text:
WestwoodWestwood Week kicked off it’s week-long event series with An Italian Affair at the Hollywood Roosevelt Hotel hosted by LPA’s Pia Baroncini. The carb-centric soiree was filled with delicious pizza straight from the over, which was set up in the Hollywood Roosevelt parking lot, from Jon & Vinny’s. Guests enjoyed Peroni beer and Ramona wine spritzer’s with Tunes throughout the night were provided by DJ Yimmy Yayo and them jeans.
RSVP – Celebrate the release of REPRESENT CLOTHING’s S/S 16 Collection at The Well with hosted bar from Peroni.
Ticketed – Join us for an extraordinary IVY Winter Gala, as we toast to a wonderful year of new friendships and amazing memories! This special black-tie celebration will take place in the heart of Beverly Hills at the stunning ACE GALLERY.
Peroni is hosting a game night at the Spare Room filled with fun. The evenings begin at 9pm, and this weeks game is staring contest, so get your eyelids ready.
Watch the Grammys live for the East Coast telecast with Guest Host Jeremiah Red + open bar + pizza + suprises.


text:
Creator: Unknown. January 15, 1881.
The Comanche Chief. (Comanche, Tex.), Vol. 8, No. 8, Ed. 1 Saturday, January 15, 1881, newspaper, January 15, 1881; Comanche, Texas. (https://texashistory.unt.edu/ark:/67531/metapth904039/: accessed April 24, 2019), University of North Texas Libraries, The Portal to Texas History, https://texashistory.unt.edu; crediting Comanche Public Library.

industry_corpus2: Subject Education


text: Tobii is the global leader in eye tracking. Our vision is a world where all technology works in harmony with natural human behavior. Tobii operates through three business units: Tobii Dynavox makes specially designed computers that are controlled by eye movement or touch screens for use by people with special needs due to spinal cord injuries, CP, ALS or other medical conditions. Tobii Pro develops and sells eye-tracking equipment and services used today by more than 3,000 companies and 2,000 research institutions, including all of the world’s 50 highest ranked universities. Tobii Tech further develops Tobii’s technology for new volume markets, such as computer games, personal computers, virtual reality, augmented reality and smartphones. Tobii is headquartered in Sweden and is listed on Nasdaq Stockholm (TOBII). The group has about 1,000 employees. For more information, visit www.tobii.com.

