induce apoptosis than wild-type p53 , but was not induced by R175H  (P = 0.87) (P < 0.05) (P < 0.01)   . In SF12 = 0.87) . Next, w < 0.05) . Similar < 0.01) . These rctively) . These rDRAM shRNA were confirmed by fluorescence microscopy for GFP (data not shown). The DRAM mRNA levels were analyzed by qRT-PCR. We confirmed that the DRAM mRNA levels were downregulated in Saos-2Tet-p53-S121F-shDRAM and SF126Tet-p53-S121F-shDRAM compared with noninfected cells (no shRNA) or NSC   or NSC . Using tctively) . We alsoDRAM mRNA levels were analyzed by qRT-PCR. We confirmed that the DRAM mRNA levels were downregulated in Saos-2Tet-p53-WT-shDRAM and SF126Tet-p53-WT-shDRAM compared with NSC clones (P < 0.05 for both) .Next, we examined whether DRAM knockdown affected the ability to induce autophagy and apoptosis by wild-type p53. As previously mentioned, we established endogenous DRAM knockdown cell lines in wild-type p53. The C clones . The relor both) . These rIn this study, we performed gene expression microarray analysis using a Dox-inducible p53 expression system to test the hypothesis that the mechanism of S121F-dependent apoptosis involves increased expression of genes of known or unknown functions.DRAM has recently been reported as a p53 target gene and has been shown to be involved in apoptosis and autophagy induced by p53 [First, the results suggested that DRAM was involved in the potent ability of S121F to induce apoptosis. d by p53 .DRAM increases and contributes to apoptosis induction, and more potent expression of DRAM is expected to further enhance wild-type p53-dependent apoptosis.Next, we showed that DRAM enhanced wild-type p53-dependent apoptosis. When DRAM alone was expressed, the influence on the ability to induce apoptosis was unchanged or minimal, but in the presence of wild-type p53, a significant enhancement was observed using flow cytometry. When wild-type p53 is expressed, the expression of the target gene However, the mechanism of DRAM to enhance wild-type p53-dependent apoptosis remains unclear. One hypothesis is that DRAM localized to lysosomes acts in concert with the activation of the ability to induce apoptosis mediated by lysosome-dependent pathways resulting from p53 expression. Phosphorylated p53 is known to be localized in lysosomes and may contribute to enhanced apoptosis . In addiDRAM expression more strongly than wild-type p53, no changes in the ability of S121F to induce apoptosis were noted when endogenous DRAM expression was suppressed. Several p53 target genes have been reported, and with regard to the transcription-dependent pathway, Bax, Puma, Noxa, p53AIP1, and Killer/DR5 have been mentioned in the mechanism of p53-dependent apoptosis. In addition, when the transcription-independent pathway in which p53 directly acts in mitochondria and lysosomes to induce apoptosis is also included, apoptosis induction by p53 is expected to occur through multiple mechanisms. When the expression of one of the multiple p53 target genes is suppressed, there may be a compensatory increase in the expression of another target gene.Although DRAM overexpression enhanced the ability of wild-type p53 to induce apoptosis and S121F induced Crighton et al. reported that DRAM was critical for p53-dependent apoptosis . In theiDRAM expression, the antitumor activity of wild-type p53 may be enhanced. In other words, if DRAM expression levels in cancer tissues can be increased by stimulating DRAM transcription or inhibiting DRAM degradation, the therapeutic effects of anticancer drugs and radiation therapy may be further enhanced.About half of all human cancers retain wild-type p53, so that by further increasing


Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES. Ewing sarcoma (ES) is an aggressive malignancy affecting children and adolescents. The presence of metastases is the most adverse prognostic factor, with a 3-year event-free survival at 27% for patients with metastatic tumors . This poThe fraction of CSCs often increases in hypoxia, leading to disease dissemination and resistance to therapy -5. In ESin vitro, is elevated in tissues from metastatic ES  thymidine [ES cells were grown in HMVEC culture media, which was collected 24h later. HMVECs were plated onto 96-well plates, growth-arrested for 24h, and then treated for 24h with NPY  using Dunnett's method was used for data comparison and analysis. All experiments were repeated at least three times. Data is presented as mean ± standard errors.