industry_corpus2: Technology & Research


text:
Unmethylated cytosine-guanine (CpG) motif-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists of Toll-like receptor 9 (TLR9). ODNs with a phosphorothioate (PTO) backbone have been studied as TLR9 agonists since natural ODNs with a phosphodiester (PD) backbone are easily degraded by a serum nuclease, which makes them problematic for therapeutic applications. However, ODNs with a PTO backbone have been shown to have undesirable side effects. Thus, our goal was to develop nuclease-resistant, PD ODNs that are effective as human TLR9 (hTLR9) agonists.
The sequence of ODN2006, a CpG ODN that acts as an hTLR9 agonist, was used as the basic CpG ODN material. The 3'-end modification of ODN2006 with a PD backbone (PD-ODN2006) improved its potential as an hTLR9 agonist because of increased resistance to nucleolytic degradation. Moreover, 3'-end modification with oligonucleotides showed higher induction than modification with biotin, FITC, and amino groups. Further, enhancement of hTLR9 activity was found to be dependent on the number of CpG core motifs (GTCGTT) in the PD ODN containing the 3'-end oligonucleotides. In particular, ODN sequences consisting of two to three linked ODN2006 sequences with a PD backbone (e.g., PD-ODN2006-2006 and PD-ODN2006-2006-2006) acted as effective agonists of hTLR9 even at lower concentrations.
This study showed that PD-ODN2006-2006 and PD-ODN-2006-2006-2006 can be used as potentially safe agonists for hTLR9 activation instead of CpG ODNs with a PTO backbone. We propose these CpG ODNs consisting of only a PD backbone as a novel class of CpG ODN.
Unmethylated cytosine-phosphate-guanine (CpG) motifs are considered to be pathogen-associated molecular patterns because of their abundance in microbial genomes but scarcity in vertebrate ones . Short, single-stranded, synthetic oligodeoxynucleotides with CpG motifs (CpG ODNs) can induce the Th1-type immune response through interaction with Toll-like receptor 9 (TLR9), a member of the Toll-like family of pattern-recognition receptors [2–4]. Because Th1-type cytokines such as IFN-γ and IL-12 can inhibit the Th2-type response [5, 6], CpG ODNs are thought to have potential for various immune therapies such as for cancer, asthma, pollinosis, and infectious diseases [7–9].
To date, at least four different types of immune-stimulatory CpG ODNs with distinct structural and biological characteristics have been reported . The class A CpG ODNs consist of phosphodiester (PD), CpG-containing, palindromic motifs with phosphorothioate (PTO), poly-G motifs at both the 5' and 3' ends. CpG ODNs in this class can induce high levels of interferon-α (IFN-α) production in plasmacytoid dendritic cells (pDCs), but are weak stimulators of TLR9-dependent NF-κB signaling . On the other hand, the class B CpG ODNs consist entirely of PTO backbones and do not form palindromic structures. These ODNs can strongly activate B cells through NF-κB signaling but show no effect on IFN-α production [12, 13]. The class C CpG ODNs show an intermediate immune property between classes A and B, since these CpG ODNs have CpG-containing palindromic motifs consisting of only a PTO backbone . Recently, a novel type of CpG ODN called the P class, which has two palindromic motifs with a PTO backbone, was reported to show higher potential to produce IFN-α and activate NF-κB .
Natural PD-ODNs are susceptible to nuclease degradation, which renders them inactive for TLR9 activation in the free form. Therefore, all CpG ODNs classified above partially or completely contain a PTO backbone to increase resistance to nuclease degradation in the serum [16–18]. However, the PTO backbone has been reported to cause unwanted side effects such as unspecific binding to various proteins  and renal damage . Therefore, because of safety considerations, the development of novel natural CpG ODNs consisting of an entirely PD backbone is desirable. Furthermore, these natural CpG ODNs should be able to strongly activate TLR9-dependent NF-κB signaling. To develop such a CpG ODN, we examined the effects of the sugar backbone, the sequence of the CpG motif, and modification of both terminals in CpG ODNs on NF-κB activation. We found that a series of linked PD sequences of ODN2006 (a class B agonist of hTLR9) has the potential to resist nuclease degradation and to activate NF-κB through TLR9 at even lower concentrations than are known for PTO ODNs.
To develop natural CpG ODNs with a PD backbone that are capable of strong human TLR9 activation, the ODN2006 sequence, a well-known class B agonist of hTLR9 , was used as a basic sequence for designing CpG ODNs. TLR9 activation was assessed by relative NF-κB activation in human TLR9-expressing cells (293XL-hTLR9), since TLR9 activation by CpG ODNs leads to activation of NF-κB . We synthesized an ODN2006 sequence consisting of either a PD or PTO backbone (PD-ODN2006 and PTO-ODN2006, respectively) (Table 1) and examined the effect of the backbone composition on NF-κB activity. PD-ODN2006 showed little potential for activating TLR9, as demonstrated by the increased NF-κB activity, whereas PTO-ODN2006 significantly activated NF-κB in a dose-dependent manner (Figure 1A). Identical to previous reports on the susceptibility of natural PD ODNs to nuclease degradation [16–18], PD-ODN2006 likely showed low potential for activating TLR9 because of degradation in the serum (Figure 1B). In contrast, PTO-ODN2006 still remained as clear band after serum treatment, but a high-molecular-weight band was also observed (Figure 1B). This band could be attributable to proteins binding to the PTO backbone rather than being specifically associated with the PTO-ODN2006 sequences, since Brown et al.  also reported binding of proteins to ODNs with PTO backbones. Our result suggests that PTO-ODN2006 resists nuclease degradation, but that an unfavorable reaction may also be occurring in the serum. Therefore, novel GpG ODNs that can stimulate TLR9 activity need to resist nucleolytic degradation as well as not react with serum proteins.
ODNs: oligodeoxynucleotides; PD: phosphodiester; PTO: phosphorothioate. Capital and lowercase letters in sequences indicate PD and PTO backbones, respectively. Bold sequences indicate the PD-ODN2006 sequence.
Effect of ODN backbones and CpG motif on NF-κB activation. (A) 293XL-hTLR9 cells transfected with hTLR9-signaling reporter plasmid were stimulated by PD-ODN2006 and PTO-ODN2006 with various concentrations. n = 3, mean (sd). Statistical significances are denoted as follows: *P < 0.05, **P < 0.01. (B) Stability of PD-ODN2006 and PTO-ODN2006 in aqueous solution with/without serum. The arrow indicates PTO-ODN-protein complex. (C) PD-ODN2006 and PNA-ODN2006 (0.2 μM) were applied to cells with/without LyoVec. n = 3, mean (sd). Statistical significances are denoted as follows: *P < 0.05, **P < 0.01. (D) 0.1 μM of PTO-ODN2006 mutants (1: PTO-ODN2006, 2: PTO-ODN2006-GC, 3: PTO-ODN2006-1GC, 4: PTO-ODN2006-2GC, 5: PTO-ODN2006-3GC, 6: PTO-ODN2006-4GC, 7: PTO-ODN2006-2,3,4GC, 8: PTO-ODN2006-2,3GC, 9: PTO-ODN2006-2,4GC, 10: PTO-ODN2006-3,4GC) were applied to cells. n = 3, mean (sd). All P values are shown relative to the PTO-ODN2006, *P < 0.05, **P < 0.01.
Peptide nucleic acids (PNAs), which have backbones composed of repeated N-(2-aminoethyl)-glycine units linked by peptide bonds, have been developed to solve the problem of biological instability in short-stranded DNA or synthetic ODNs . Thus, we examined the potential of ODN2006 consisting of a peptide backbone (PNA-ODN2006) for TLR9 activation. However, no enhancement of TLR9 activity was observed (Figure 1C). Interestingly, PD-ODN2006 showed higher potential when LyoVec, a cationic lipid, was used as a carrier to prevent nucleolytic degradation, but the carrier had no effect for PNA-ODN2006. This indicates that the lack of TLR9 activation by PNA-ODN2006 is attributable to PNA and that the sugar backbone of CpG ODN is essential for TLR9 activation.
Next, we tried to design novel CpG ODN sequences with a higher potential for activating TLR9 than ODN2006. To determine if mutation or deletion of CG units could improve CpG ODN design, we prepared different mutants of PTO-ODN2006 (Table 1) by replacing CG unit(s) with GC unit(s) and then examined their potential. NF-κB activity was dramatically decreased by PTO-ODN2006-GC, an ODN in which all four CG units had been replaced with GC ones (Figure 1D), indicating the importance of the CG units for TLR9 activation. Interestingly, the third CG unit in PTO-ODN2006 appeared to be the most important one, since lower activity was observed for PTO-ODN2006-3GC than for the three other ODNs with a single GC substitution (Figure 1D). Similarly, lower activity was also observed for PTO-ODN2006-2,3,4GC, PTO-ODN2006-2,3GC, and PTO-ODN2006-3,4GC, than for PTO-ODN2006-2,4GC. This suggests that the position and number of CG units in ODN2006 are critical for TLR9 activation, and that neither mutation nor deletion of the CG units can enhance activity.
Exonucleases cleave nucleotides one at a time from the terminal end of a polynucleotide chain. Thus, we modified the PD-ODN2006 5' and 3' terminal ends using biotin, FITC, and amino groups (-NH2) to examine whether these changes would improve the potential for TLR9 activation. As shown in Figure 2A, modifications at the 3'-end of PD-ODN2006 enhanced NF-κB activity relative to no modification of PD-ODN2006. However, no significant change was observed by modifying the 5'-end. To clarify why the 3'-modifications improved the activity, we incubated the 3'-modified PD-ODN2006s in serum and found that they showed improved resistance to degradation (Figure 2B).
Effect of terminal modification and linker sequences in PD-ODN2006 on NF-κB activation. (A) 293XL-hTLR9 cells transfected with hTLR9-signaling reporter plasmid were stimulated by modified PD-ODN2006s (1: PD-ODN2006, 2: PD-ODN-3'-Biotin, 3: PD-ODN-5'-Biotin, 4: PD-ODN-3'-FITC, 5: PD-ODN-5'-FITC, 6: PD-ODN-3'-NH2, 7: PD-ODN-5'-NH2, 8: PD-ODN-3'-A, 9: PD-ODN-5'-A, 10: PD-ODN-3'-B, 11: PD-ODN-5'-B) at a concentration of 0.5 μM. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006, *P < 0.05, **P < 0.01. (B) Stability of modified PD-ODN2006s in aqueous solution with/without serum. (C) 293XL-hTLR9 cells transfected with hTLR9-signaling reporter plasmid were stimulated by series connection of PD-ODN2006 with/without linker sequences. n = 3, mean (sd). (D) Stability of these PD-ODNs in aqueous solution with/without serum.
We further modified the PD-ODN2006s by adding two 14-mer oligonucleotides (called A and B) consisting of a PD backbone (PD-ODN2006-5'/3'-A and PD-ODN2006-5'/3'-B, respectively) (Table 1). These ODNs showed enhanced NF-κB activity relative to the unmodified PD-ODN2006 (Figures 2A and 2B). Interestingly, modification at the 5'-end also improved activity (Figure 2A). This seemed to be due to the presence of CG units remaining after degradation, since molecules were detected even after incubation in serum (Figure 2B).
Since the addition of the 14-mer oligonucleotides to the 3'-end of PD-ODN2006 significantly improved stability and led to increased NF-κB activity, we examined the effect of adding other oligonucleotide sequences to the 3'-end of PD-ODN2006. Three types of modified PD-ODN2006 sequences were designed. PD-ODN2006-3'-A-2006 and PD-ODN2006-3'-B-2006 were constructed from an ODN2006 sequence extension at the 3'-end of PD-ODN2006-3'-A and PD-ODN2006-3'-B, respectively (Table 1). For these ODNs, oligonucleotides A and B were linkers between the two PD-ODN2006s. As the third modified sequence, PD-ODN2006-2006 comprised two directly linked PD-ODN2006s. All three structures increased NF-κB activity in a dose-dependent manner (Figure 2C). Although PD-ODN2006-3'-A-2006 and PD-ODN2006-3'-B-2006 enhanced activity to a further extent compared to PD-ODN2006-3'-A and PD-ODN2006-3'-B at the same concentration (0.5 μM) (see Figure 2A), PD-ODN2006-2006 was more effective than PD-ODN2006-3'-A-2006 and PD-ODN2006-3'-B-2006 at every concentration. All three 3'-modified PD-ODN2006s with oligonucleotides resisted nucleolytic degradation as well as the other 3'-end modified PD-ODN2006 (Figure 2D).
It has been reported that the GTCGTT sequence is a core CpG motif for hTLR9 activation . Thus, the effect of the 3'-end extension of PD-ODN2006 by this core motif was examined. PD-ODN2006-3'-TCGTCGTT and PD-ODN2006-3'-TCGTCGTTTTGTCGTT contain one and two core motif(s) at the 3' end, respectively (Table 1). As the PD-ODN2006 sequence contains three core motifs, these CpG ODNs represented a total of four and five core motifs. PD-ODN2006-2006 and PD-ODN2006-2006-2006 also have six and nine core motifs, respectively (Table 1). As shown in Figure 3A, the potential for TLR9 activation depended on the number of core motifs. In particular, PD-ODN2006-2006-2006 enhanced the activity even at a concentration of 0.01 μM. Moreover, PD-ODN2006-2006 showed much higher potential than PD-ODN2006-3'-TCGTCGTTTTGTCGTT at concentrations below 0.1 μM.
Effect of novel CpG-ODNs on NF-κB activation in 293XL-hTLR9 cells and 293XL-null cells. (A) 293XL-hTLR9 cells transfected with hTLR9-signaling reporter plasmid were stimulated by PD-ODN with an elevated number of CpG motifs. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006 with the same concentrations, *P < 0.05, **P < 0.01. (B) Stability of PD-ODNs in aqueous solution with/without serum. The arrow indicates supramolecular structure of PD-ODN2006-G5. (C) NF-κB activation by PD-ODNs (1: PD-ODN2006, 2: PD-ODN2006-2006, 3: PD-ODN2006-2006-2006, 4: PD-ODN2006-G5, 5: PTO-ODN2006) in hTLR9 deficient cells (293XL-null) and hTLR9 expressing cells (293XL-hTLR9 cells). Statistically significant results are denoted as follows: *P < 0.05, **P < 0.01.
Linked series of PD-ODN2006 (e.g., PD-ODN2006-2006-2006) also showed higher potential than that reported for PD-ODN2006-G5, the backbone of which consists of PD entirely [24, 25]. These differences were especially significant at the lower concentrations (Figure 3A). A higher molecular band was observed for PD-ODN2006-G5, which may be attributable to a supramolecular structure formed by the poly-G tails . However, none were observed for the ODNs consisting of PD-ODN2006 in a series (Figure 3B). To examine whether NF-κB activation by PD-ODNs is mediated by TLR9, we used 293XL-null cells. These TLR9-deficient cells did not show any significant increase in NF-κB activity when stimulated either by PTO-ODN2006 or our novel PD-ODNs (Figure 3C).
We also assessed the potential of PD-ODNs using B lymphocyte Ramos-Blue cells. PD-ODN2006-2006 had similar potential to PD-ODN2006-G5, but less than PTO-ODN2006. However, the potential of PD-ODN2006-2006-2006 was much higher than that of PD-ODN2006-G5 and almost the same as PTO-ODN2006 (Figure 4A). In addition, PD-ODN2006-2006-2006 had higher potential to induce IL-6 secretion in peripheral blood mononuclear cells (PBMCs) than PD-ODN2006-G5 and PTO-ODN2006, although no significant change was observed (Figure 4B). We also examined the IL-6 secretion in plasmacytoid dendritic cells (CAL-1). For PD-ODN2006-2006-2006, the secretion of IL-6 was higher than PD-ODN2006-G5 and PTO-ODN 2006, which was consistent with what was observed in the PBMCs. However, the secretion of IL-6 by the stimulation of PBS and PD-ODN2006 was not detected in this cell line (Figure 4C).
TLR9 activation by B lymphocytes, PBMC, and CAL-1 cells. (A) Activation of NF-κB in human B lymphocyte cell lines, Ramos-Blue cells, stably transfected with NF-kB/AP-1-inducible SEAP reporter gene. n = 3, mean (sd). All P values are shown relative to the PD-ODN2006: *P < 0.05, **P < 0.01. (B-C) IL-6 production induced by CpG-ODNs (1: PD-ODN2006, 2: PD-ODN2006-2006, 3: PD-ODN2006-2006-2006, 4: PD-ODN2006-G5, 5: PTO-ODN2006). n = 3, mean (sd). From human PBMCs (B) and human plasmacytoid dendritic cells CAL-1(C). The symbol # indicates that no cytokine secretion was detected.
Synthetic oligonucleotides in recent use are usually protected from degradation by modification of the backbone, whereas natural CpG ODNs are readily degraded by ubiquitous nucleases both in vivo and in vitro [16–18]. The CpG ODNs developed in this study consist of only a natural PD backbone, which may eliminate the side effects introduced by the PTO backbone modification. Moreover, by connecting two or three simple ODNs, we obtained novel CpG ODNs with better nuclease tolerance and higher immunostimulatory potential in hTLR9-dependent NF-κB activation.
We used the sequence of a typical class B CpG ODN (ODN2006) to develop novel agonists for human TLR9. In our in vitro model system, it was shown that natural PD-ODN2006 likely had very little potential for activating NF-κB due to degradation by serum nucleases. TLR9 activation requires the internalization of CpG ODNs into the late endosome/lysosome of TLR9-expressing cells for recognition. Therefore, protection of the ODNs from degradation is an initial step in maintaining their activity. Hence, PTO backbones are commonly used to protect the open ends of linear oligonucleotides. Although PTO-ODN2006 demonstrated much higher potential for TLR9 activation than PD-OD2006, it was not a perfect candidate as an immunotherapeutic agent. The PTO backbone modification led to nonspecific interactions between PTO-ODN2006 and serum proteins, as shown with the high-molecular-weight band for this ODN, whereas no bands were obtained for PD-ODN2006. This result also suggests that the side effects of PTO-ODNs may be a product of the backbone modification and may not be related to the increased half-life.
PNA is a DNA analog in which the natural phosphodiester backbone of DNA is replaced by a 2-aminoethyl-glycine linkage. PNA modification is widely used to increase the biological stability of short-stranded DNA when developing antisense technology . We examined the effect of the PNA-ODN2006 sequence, but no significant increase in activity was observed. Haas et al. suggested that the DNA sugar backbone (2' deoxyribose) determines TLR9 activation because they found that the corresponding RNA ribo-oligonucleotides could not activate TLR9 . In our study, the inhibitory effect of PNA-ODN also supports the suggestion that the 2' deoxyribose in the DNA plays a crucial role in TLR9 activation.
We concluded from the decreased activity of the mutated PTO-ODN2006 that all the CG units in ODN2006 were essential for activation. Moreover, the third CG seemed to be the most important unit for TLR9 activation. Hence, protection of the CG units from degradation in natural PD-ODN2006 is crucial for maintaining its immunostimulatory activity. Modification of ODN2006 with either organic molecules at the 3'-end or oligonucleotides at the 3'- or 5'-ends enhanced the stimulatory activity. This enhancement can be explained by the increased resistance of the ODNs to nuclease degradation. Although it has been reported that free 5' ends in CpG ODNs with a PTO backbone are required for the activation of TLR9 [28, 29], loss of activity in 5'-modified PD-ODN2006 in this study was attributed to degradation. When we modified PD-ODN2006 with 14-mer oligonucleotides at the 5'-end, there was still some potential to activate NF-κB, with some bands still present after serum degradation.
To further increase the immunostimulatory activity of the 3'-modified PD-ODN2006, we next prepared a series of linked PD-ODN2006 sequences (PD-ODN2006-3'-A-2006 and ODN2006-3'-B-2006), which increased the number of CG motifs in each oligonucleotide. Surprisingly, PD-ODN2006-3'-B-2006 led to higher activity than PD-ODN2006-3'-A-2006 at every concentration, which indicated that the linker sequence can affect the potential of CpG ODNs for NF-κB activation. Finally, we removed the linker, generating PD-ODN2006s such as PD-ODN2006-2006 and PD-ODN2006-2006-2006. The enhanced potential for NF-κB activation obtained by CpG-ODN2006-2006 compared with ODN2006-A/B-2006 demonstrated that the linker sequences used in this study inhibited the activation of TLR9. Moreover, PD-ODN2006-2006-2006 showed much higher activity than PD-ODN2006-2006, indicating that the number of CG motifs played a key role in the activity. To verify that the ODN2006-linked sequences activate NF-κB activation through the TLR9 pathway, we applied PD-ODN2006-2006 into a 293XL-null cell line that does not express hTLR9. Consequently, we observed no NF-κB activation in this cell line. Therefore, PD-ODN2006-2006 is thought to indeed activate NF-κB through the TLR9 pathway. Hence, these series of PD-ODN2006 sequences might be used as novel and effective TLR9 agonists.
To our knowledge, there are a few reports on nuclease-resistant immunostimulatory CpG ODNs with entirely PD backbones. Lee et al.  reported that the conjugation of poly-guanosine (poly-G) sequences at the 3'-end of PD-CpG ODNs significantly enhanced TNF-α and IL-12 production from mouse splenic dendritic cells. Bartz et al.  modified PD-ODN2006 with poly-G sequences and increased the cellular uptake and stimulatory activity of PD-ODN2006 in human leukocytes. They suggested that the increase in the activity resulted from the enhanced cellular uptake, which was attributed to the formation of ODN tetrads, G-Quadra structure. We also observed a supramolecular structure for PD-ODN2006-G5, as indicated by the presence of a high-molecular-weight band from agarose electrophoresis. However, no high-molecular-weight aggregate was formed for PD-ODN2006-2006. Atomic force microscopy also revealed that class A CpG ODNs with poly-G sequences form a particle structure, while class B PTO-ODN2006 forms a linear structure . Clear bands of PD-ODN2006-2006 and PD-ODN-2006-2006-2006 remained after serum degradation, indicating that the series of natural PD-ODN2006s preserved the potential for TLR9 activation due to enhanced stability. We also speculate that the increase in the number of CG motifs in the oligonucleotides played an important role in the enhanced TLR9 activation potential. Our novel PD-ODNs demonstrated higher NF-κB activity in not only the 293XL-hTLR9 model cell lines but also B lymphocyte cell lines (Ramos-Blue) when stimulated with PD-ODN2006-2006 and PD-ODN2006-2006-2006.
In this study, we examined the production level of IL-6 because our CpG ODNs are considered to have the same linear structure, which is proved to activate only B cells but not dendritic cells that produce IL-12 and type I interferon, as class B CpG ODNs [12, 13]. Our PD-ODNs also showed a higher potential for the production of IL-6. Therefore, our CpG-ODNs are suitable for immunotherapeutic use.
The purpose of this study was to develop safe and effective CpG ODNs consisting of a PD backbone for hTLR9 activation. Our data indicate that 3'-modified CpG ODNs consisting of a PD backbone are effective at resisting nucleolytic degradation, and that the number of CpG core motifs strongly influence their potential as hTLR9 agonists. In particular, CpG ODNs with linked PD-ODN2006 sequences (e.g., PD-ODN2006-2006 and PD-ODN-2006-2006-2006) can act as effective and potentially safe agonists of hTLR9 at low concentrations.
293XL-hTLR9 cells stably expressing hTLR9 and 293XL-null cells were purchased from Invivogen (CA, US). Cells were grown in high-glucose, Dulbecco-modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin, 50 mg/mL streptomycin, and 10 μg/mL blasticidin at 37°C in humidified air containing 5% CO2. Cells were seeded in 48-well culture plates for transfection and cell stimulation. A B lymphocyte cell line that stably expresses an NF-kB/AP-1-inducible secreted embryonic alkaline phosphate (SEAP) reporter gene, Ramos-Blue cell line (Invivogen), was cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (FBS), 2 mM L-glutamine, 50 units/mL penicillin, and 50 mg/mL streptomycin. Peripheral blood mononuclear cells (PBMCs) were purchased from Cellular Technology Limited (OH, US) as frozen cells. The procedures for the thawing of PBMCs were adapted from the manufacturer's protocol. The plasmacytoid dendritic cell line, CAL-1 was maintained in RPMI 1640 medium supplemented with 10% FBS as indicated in the report .
ODNs consisting of phosphodiester (PD) and phosphorothioate (PTO) backbones, and ODNs modified with biotin, FITC, or amino groups (-NH2) were purchased from Fasmac Inc. (Kanagawa, Japan). The ODNs were diluted in sterilized water and stored at -20°C. Peptide nucleic acid was purchased from Panagene (Daejeon, Korea).
To monitor transient NF-κB activation, 293XL-hTLR9 cells and 293XL-null cells were seeded at 1 × 105 cells per well and transiently transfected with pNiFty-luc (a TLR9-signaling reporter plasmid, Invivogen) and pGL4.74 (a Renilla luciferase gene containing plasmid, Promega, WI, US), using LyoVec (Invivogen). After 24 h, cells were stimulated with oligonucleotides. These cells were lysed using a passive lysis buffer after 24 h, and lysates were assayed for luciferase activity using a luminometer (TD-20/20, Promega) according to the manufacturer's instructions. The data were represented as relative NF-κB activity compared with that of non-stimulated control cells.
B lymphocyte Ramos-Blue cells were seeded at a density of 2 × 106 cells/mL, and the cells were immediately stimulated with 0.5 μM ODNs. Cell supernatants were collected, and NF-κB activation was examined by the reporter gene SEAP expression levels using a spectrophotometer at 620 nm.
Twenty microliters of ODNs at a final concentration of 10 μM was incubated in an aqueous solution containing 20% FBS at 37°C for 16 h. All samples were subsequently treated with 2 μL of 250 mM EDTA for 2 min at 80°C to quench the digestion reaction. The integrity of the ODNs was then analyzed by gel electrophoresis using a 4% agarose gel.
After thawing, PBMCs were seeded in serum free medium, CTL-test medium (Cellular Technology Limited), at a density of 1.0 × 106 cells/mL. Cells were immediately stimulated with 0.5 μM ODNs. After 24 h of incubation at 37°C, cell supernatants were collected and stored at -20°C until further analysis. CAL-1 was seeded at a density of 5.0 × 106 cells/mL and stimulated with 1.5 μM ODNs for 48 hours. Supernatants were collected and stored at -20°C until further analysis. The level of human IL-6 in the media was determined by enzyme-linked immunosorbent assay (ELISA) using the Ready-SET-Go! Set (eBiosciences, San Diego, CA). The procedures for the ELISA for IL-6 were adapted from the manufacturer's protocol.
Stoic analysis was performed using Student's t-test. A p-value less than 0.05 was considered to be statistically significant.
We thank Dr. Takahiro Maeda for supplying the plasmacytoid dendritic cell line CAL-1. This study was supported by a Grant-in-Aid for Scientific Research (C-22560777) from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
WM carried out most of the experiments including the NF-κB luciferase assay and DNA degradation experiments, performed the statistical analysis, and drafted the manuscript. YT participated in the design of the study and the statistical analysis, contributed to most of the experiments and helped draft the manuscript. NY participated in the preparation of plasmids and cell maintenance. HN initiated and supervised the study and revised and finalized the manuscript. All authors read and approved the final manuscript.