The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP).The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially.This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible. Recycling of wastepaper has gained momentum over the past decades due to the severity in the demand of green plants being imposed by the paper industry throughout the world The conventional methods of deinking utilize chemicals such as sodium hydroxide, sodium silicate, hydrogen peroxide, chlorine based chemicals and other chelating agents which are environmentally hazardous Physical methods such as sonication and high temperature are pretreatment stages for deinking owing to their ability to reduce chemical consumption and enhance physical as well as optical properties of recycled papers This study employed bacterial laccase and xylanase enzymes, sonication and microwaving, alone and in combination, for the deinking of ONP pulp. The parameters for enzymatic deinking were optimized statistically. The optical and physico-mechanical properties of deinked pulp and reduction in chemical consumption were evaluated. The surface properties of enzymatically treated pulp were studied by X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy.The different pulps used were made from locally procured wastepapers viz. old newsprint, magazines, laser, inkjet and xerox with initial brightness of 36, 42, 65, 60 and 58% ISO respectively.Bacillus halodurans FNP 135 soil isolate by solid state fermentation of wheat bran under optimized conditions viz. 2.31% Na2CO3 (w/w), 80% moisture content (v/w), 68 h of fermentation at 37°C and assayed using birchwood xylan as substrate A cellulase-free, thermo-alkali-stable and halo-tolerant xylanase with optimum activity at pH 8–9.5 and 65°C was produced from gamma-proteobacterium, a novel bacterium isolated from industrial effluent Rheinheimera species g, 4°C for 10 min and the supernatant was used as crude enzyme preparation. Laccase activity was determined using 2 mM guaiacol, at 55°C in 0.1 M phosphate buffer (pH 6.5) −1cm−1). Unit of laccase was expressed in nkat (n moles of substrate converted/sec/ml of enzyme).A cellulase and xylanase-free laccase with optimum activity at pH 8 and 55°C was produced from Waste papers were shredded and soaked overnight in tap water at room temperature. The soaked papers were washed several times and were disintegrated with the help of a grinder to obtain soft cottony pulp. It was then squeezed to remove absorbed water and oven dried. This oven dried pulp (odp) was used for further experiments.2O2 (1%) at 50°C and 200 rpm for 15 min. To facilitate removal of ink particles detached from fibers, pulp was thoroughly washed with distilled water and was used to make hand sheets to determine brightness of the pulp.All treatments were carried out in triplicates at least. A standard deinking sequence (P-C) was performed in the laboratory. P refers to the pulp preparation stage and C refers to chemical bleaching stage where pulp at 5% consistency was treated with sodium hydroxide (2%), sodium silicate (2.5%), DTPA , Triton X-100 (1.2%) and HEnzymatic and physical pretreatments were evaluated using the standard sequence (P-C) by incorporating (a) xylanase (X) stage (P-X-C), (b) laccase (L) stage (P-L-C), (c) L stage along with mediators/inhibitors (M/I) (P-L+M/I-C), (d) X followed by L stage (P-X-L-C), (e) sonication followed by XL stage (P-S-X-L-C), (f) microwaving (MW) followed by XL stage (P-MW-X-L-C), and (g) S followed by MW followed by XL stage (P-S-MW-X-L-C). In all cases, standard sequence (P-C) was used as a control. Both xylanase and laccase treatments were carried out with 10 g (dry weight) of pulp at 5% consistency at different enzyme doses, temperatures and pH for optimization of various parameters for deinking using Response Surface Methodology. The treatments were carried out in flasks, at 160 rev/min and 65°C and 55°C for xylanase and laccase respectively. Laccase mediator and inhibitor treatments were carried out with 10 g (dry weight) of pulp at 5% consistency at optimum pH, enzyme dose and incubation time using 2 mM of 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1-hydroxybenzotriazole (HBT), syringaldehyde (Syr) as mediators and 1 mM of sodium azide (SA) as inhibitor. Sonication The color removal from the pulp was determined spectrophotometrically from λ 200 nm to λ 800 nm. The reducing sugar released was measured by the dinitrosalicylic acid (DNS) c) was calculated from the X-ray diffraction