industry_corpus2: Biopharmaceuticals


text:
As anyone who follows the Hugo Awards will already be aware, the 2016 ballot was decided in large part by one man: Vox Day. As part of his Rabid Puppies campaign, the self-styled “national libertarian” provocateur sent his followers to vote for a slate that he himself assembled. The Rabid Puppies also affected last year’s Hugos; the campaign was in turn inspired by the less extreme Sad Puppies, which began in 2013. But previously, neither movement showed much interest in comics. Before 2016, the Sad and Rabid slates contained just two Best Graphic Story entries between them, both webcomics: Howard Tayler’s Schlock Mercenary and Carter Reid’s The Zombie Nation. Things changed this year, as Best Graphic Story is one of four Hugo categories to be dominated entirely by Vox Day’s choices.
In 2015, the Worldcon voters reacted to the Puppies by placing almost every slated finalist beneath the “No Award” option. When assembling his 2016 slate, Vox Day preempted this response by including a few popular choices that would likely have been nominated anyway. These include Andy Weir for Best New Writer, The Martian for Best Dramatic Presentation (Long Form), and File 770 for Best Fanzine.
Such picks have been dubbed “human shields” by commentators, as Day was presumably intending to leave his opponents with a dilemma. Should they vote for these nominees, as they would have done ordinarily, and thereby, allow the Rabid slate to “win?” Or should they continue to place the Puppy picks below No Award, even if it meant depriving their favoured nominees of victory?
In Best Graphic Story, the human shield was clearly The Sandman: Overture by Neil Gaiman and J. H. Williams III. After all, Gaiman had already won four Hugo Awards, and the return of his most iconic creation was not something that Worldcon’s voting base would pass by.
But with the book nominated on the back of the Rabid Puppies campaign, some of Gaiman’s admirers expressed hope that, having previously won the award multiple times, he would withdraw the nomination and allow a non-Rabid candidate to take his place on the ballot. Gaiman expressed resentment at being on “that creepy racist’s slate,” but stressed that The Sandman: Overture was a collaborative work and, consequently, not his to withdraw.
The ballot includes two other graphic novels. One of these is Invisible Republic Volume 1 by writer-artist Gabriel Hardman, co-writer Corinna Bechko, and colourist Jordan Boyd.
A twenty-ninth century journalist explores the remains of a failed dictatorship and a future so run-down that it resembles the present. He comes across a journal that once belonged to Maia Reveron, cousin of the former dictator Arthur McBride. The story then splits into two branches: one follows the reporter as mysterious pursuers try to obtain the journal from him, while the other follows Maia Reveron as she witnesses her cousin transform from fugitive to Che Guevara-like icon.
Invisible Republic Volume 1 collects the first five issues of the series. Taken together, these cannot be said to constitute a complete narrative arc—the mystery story still has more questions than answers at the volume’s end—but they certainly make for a promising start. Between its stark, noir-inflected artwork and a gripping storyline about the nature of revolution, Invisible Republic is very much a series to keep an eye on.
The last of the three graphic novels on the ballot is The Divine by writer Boaz Lavie and artists Asaf and Tomer Hanuka. The story was inspired by a photograph taken in Thailand in 2000, showing a pair of child soldiers: twelve-year-old twins Johnny and Luther Htoo, one weary-faced, the other smoking a cigarette. Leading the God’s Army guerrilla organisation on the border between Thailand and Burma, the two children gave rise to a set of local legends alleging that they had supernatural powers.
The Divine’s treatment of these legends follows the general template of Joseph Conrad’s Heart of Darkness and Francis Ford Coppola’s Apocalypse Now, accompanying a Western traveler on his journey to an exotic land of mystery and horror, which causes him to have second thoughts about his own culture.
The protagonist, Mark, is an American explosives expert who accepts a risky job in the fictional Southeast Asian nation of Quanlom so that he can afford better prospects for his newly-pregnant wife. While there, he encounters the Divine: a pair of nine-year-old twins who resemble a cross between the Htoo brothers and the parchment-faced children from Katsuhiro Otomo’s Akira. Mark learns that the operation was not as humane as he had assumed and stands alongside the two children as they use magic to defend their homeland.
Boaz Levie’s straightforward story of magical Asians fighting macho bigoted Americans is brought to life by the Hanuka brothers’ artwork. The characters are well-observed, the Southeast Asian backgrounds convincing, and the occasional forays into body horror (again recalling Akira) suitably jolting. Most striking of all is the comic’s colouring: the palettes are kept generally muted, but with sudden splashes of brighter hues for punctuation, particularly during the supernatural scenes. The results are thoroughly effective.
Full Frontal Nerdity is a geek-humour strip in the vein of Penny Arcade, with a general emphasis on roleplaying. I will have to admit that this genre has never been my bag, and Nerdity does nothing to convert me. An in-depth familiarity with roleplaying seems necessary to understand the build-up of most strips, let alone the punchline, while the dialogue is decidedly long-winded. Brevity is vital to a three-panel gag-strip, and there is little space for sentences like “I was in California, beta-testing the new theme park attraction based on the Mass Effect video games.” That said, given that Full Frontal Nerdity maintains a loyal enough audience to have been running regularly since 2003, it seems to have hit the spot within its target demographic.
Erin Dies Alone is more interesting. It stars a lonely young woman who is suddenly joined in her run-down flat by Rad Panda, a Sonic-like character from a video game she played as a child. As the protagonists segue in and out of game-worlds, the strip spoofs popular franchises such as Halo and Final Fantasy while also probing the clearly troubled mental state of its heroine.
The comedy in Erin Dies Alone once again comes from the tradition of the geek humour strip, but the surrounding story is adds a little more psychological depth than is typical for the genre. The result is worth a look, although I am not sure that I would not call it award-worthy.
As the above ballot indicates, comic readers of 1941 had a vintage year to look back upon. The first issue of Batman had hit the newsstands, while three other long-lasting heroes—Captain Marvel, the Spirit, and the Spectre—each made their debut. Meanwhile, Flash Gordon (still drawn by his original creator, Alex Raymond) was exploring the Ice Kingdom of Mongo.
Each of these characters maintains an enduring appeal among comic fans, and the amount of mud-flinging and dirty tricks that surround this year’s Hugo Awards should remind us why.
Note: An earlier version of this article incorrectly stated that The Zombie Nation was the only comic to appear on a puppy list prior to 2016; in fact, it shares this honour with Schlock Mercenary. This error has since been fixed.

industry_corpus2: Film & Entertainment


text:
The three major credit agencies now allow you to freeze your credit for free.
A reporter was recently confused for a serial felon when a landlord did a tenant screening on him.
A federal jury ordered Equifax to pay an Oregon woman $18.6 million after her two-year fight to correct her score.

industry_corpus2: Finance & Economics


text:
Oakwood Massage and Bodywork is here to offer restorative massage and bodywork to all.
After bilateral total knee replacements, i have suffered with stiffness and range of motion issues. Nadia has helped me tremendously. I have had a few massages from her and she is now, by far, the best therapist i have had. She has great skill but also an intuitive healing touch. Always professional and prepared, my experience with her has always been great. I would highly recommend her.
50 minutes of hands on massage and bodywork of any modality.
Welcome to Oakwood Massage and Bodywork where restorative, relaxing, and rejuvenating massage is offered to all. Equipped with the training to do a variety of massage and bodywork modalities, I focus on treatment based, individualized sessions to meet each client's needs. Whether you're searching for some much needed pampering or needing recuperative bodywork for a serious ailment or injury, you've come to the right place. I invite you to book a session today and take the edge off!
Service Received: 60 minute session with Nadia P.

industry_corpus2: Health & Medicine


text:
In the 2012 elections for the House of Representatives, Republics won 54% of the races despite receiving fewer votes nationwide than the Democrats. In 2016, the Republicans eked out a narrow victory in the nationwide popular vote, 51% to 49%, but ended up controlling 55% of the seats.
The Senate: The Vestigial House?
We often hear the United States Senate referred to as “the world’s greatest deliberative body.” That expression was coined by the fifteenth President of the United States, the otherwise unremarkable James Buchanan. You could say that the United States Civil War was just Abraham Lincoln cleaning up the mess Buchanan left behind. Since Buchanan was himself once a Senator, calling a body of which he was a member “the world’s greatest” anything smacks of shameless self-promotion.

industry_corpus2: Politics & Administration


text:
What you gonna clamp together?
These are to hold things in place while I prep the forms for concrete.
I like cast aluminum clamps. Light weight and no rust except for the threaded steel shaft.
When I use to do construction by myself, clamps were my 3rd 4th and 5th hands.
I C what you did there.

industry_corpus2: Real Estate & Construction


text:
The Town of Orono map viewing site is built with modern web technology, so the controls and how it works should be familiar because they are like other websites you visit. The site and maps can be used on iPhones and Android phones, tablets, and desktop/laptop computers. The site can be used to display different layers (data) about the town, including parcels, rental registration, trails, soils, open spaces, and zoning. The data are maintained by the Town of Orono.
The maps are intended for planning purposes. Please contact the Town of Orono with questions.
This Spatial IT/Geographic Information System (GIS) site was built and is managed by Corson GIS Solutions.
A number of maps showing the natural resource characteristics of Orono are available here. These should be used for planning purposes only. They are only approximate at the parcel or small area level. Mapping required for permits needs higher resolution, site-specific surveys.
You can link to the Tax Assessor's property maps online.

industry_corpus2: Computing & Telecommunications


text:
Join Makin' it Happen on February 19th for Hear it Here!
Addiction Treatment & Recovery – Where? How? What works?
Focused on educating the community about potential warning signs, when to worry, and what to do if you have concerns. We want the public to be aware of the innovative and collaborative work being done to better understand and treat substance use disorders. We want to help you to understand the types of treatment and recovery support services available, how to access them.


text:
what some may call my deathbed.
Is there a faint whisper?
I hold my breath to hear it.
a catching on a thought.
given a few more days on earth.
as I lay complexities aside.
What is this long-lost feeling?
this air will not be breathed.
toward our one and only Light.
Copyright © 1999 by Alan Harris. All rights reserved.

industry_corpus2: Literature & Emotions


text:
This leaflet answers some common questions about Eleuphrat.
All medicines have risks and benefits. Your doctor has weighed the risks of you using Eleuphrat against the benefits it is expected to have for you.
The name of your medicine is Eleuphrat. It contains the active ingredient called betamethasone dipropionate.
It is a type of cortisone and belongs to the group of medicines called corticosteroids. Eleuphrat is classified as a high potency topical corticosteroid.
Your doctor may have prescribed Eleuphrat for another reason.
Ask your doctor if you have any questions about why Eleuphrat has been prescribed for you.
Do not use Eleuphrat just before having a bath, shower or going swimming. If you do, you may reduce the effectiveness of Eleuphrat.
Do not use Eleuphrat after the expiry date (EXP) printed on the pack has passed. If you use it after the expiry date has passed, it may not work as well.
Do not use Eleuphrat if the packaging shows signs of tampering.
Your doctor will tell you if you can use Eleuphrat during pregnancy or while you are breast feeding.
Apply a thin film of Eleuphrat Cream or Ointment to the affected skin twice daily. Massage gently until it disappears. For some patients, once daily application may be enough for maintenance therapy.
It is important to use Eleuphrat exactly as your doctor has told you. If you use it less often than you should, it may not work as well and your skin problem may not improve.
Do not use Eleuphrat for more than four weeks at a time unless your doctor tells you.
If you forget to use Eleuphrat, use it as soon as you remember and then go back to your normal time for applying Eleuphrat. Do not try to make up for the amount you missed by using more than you would normally.
Telephone your doctor or Poisons Information Centre (Phone 13 11 26) or go to the accident and emergency centre at your nearest hospital immediately if you think that you or anyone may have swallowed Eleuphrat.
Tell all doctors and pharmacists who are treating you that you are using Eleuphrat.
Tell your doctor if you feel that Eleuphrat is not helping your condition or if your skin condition worsens or seems infected.
Tell your doctor if, for any reason, you have not used Eleuphrat exactly as prescribed. Otherwise, your doctor may think that it was not effective and change your treatment unnecessarily.
If you become pregnant while using Eleuphrat, tell your doctor.
Do not use Eleuphrat under dressings or on large areas of skin unless your doctor tells you.
Do not use plastic pants or tight fitting nappies if Eleuphrat is to be used on the nappy area of young children.
Do not use Eleuphrat in or near the eyes.
Do not give Eleuphrat to anyone else even if their symptoms seem similar to yours.
Do not use Eleuphrat to treat other conditions unless your doctor tells you. Your doctor has prescribed Eleuphrat specially for you and your condition. If you use it for another condition, it may not work or make the condition worse.
Only use Eleuphrat on skin areas that rub together such as under the arm or in the groin area if your doctor tells you.
Only use Eleuphrat on the face if your doctor tells you.
If improvement does not occur within one week, tell your doctor.
Tell your doctor if you do not feel well while you are using Eleuphrat. Eleuphrat helps most people with skin problems but it may have unwanted side effects in a few people.
Eleuphrat may cause other side effects.
Side effects that may happen with oral or injectables corticosteroids may also occur with corticosteroids used on the skin, especially in infants and children.
Keep Eleuphrat in a cool dry place where the temperature stays below 25°C.
Do not store Eleuphrat or any other medicines in the bathroom or near the sink. Do not leave it in the car or on window sills. Heat and dampness can destroy some medicines.
Keep Eleuphrat where children cannot reach it. Keep the medicine away from pets. A locked cupboard at least one-and-a-half metres above the ground is a good place to store medicines.
If your doctor tells you to stop using Eleuphrat or it has passed its expiry date, ask your pharmacist what to do with any that is left over.
Eleuphrat Cream is a white to off-white cream. It is packed in 15 g tubes.
Eleuphrat Ointment is a white to off-white ointment. It is packed in 15 g tubes.


text: In February of 2016, the Foundation welcomed Lisa Jennings as the new Executive Director. A native of Winchester, Lisa had previously served on the Board of Directors of the Community Foundation as Treasurer. She has a B.A. in Entrepreneurship and Management Information Science from Ball State University, as well as a B.S. in Accounting. Prior to joining the Foundation as Executive Director, Lisa worked in the Wealth Management Divisions of First Merchants Trust Company and Old National Trust Company for a combined 15 years. She is married to David Jennings and together they have twins, Ashley and Alex.


text:
The Mission for Biblical Literacy continues its efforts to "Stamp Out Biblical Illiteracy" while also offering "Hands On" Biblical Service through International Mission Project: MBL is currrently serving in Russia, Armenia, Nicaragua, and Haiti.
Since its inception, MBL has been in partnership with the Christian College of Georgia, a Disciples of Christ Specialized Institution of Higher Education. Both ministries place a high priority on Biblical Literacy.


text: With this latest in the The Worst Case Scenario Handbook series, readers can indulge their morbid fantasies and action hero daydreams while maybe even picking up a helpful tip for the next time they get stuck in quicksand, ride an out-of-control camel or suffer frostbite. Piven and Borgenicht turn survival into a fun guessing game: with over 200 illustrated dilemmas in multiple choice quiz format, each with a level of difficulty rating, readers can test themselves (or their friends) to see how they might fare if caught in harm's way. From the outback (facing down a mob of kangaroos) to the urban jungle (falling through a Manhattan subway grate), the authors sketch out an array of dilemmas and solutions. Some of the situations cleverly combine titillating action with useful advice: if diving out a window into a dumpster is ever your only escape route from an intruder, be sure to ""jump straight down, executing a three-quarter somersault to land on your back so as not to break bones."" Other scenarios (eating poison berries, suffering severe dehydration) are more mundane and perhaps more likely. But the humorously far-fetched dilemmas (battling a sword-wielding renaissance fair actor, swimming through piranha) make for compulsive reading. Scattered throughout are tidbits including ""Which Is Your Worst Case?"" sections, Hobson's choice-style dilemmas (blizzard or a sandstorm?) with helpful facts to aid your decision. Of course this entertaining volume is hardly a practical survival guide, but readers will be compelled to test their survival know-how-just in case.