patterns by the following equation Crystallinity of cellulose was determined for both untreated and XL treated samples by X-ray diffraction. After freeze drying, samples were analyzed using the X-ray diffractometer with X-ray generator from 2 to 50 of 2θ (scattering angle). The crystalline index of cellulose (X0 0 2 is the peak intensity from the (0 0 2) lattice plane (2θ = 22.6°) and Iam is the peak intensity of amorphous phase (2θ = 19.0°). Apparent crystallite size (ACS) was estimated through the use of the Scherrer equation Where I−1 over the wave number range of 4000–400 cm−1, using 32 scans per sample. Empirical cellulose crystallinity index (CI) was calculated by dividing absorbance intensities of IR bands at 1430 and 897 cm−1FTIR spectra for pulp samples were recorded with a resolution of 4 cmFTIR spectrum was analyzed using the FTIR database The fibers were washed thrice with deionized water and were gradually dehydrated with acetone gradient between 30 and 90% and finally suspended in 100% acetone; small pieces of fibers were air dried and placed on the stubs, mounted with silver tape, and sputter coated with gold using fine coat  and examined at 10 KV.All physical, chemical and residual chlorine measurements were carried out by following the TAPPI standard methods for brightness and whiteness (T-452 OM-87 and T-1216 OM respectively with a Minolta spectrophotometer CM3630), kappa number (T 236), tear factor (T-414 OM-98), burst factor (T-403 OM-97), breaking length (T-404 OM-92) and viscosity (T-230 OM-82). The effective residual ink concentration was measured with the Elrepho ERIC tester using Lorentzen and Wettre software.Central composite design with three factors - pH, enzyme dose and incubation time at five levels was employed to investigate the first and higher order main effects of each factor and interaction amongst them for deinking of ONP pulp using xylanase and laccase enzymes . With xyDifferent laccase mediators were tried to further enhance deinking . Except Chemical deinking causes removal (dislodging) of ink particles from pulp fiber only whereas laccase enzyme may have caused either decolourization only or both decolourization and dislodging of ink particles. This was further supported by the fact that on comparing the absorbance scans of effluents (λ 200–800 nm) to detect the release of colored compounds, chemically treated pulp effluent showed high absorbance whereas laccase treated and untreated (control) pulp effluents were colourless .This is the first report on deinking of ONP pulp with a bacterial laccase without the need of a mediator, thus making the process cost effective and eliminating mediator-linked enzyme toxicity problems. Furthermore, no deinking effect was observed with laccase in the presence of laccase inhibitor sodium azide which confirmed the role of laccase in deinking .The present work demonstrated that an improved level of deinking was achieved by using xylanase and laccase sequentially as compared to xylanase or laccase alone. Pretreatment of ONP pulp with xylanase, followed by laccase, obtained a brightness of 59.6±0.8% ISO which was 8.5, 11.4 and 21.6% higher than that of the P-L-C, P-X-C and untreated pulp . ERIC fo002 intensities (in cm) at 2θ = 22.6° were 2.4 and 8.4, Iam intensities (in cm) at 2θ = 19° were 1.7 and 2.9, half height width (in cm) of the peak angle of the (002) reflection at 2θ = 22.6° was 1.9 and 1.1 for the P-C and P-X-L-C pulps respectively. XRD analysis revealed that P-X-L-C treatment increased the crystallinity index of the ONP pulp from 29.16% (untreated) to 65.47% while the cellulose crystallinity index increased by 10.3%  treated ONP pulp is shown in by 10.3%  which me−1 depict –OH stretching of hydrogen-bonding. The increase in their relative intensity after enzymatic treatments is attributed to the increase in cellulosic content of the pulp. The decrease in relative intensity of bands at 2921–2917 cm−1 and 617 cm−1, assigned to CH asymmetrical stretching vibration in CH3, CH2, CH, in P-X-L-C treated pulp indicated the degradation of aliphatic side chains. Relative intensity around 1642–1639 cm−1 increased in P-X-L-C treated pulp which is attributed to release of free carbonyl groups (C = O) due to action of enzyme on lignin’s aromatic ring −1, a band assigned to aromatic skeletal vibrations combined with –CH3 in-plane deformations, showing that some methoxyl groups were removed during the enzymatic treatment. The band at 1374–1372 cm−1 was assigned to aliphatic C–H stretching in CH3 (not in –OCH3) and phen–OH. The decrease of its relative intensity illustrated that either the side chains or phen–OH of lignin decreased after the enzymatic treatment. The decrease in relative intensity at 1266–1252 cm−1 indicates degradation of guaiacyl groups. A new band that appeared at 1737 cm−1 in P-X-L-C treated pulp was assigned to C = O stretching vibration in β-C = O, COOH, ester indicating that residual lignin after an P-X-L-C treatment was enriched in these types of functional groups. New band at 1162 cm−1 in P-X-L-C treated pulp indicated degradation of syringyl groups. The changes in pulp crystallinity derived from A1430/A897 ratio in accordance with the X-ray diffraction studies.FTIR spectra for xylanase–laccase (P-X-L-C) treated pulp samples showed several characteristic and prominent changes . The P-XAs can be observed from The parameters for the effect of sonication and microwave pretreatments alone and in combination with P-X-L-C pretreatment of ONP were optimized . MaximumWhen used in combination with P-X-L-C pretreatment, further 3 (P-S-X-L-C) and 3.5% (P-MW-X-L-C) increase in brightness to 61.45±0.75 and 61.73±0.33% ISO was observed. Sonication causes swelling of fibers thus opening them which in turn increased the surface area of contact between fibers and enzymes or chemicals allowing them to reach protected inertial sites When all the three pretreatments (P-S-MW-X-L-C) were used together, the combination resulted in 62.13±0.5% ISO brightness (28.8% increase), 7.4±0.66 kappa number (22% decrease) and 139.04±22 ppm ERIC (73.9% reduction) as compared to brightness and ERIC of P-C pulps. Substantial reduction in ERIC to this level with the use of a new sequence combining enzymatic and physical methods for efficient deinking of ONP pulp has not been reported before. Though P-S-MW-X-L-C deinked pulp had lower strength properties than P-X-L-C deinked pulp, yet these were better than those of conventional chemically treated pulp. The breaking length of P-X-L-C deinked pulp was 26, 8.6 and 11% higher; burst factor was 0.6, 1.7 and 0.06% higher and tear factor was 12.1, 3.3 and 5.2% higher than P-X-C, P-L-C and P-S-MW-X-L-C deinked pulp. As none of the enzyme preparations had cellulase activity, a 13% increase in viscosity was observed in P-X-L-C treatment. P-L-C treatment caused 12% reduction in kappa number as compared to P-C, indicating the role of laccase in delignification.Different wastepaper pulps were effectively deinked by using a combination of xylanase and laccase enzymes . MaximumLaccase and xylanase effectively deinked wastepaper pulps without mediator supplementation for laccase activity. XL-pretreatment resulted in better optical and strength properties, highly reduced ERIC, higher cellulose crystallinity, more fibrillar surface and formation of lignin-degradation and conjugated carbonyl groups on the surface. A 50% reduction in chemical consumption for ONP pulp was also achieved. Sonication and microwaving contributed further to the improvement in the deinking of pulp. This work reports for the first time a new sequence combining biological and physical methods for effective deinking of old newsprint yielding better quality paper and substantially reduced requirement of chemicals.Figure S13D graph of combined effects of (A) Time and Enzyme dose (B) Time and pH (C) Enzyme dose and pH, on brightness when third factor was kept constant and its optimal level for xylanase treatment.(TIF)Click here for additional data file.Figure S23D graph of combined effects of (A) Time and Enzyme dose (B) Time and pH (C) Enzyme dose and pH, on brightness when third factor was kept constant and its optimal level for laccase treatment.(TIF)Click here for additional data file.Figure S3Wavelength scan results for chemically treated (1), laccase treated (2) and untreated (3) ONP pulp.(TIF)Click here for additional data file.Table S1Experimental range and levels of independent test variables used in central composite rotary design for optimization of deinking of ONP pulp with xylanase or laccase.(DOC)Click here for additional data file.Table S2Central composite rotary design matrix with experimental values of brightness for optimization of pH, incubation time and enzyme dose for deinking of ONP pulp using xylanase and laccase enzymes.(DOC)Click here for additional data file.Table S3Response surface quadratic model analysis of variance (ANOVA) for deinking of ONP pulp using xylanase and laccase enzymes.(DOC)Click here for additional data file.