text:
Durban rap artist Judas takes us to Abu Dhabi with his new smash single. Things are looking up for this one, cant wait to see the video.
Dreamteam have been quiet for a while now but seems like they are back to please they fans.
Lack inspiration lately? Here’s a nice set of quick tips that should help you find inspiration in the studio.
1. Using reverb can push elements towards the back of the soundstage. If you want to add some reverb, but still keep the sound “up front”, try increasing the amount of pre-delay on the sound. This will detach the sound from its reverb, so your element remains more “up front” while you still have a nice reverb effect.
2. When using reverb/s, make use of busses with the reverb/s on them, and send your channels to the busses. This not only saves on CPU, but also makes the reverb much easier to control. Just moving a couple of faders on our busses can make the overall mix “wetter” or “dryer”.
3. As a general rule, leave all of your mix automation somewhere towards the end of the mixing process. Get your mix and arrangement sounding good, then go in and add your automation tweaks.
4. If you are just starting to train your ears consider including a spectrum analyzer on your master bus and a great track that you’d like to emulate as part of your autoload. As you go along making your track, try A/B-ing your track against the track you want to emulate and analyzing each with the spectrum analyzer. NOTE: simply copying what you see on the Pro track does not mean that your mix is going to be great – things like the key of the track make a difference. However, if you do this with different reference tracks, you’ll start to notice general similarities in the spectrum of highly produced tracks in your genre. Notice things like where the low end is rolled off, how the mids look in relation to the highs etc. Above all use your ears!
5. Don’t get caught up in the “I must have every new plugin” game. Find ones that you really like and focus on mastering them before you add more. Learn your craft.
6. If you like to gate things to your drums, create a dedicated sidechain trigger track – so you can keep those nice pumping effects even when your main drums are muted. This can be as simple as copying your drums to another channel with no output (or muted) and having things sidechained to that channel.
7. It’s often a good approach to lay down the musical elements of your track before writing any basslines – the musical elements will help give your bassline direction and ensure that they all fit together nicely.
8. Using vocal samples? Pay attention to the key and transpose them so that they match the key of your track – if you aren’t sure about this see our post on understanding key. Once you’ve found a nice fit, a good trick on energetic tracks is to then tune the sample up by 10-20 cents. The slightly sharper tone with give your vocals a bit more punch, as many vocalists tend to sing slightly flat.
9. Want to fatten up that synth sound? Tune one of the oscillators on your synth down an octave (12 semitones). Tuning an oscillator up 7 semitones also gives you a nice open-5th sound, which works great on big pads or leads.
10. Planning on taking your Ableton track somewhere else to work on it? Don’t forget to use the “Collect all and save” feature when saving it. This will make sure that all of your project files are collected from wherever they may be on your computer and packed nicely into a handy folder that you can throw on your drive. No more getting there and realizing that you’re missing a few of the key audio clips!
11. If you’re looking to fatten your synth sounds, don’t overlook the “unison” button (you’ll find this on the ES2 for example, as well as many other hardware and software synths) – try playing around with this to see how the additional voices thicken things up quickly.
12. Try using a delay send-effect to simulate loops playing at different tape speeds. The delay time should NOT be synced to tempo, but just increase linearly over the duration of the process (you can just draw this automation in). You’ll get some interesting results at various stages, which you can again bounce down – and this works nicely for things like vocals and pads as well.
13. Looking for some interesting twists on your drum loops? Try copying a percussive loop to two tracks. Shorten one of the loops by a 16th note, and then repeat both loops for 16 measures. The shorter note will fall behind a 16th each measure and then , at measure 16, be back in sync. Among these 16 bar segements, you’ll find some interesting variations, which you can then bounce down as a new loop. (You can also shorten one loop by a 16th note triplet and get some interesting syncopation and swing into the pattern).
14. If you are using Ableton in a studio setting, you may want to optimize it’s audio quality when bouncing your track down.
15. Have your drums channels mixed the way you like them? Then group them, so that you can adjust the overall drum level in the mix while keeping the relative drum channel levels the same. In Ableton, this is as simple as shift-clicking on all your drum tracks and then hitting Cmd-G or Ctrl-G.
16. If you are mixing in loops and rhythm elements from a lot of different sources, it helps to use a frequency analyzer when mixing them and keep an eye on the spectrum. You can use a little EQ to even out any frequencies that stand out – generally you are aiming for a fairly flat spectrum.
17. Most people just use the default settings for quantization in logic, or import templates – but the extended quantize parameters in logic are extremely versatile and useful. These include things like Q-Swing, Q-Strength, Q-Range and Q-Flam. The key command for bringing up these extra quantization parameters in Logic is “P” – or you can find these in the extended parameters window which by ‘Right Clicking’ or ‘Control Clicking’ on “Quantize” and selecting ‘Extended Parameters’.
18. If you are using Logic’s ES2 for Bass sounds, don’t overlook the “Sine Level” pot. Adding a bit of this can give your patch a touch more bass end as it is a sine-wave generator tuned at oscillator 1’s pitch and reinforces the original signal. Also, play around with putting the ES2 into Unison mode, set to mono, and adding more voices. Watch your ears and monitors!
19. Did you know that best practice is to bounce down to the format that is specific to your platform? If you are on a MAC it’s probably safest to bounce to AIFF and if you are on a PC bounce to WAV to get the best results.
-Control-click or right-click the Sine label, and choose a waveform from the pop-up menu.
-Click-hold the Sine label and drag the mouse vertically.
-Shift-click the menu, and type in a value, to select the Digiwave numerically.
21. After some interesting random percussive rhythms to apply on top of a beat in Logic? Try loading something like a single Tom sound mapped across your keyboard in the EXS24. Once you’ve done this, draw in an empty MIDI region and draw in a trigger on each 16th for your looping area. NOW go to Functions/Transform and select “Reverse Pitch”. On the “Flip” button select “+/-Random” and increase the amount to 24 (basically this will randomize each 16th by pitch randomly over two octaves). The select “Operate”. Try it!
22. Have a muddy mix? A high-pass filter applied to every channel can really help clean up a busy mix and give the kick and bass some room to move.
23. If you are looking for interesting effects, try adding sending an element to a bus with chorus on it, set to “pre” and then panning the effected sound to the rear and reducing the volume. If you do this while keeping the original signal in the front you can get some interesting effects.
24. Using Cubase and looking for that “disco re-edit” sound or a more live feel generally in your original track? Try looking beyond quantization swing and play around with the Tempo record function. Within the Tempo window (Alt/Cmd-T), just move the slider to your desired tempo while the track is playing back. Maybe leave the intro and outro nicely locked for mixing and add a little fluctuation in the middle to give the whole track some new swing?
25. When using Pro Tools, adjust the Hardware Buffer Size appropriately in the Playback Engine set up window. As a general rule, keep it low (up to 128 samples) for recording to avoid latency, unless you use the low – latency monitoring options, in which case, any processing on armed tracks will be bypassed. Use a high buffer size for mixing, to maximise processing.
26. Having Plug-in Buffer problems in Logic? Be aware that plug ins like the Adaptive Limiter will produce their own latency problems, so don’t use these until all recording is complete. The Multipressor will cause similar issues as will the use of too many ‘regular’ plug-ins. Bounce a file of your track if you need to record audio over a ‘busy’ backing track.
27. Did you know that if you drag a quick-time movie with audio into arrangement view in Live the video will pop open and you see the movie audio just like any other audio sample? You can then use the “Loop Brace” to create movie loops and use warp markers to radically speed up and slow down the movie.
28. Don’t overlook the time saving features of Key Command Macros in cubase. Want to quantize, trim note legnth, and fix velocity of your MIDI in one go? Create a new Macro in your key commands window and then add “MIDI – fixed lengths”, “MIDI – fixed velocity”, and “MIDI – Quantize”. Then select your MIDI part and apply the Macro (bottom of the edit window). Figure out which things you might frequently do and create useful macros for yourself.
29. Your computer will run better when it’s plugged into the mains. So, if you’re doing CPU intensive work, whenever possible make sure you are plugged up.
30. If you are using Reason and want that great reversed vocal or drum sound before the original, there’s a nice one-key way to do this. Open your sample in the audio editor and reverse it – then set the playback mode to “Forward + Backward” and you’ll hear the sound nicely swell into itself.
31. No matter what your DAW, the smallest buffer size possible should be used when recording to prevent latency – but when on playback you can increase this to avoid system overload messages.
33. It is advisable not to compress your whole mix prior to mixdown but just to concentrate on getting a good mix. Leave worrying about power and loudness for when you pull the bounce into a new mastering session. This way, if your track is picked up, you’ll have a nice raw track so send to a proper mastering engineer.
34. If you are tired of waiting for Live to analyze your files when you drop them into your sets, just browse to the folder and control/right-click on the folder and choose “Analyse”. Now when you drop one of the files into your set, it’s ready instantly!
35. Want to know how to make a simple major chord from any key on your keyboard? Easy. Put your finger on any key, count up four keys (whites and blacks) and put your second finger there. Lastly, count up 3 more keys from your second finger. There you have it, a basic major chord.
36. Want to know how to make a simple minor chord from any key on your keyboard? Easy. Follow the instructions above for a major chord. Then drop your middle finger down one key. There you have it, a basic minor chord.
37. Want to quickly automate more than one element in Logic? Just group the faders, then any automation moves will be written en masse.
38. If you always want to use the high-quality setting for your EQ8 in Live, just right-click on the EQ8 and select high-quality as the default setting.
39. If you are limited in terms of your investment capital for your setup, at least get a quality set of monitors. Having a decent set of monitors to reference your music on will change the way you produce.
40. If you’re creating your own loops from chopped material, recorded material etc, make sure that you prep them before getting too far into producing with them. Make sure they don’t have unwanted silence on the front end, which might make them out of sync with the rest of your material. Also, be sure to check that the start and end points fade nicely at the zero crossing. This will ensure you don’t get left with nasty little pops and clicks.
41. If you’re running out of room on the default drive you loaded your Live library to, you may want to move it to an external drive to free up some room. Go into your preferences and choose Library>Location. You can then move your library to another location.
42. After some interesting rhythmical textures? Right/Cntrl-click any random audio in Live and select “Slice to New MIDI Track”. The try reordering the generated MIDI to work with your drums, or even integrate them into your drum rack by dragging the drum pads over.
43. You can get some interesting effects side-chaining the filter in Live. A nice little feature.
44. If you you have a complex MIDI part in Cubase and you want to separate its contents or delete some notes you can speedily do this by choosing MIDI>Dissolve part with the even selected. Each note can then be split off to a separate even or even split by channel.
45. If the machine you are using for music production is also connected to the web, it’s probably a good idea to turn off automatic updates for your machine. By doing so, you’ll make sure that any incompatibilities that updates might create with your DAW are avoided. You can always select to download updates but manually install them – giving you time to ensure that you have properly backed up your machine before installing updates.
46. Sorry, did you say that you don’t regularly back up your machine/sample library? Are you a glutton for punishment? Back up, back up, back up – or you’ll eventually regret it.
47. The EVOC (Logic’s vocoder) can be used for much more than the traditional vocoder effect – especially if you make use of side-chaining. Try setting it up in it’s synth mode and play in a chord/note that works with your track. Then side chain it to something like your drums to get an interesting rhythm that sits well with your drums. Try delaying this to get some interesting accompanyments that compliment your drums.
48. If you’re using pads to fill out your track, try using a band-pass filter on the sound and modulate it in some way to give your pads movment – this could be something like LFO’s, envelopes, or even just track automation of the filter itself. 49. Some limiters will let you push their output to 0db but some will let you push past that. It’s good practice to set the output level below zero (-0.1 or -0.2 for example) to avoid any potential clipping or errors.
50. Compression and limiting are nice but don’t squash the life out of your track. It results in no dynamics and a tiring feel…if your waveform basically looks like a solid block, you’re probably pushing way too hard.
“…files should be in WAV or AIFF format and should not have been converted to a lesser quality (ie resolution or sample rate) – please do not send us MP3 versions! Your files should be as raw as possible please send with with no effects, compression or maximisation. It helps to ensure no plug-ins used have over-levelled at any point and the file sent has had no channels touching the red, lastly a good 6db or so of headroom on the finished file is essential to allow us to do our thing and obtain the best possible audio result.
52. When you are layering kicks to get that perfect sound, pay attention to phase. If you are layering a couple of kicks, say one with a low end and one with a more punchy mid/top, try switching the phase on one of the channels. This can sometimes result in a more pleasing sound. Another option is to play with the track delay. Moving one of them slightly in relation to the other can often give you a nice result.
53. When trying to brighten a sound, it’s often as useful to remove some of the lower mids as it is to try and increase the higher frequencies with EQ. Just be careful not to remove too much as you will loose punch.
54. Try sending all of your drum elements, minus the kick and snare, to a bus and add a compressor to the bus. If you side-chain your kick and snare to the compressor on this bus and adjust the send levels of the kick and snare to the bus, you will be able to push up all the elements in your drums but still keep the main driving elements – kick and snare – prominent.
55. If you can’t seem to find that right percussive sound and you’ve gone through your library, don’t forget about adjusting the transients on what you have. This, and detuning, can seriously alter your drum library sounds and you’ll likely find what you are after. For a more percussive sound, use short attacks, low sustains and quick decays. For more smooth and extended sounds it’s the opposite.
56. After struggling to work this out at home, I came across this really informative video courtesy of makemorenoise that covers how to easily copy track automation in Logic Pro using very simple key commands.
57. Use the Flutter control to add pitch inconsistencies to the output of the Tape Delay plug-in. Interestingly, this can work particularly well with ‘fixed pitch’ sounds (like a Fender Rhodes piano, for example) as the pitch modulations add an extra touch of chorus-like warmth.
58. When applying delay, experimenting with feedback can create interesting effects. Features to use in conjunction with feedback are the Low Cut and High Cut controls. Delay always seems to work best when it has a different timbral colour from the original input, arguably helping the ear to make a distinction between the dry and wet signals as well as mimicking the tonal colouration of an ‘echo’ that might occur in the real world. Using these filtering controls (which are positioned as part of the feedback circuit) you can make the delay darker (using High Cut), thinner (using Low Cut) or narrower (using a combination of both filters).
59. You can give your drums a pulsing feel if you apply an LFO controlled low-pass filter to them. Add the filter to your drum track and lower the frequency until you start hearing it filter the hi-hats. Then, set the LFO amount nice and low and sync the rate to a 1-bar cycle. You can then offset the LFO to taste.
60. Off beat hi-hat patterns can make your patterns more interesting if you loop them for 3 beats rather than the usual four. This way, they cycle at a different rate to the main loop, but still stay in time. Give it a try and see how it sounds.
61. DAW’s will store your audio files, unless you deliberately delete them. Having masses of audio in your project file will clog up your hard drive un-necessarily when your project is finished. So, in Logic go to your Audio Bin and find the ‘delete un-used audio’ function.
62. If you’re having trouble with hard drive space, think about recording in MIDI instead of audio. This will allow you to record lots of alternate takes without having to worry too much about space. Perfect for laptop producers, who’s hard drives may not be as large.
63. Confused about when to use insert effects or send to a buss with an effect on it? As a general rule, you should use inserts on channels that you want to process independantly, but use busses if you want to process a group of channels collectively. For example, you might have a group of drum tracks – kick, snares, hats, shakers, that you want to EQ and compress. You might put an individual EQ on each of the channels, and compress each channel differently to get the desired sound. Then, you could send all of these channels to a buss with another compressor on it that compresses the collective tracks to “glue” them together.
64. Using Reverb and Delay as buss effects is often useful as it creates a common sense of space for all of your track parts. If you use different Reverb and Delay settings for each track as an insert, this can create a sense of non-conformity that your brain feels is strange – afterall, we are used to listening to music in one type of environment, which colours the whole sound.
65. Even though you remove a file from your session in Ableton, Ableton’s file management system still keeps any previously used files in the project folder. This can end up taking up a lot of unecessary space on your drive. You can use the “manage files” function.
-then click show in finder (make sure that the headphone sample preview is off. Select all and delete…Bam free hard drive space.
66. One way to grab vocals off a recording is to eq out the drums but this often has some undesireable effects on the vocal as well. If you happen to have the instrumental version, you can try another technique – phase cancellation. Here’s a nice little video on the process.
67. Looking for that “sampled from vinyl” effect to give your tracks a little more grit? Don’t overlook the bit-crusher. Just a slight bit of crushing on some select elements can give your digital tracks that more organic feel.
68. A good way to get some movement in your mix is to use autopanners on two instruments playing the same MIDI information. Oppose the starting points so that as they pan they cross eachother as they pan. Keep on eye on your wet/dry setting on the panners though or the mix may become unbalanced.
69. An important and often overlooked part of mastering is to check your mix for mono compatibility. Many club systems have mono systems and it your track isn’t mono compatible you will get phase cancellation – removing essential parts like your bassline. Get an analyzer that checks for mono compatibility – many mastering suites have them and there are also free tools you can find on the web.
70. Use busses and group similar sounds there applying effects like reverb and compression. By creating group busses for basses and drums for example, you’ll develope a more coherent “glued together” sound.
71. Many of Ableton’s plugins have different quality modes – such as the EQ for example. Setting them to the highest quality mode will result in a better quality processing of the audio, but this also results in higher CPU use. If you are struggling with CPU load, consider setting them to a lower setting when in your arranging/jamming mode and then having your mixing defaults set to the highest setting. At this point you should have already bounced down quite a few files and will have more processing power available.
72. Be careful when using your DAW’s freeze function or exporting stems for mixing – many DAWS will bypass any sidechaning you had in place when you do this. Instead, bounce any side-chained parts down using sidechain processing individually through the master bus (set to 0dB with all plugins bypassed). Be sure you have the output of the sidechain source channels muted with still sending their signal to the bus.
73. Always keep your monitoring level at a reasonable level whilst you are working in the studio and turn it up once in a while for a short period. SAVE YOUR EAR’s – the last thing you want is to damage your ears or get tinnitus which could end your music making career for good!
74. Always save your tracks with different version names as you are writing the track. This will enable you to experiment more with the track without worries about loosing your way – if you lose your way you can always pull up an earlier version.
75. This should go without saying but many people still don’t do it. BACK UP!!! My backup external hard drive recently broke and I did not replace it straight away. 3 weeks later my hard drive died and I lost everything. 3 years of music arrangements gone. Don’t think it won’t happen to you and get caught out!
76. Name your tracks either as you go or at the end of every session. This will make it much easier when you go back to your arrangement to alter tracks easily and know what’s what. 76. An equalizer and spectrum meter are essential when you are writing a song. Analyze your tracks EQ responses to find ranges of your mix that are lacking frequencies, and fill in the gaps. You can also then use EQ to help even out your mixes. It is also a good idea to analyze some of your favourite tracks in the same genre to get a sense of what the frequency range looks like.
77. If your gain reduction meter on your compressor doesn’t return to 0dB for a few bars, you are probably over-compressing. The fact that gain reduction never returns to 0dB indicates that compression is always taking place. A constantly compressed signal – especially if it’s relating to your whole track – can sound very flat and lifeless, as you are squashing all of the natural dynamics out of your sounds.
78. When you are dialing in settings on a compressor ask yourself these questions each time.
79. Don’t overlook the built in track delay for each of the tracks in your DAW. By altering these slightly (1-10ms or so) you can push things forward in time or pull them back, creating a relaxed or urgent feel. Clever playing around with these will also give your track it’s own unique sense of swing.
80. If you’re using a sampler to play in percussive sounds, variation of the velocities can make them sound more human. Go further and experiment with modulating the sample starts via an LFO or the velocity – or by altering the pitch of hits subtly. These will slightly change the place of the peak transient in your groove or mimic they way a drummer strikes differently each time.
81. The way you put your effects chains together can have a profound effect on the final sound. People are often confused about when to add EQ and compression. There’s no hard-and-fast rule to this, but thinking about it in a common-sense way will help you decide. Simply ask yourself the following question: ” Do I want to EQ my compressed signal, or compress my EQ’d signal?” The answer to that will tell you where to place each in your chain in that instance. If you still don’t know the answer, experiment with both and see what pleases you most.
82. Did you know you can re-size the meters both horizontally and diagonally in Ableton to get more information and greater resolution? Try dragging the line above the meter or sideways on the track header to reveal the dB meter numbers.
83. Have a play with the EQ3 in Ableton vs. the EQ8 and try cutting a certain frequencies – say the low end for example. You’ll soon notice that the EQ3 has a very different characteristic and can be useful for your more “broad range” cuts.
84. Experiment with stacking sounds in your sampler and then sculpting the stacked sounds to create your own unique elements. If you’re using Logic for example just open up an EXS24 instrument and go to the instrument editor. Highlight the folder in the left hand “Zone” column (this will select all the zones in your instrument). Now go to the edit menu and hit “copy”. Now open up a new exs24 instrument that you think might sound interesting stacked with your first one. Go to the instrument editor as before, and the select “paste”. Your zones from both instruments will now be stacked and triggering a key will trigger both samples assigned to that key at the same time.
85. Looking for a slightly different way to add some movement to your pads other than the usual side-chained compression option? Try using a high-pass filter with an envelope following option and adding it to a pad sound. Set the sidechain of the filter to listen to the kick so that it raises the filter cut-off when the kick drum plays.
86. If your kick is lacking clarity, one fix is to subtly layer it with on-beat hats – these bring it some clarity and bite. Just mix it to taste.
87. Old schol drum machines were created so that the kick and snares in particular kits are designed to work well with eachother right out of the box, so you can use these as a foundation for your beats and then layer up with your own samples for extra flavour.
88. Give your hats some nice movement by panning the closed hats hard left and right and creating a sequence that alternately triggers left and right hats. Put an open hat in the centre.
89. If you are using the EXS24 and want to emulate the classic “choking” effect where open and closed hats cannot play at the same time just route all your hats to a single “Group”. To do this, just go to the group pull-down menu and create a new group. Set the number of voices to 1. Now, only one sound can play at once, effectively “choking” the others.
90. If you want to easily see where triplets should fall using your grid, try programming using a 24/4 quantize grid.
91. Looking for that extra bit of energy in your fills? Try switching to triplets in the fill before reverting back to 16th or 8th quantize divisions for the regular groove.
92. Looking for a more chunky sound to your hats and snares? Try some modest bit-crushing and you’ll be surprised at how this can thicken up some sounds. This will reduce some of the high range frequency, so you’ll need to compensate with a bit of upper-end EQ.
93. Working all day with a loud kick drum can tire your ears, not to mention in some cases annoy the neighbors. Try putting a low cut filter on your master channel just to give your ears a bit of rest once you’ve got your kick levels sorted. You can always turn it off periodically to test your mix levels. Your ears and your neighbors will appreciate the break.
94. Have you written in all your automation and found out you need to do some track level tweaking? Save yourself the hassle of altering your automation and instead insert a utility plugin on the rogue channel. You can dial in a negative output level and bring the gain of your channel down without having to get into the automation.
97. Fills are great for filling the space left when the vocalist or lead instrumentalist isn’t playing, and also signal changes in your arrangement. This will create tension and add interest to a track. Fills could be drum rolls, for example, or a reverse reverb from a vocal.
98. Did you know you can save clips containing just MIDI CC automation data for future use ? You could, for example, open up previous projects and save the filter sweeps, volume swells or velocity patterns. These can then easily be transferred over existing MIDI tracks on new tracks. Use something like Cubase’s Logical Editor or Logic’s Transform functions to reassign your clips to entirely new CCs. A great time saver.
99. If you ever feel like u don’t know where to go with your track, try using your DAW’s MIDI transpose function to shift entire passages up and down in pitch. Often, these transposition tools offer the option of keeping the notes relative to the key of the song. You may also want to try this without this option- you’ll be surprised at some of the interesting key change progressions you arrive at.
100. Don’t overlook MIDI control numbers for your synth. These will be listed somewhere in the manual and offer a way for you to draw in automation and assign it to a vast array of parameters on your synth. Try drawing in your own automation and assinging it to whatever MIDI parameter you want to modulate. You could assign it to the general MIDI CC 74 for the cutoff frequency or MIDI CC 7 for the volume for example. Experiment with different CC’s and see what cool effects you stumble across.
101. Looking to add some extra oomph to a chord? Try assigning a different sound to each note in the chord and play with the levels/ADSR of each to get a good fit. An easy way to do this is to play each key of your chord individually with a different synth patch for each and record the output. Then throw these sounds in a sampler and assign them to each of the appropriate keys. When you play them all together, you’ll get an interesting multi-layered chord. Very similar to the concept of stacking intstruments, but with an extra twist.
102. A lot of the space and size in my breakdowns comes from layering pads. I’ll have two or three layered and EQ them so they take care of different frequencies. So if there’s a pad that’s quite bottom-end orientated I’ll take the top out of it. Then, for another pad with really nice top end, I’ll EQ out the bottom end and so on. I also like to widen things up a little and logics sample delay is a fantastic way to get wideness to sounds. It lets you delay one of the channels by samples rather than milliseconds and is really good for widening pads.
A lot of people put too much into tracks when making trance and it can get too crowded. I always work from the peak of the track and then strip it back for the arrangement. For ambience, the Big Cave preset on logics Space Designer is great and I also use QuikQuak’s fusion field. Try raising the reverb level over time so it almost overpowers the dry signal for big build-ups.
103. Lately, I like to add a little randomness – and sometimes a lot of it – to some parameters of my instruments: pitch, pan, volume, cut-off, decay, fx levels and so on. When done subtly, it may not be too noticeable from the outside and the listener probably can’t guess what is going on. But it will start to make instruments sound a bit more lively, unique and interesting. For example, it can be done in NI Massive or any other vst through formula controller if, like me, you use FL Studio.
104. With the update of Maschine to 1.8 there are some quick new ways to select and modify notes. Hold erase+select+pad to erase notes or shift+select+pad to select them. Once you’ve selected the events on particular pad, you can modify their with the swing, timing, velocities etc.
105. We’ve probably said this before, but get used to using a spectrum analyzer on each channel to help you clean up your mixes and notch out conflicting frequencies on different tracks. You’d be surprised how much “mud” you’ll find on things you really didn’t think had any low end, and cleaning this unecessary stuff out from the start will help you get a much cleaner mix in the end.
106. The key to getting expressive unique sounds from your synth is various forms of modulation. Get to understand your Mod Matrix and the various ways you can alter the sounds over time with things like envelopes. You’ll be glad you spent the time and find yourself deviating from the standard presets in your own way.
107. It doesn’t ALWAYS have to cost a load to be good. In fact there are a ton of great free plugins on the web. Our free area at Plugin Boutique is a good place to start your hunt.
108. Looking to get a more interesting sequence from your arpeggiator? Try throwing one after another for some even stranger effects. Another great way to play with the original sequence is to alter the note velocities and lengths using your DAWs’ “Transform” or “Numerical Edit” function.
109. Modulating a synth’s filter cutoff frequency with an LFO is a great way to get a sound that evolves and works well with an apreggiator.
110. It’s a good idea to add and EQ and roll off the low end on your delay and reverb busses to keep things from getting muddy.
111. Use wide voicing and go easy on the sustain if you’re working with a Rhodes sound, to keep things from getting muddy.
112. Did you know that you can play any sample you drop into a clip in Ableton’s session view chromatically? Just drop a sample into an empty clip, click on MIDI learn, and while the clip is highlighted press the lowest and highest keys on your keyboard. Exit MIDI learn and you’ll now be able to play the sample pitched up and down on your keyboard. For good results, double click the audio file and turn of global quantization for the clip, so every time you hit a key the sample plays back immediately.
113. With the newest operating system update to Maschine, you’ve got a great way to browse your samples. Just turn on preview mode in the browser and select your sample folder. As you browse through using the hardware controller you’ll audition the sounds and if you come across something you love, you’ll be ready to load it into the sampler and start playing.
114. Did you know that if you are planning on dithering your bounced file after bouncing (during the final mastering stage) Ableton’s manual suggests you bounce at 32-bit (or above)?
These guys are so underratted, I’ve been listening and appreciating their music for a while now.
In the last year and change, Audio Push have released a ton of music. From their Inside the Vibe and The Stone Junction EPs to their My Turn series and debut album, 90951.
So much so, that they’ve decided to package up a collection of tracks they haven’t released yet and released them as The Throwaways.
Produced entirely by Larry Belafonte, The Throwaways can be heard below.
Weezy threw up the diamond sign to the packed crowd at Aebersold Recreation Center recently and mentioned that he is now part of Roc Nation, “Is it cool if I just say it? Its the Roc. You know I’m a member of that team now,”.
This is definately a good move for Weezy since he’s been having endless label issues with former label, Cash Money.
Kendrick Lamar surprises us and unveils the cover and tracklist for his highly anticipated forthcoming album titled “DAMN.” No rap features on this album and we cant wait to hear it.
Damn. is set to drop on Thursday 13th of April.


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Insight Eye Associates has been a leading provider of optometry services and vision care products in the Ft Worth community since 2010, and we want to help you achieve and maintain clear vision for years to come.
Our experienced eye doctors offer comprehensive vision examinations at our Ft Worth optometry office and specialize in the diagnosis and treatment of a wide array of eye diseases, conditions, and problems. We use advanced diagnostic technology and vision correction products and are committed to improving the quality of life of persons in the Ft Worth community through enhanced vision. Give yourself the gift of clear vision – schedule an appointment with your eye care provider today.


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Minimum quantity for "6 - Wing Back Dining Chair in Cream Leather R-567" is 6.
Wing back leather dining chair, Combining contemporary and transitional elements, this comfortable chair is attractively designed with a wing back, Kiln dried solid hardwood frame. High Density Foam with Pirelli straps. Sits amazingly well. fully assembled for commercial and residential use.
Angela Davis, Designer: This is a good chair if you want the dining area to look majestic, will go very well as set all around the table. also an excellent chair to use as captains only.

industry_corpus2: Manufacturing


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HAWTHORNE, Fla. (WCJB)-- Hawthorne residents may soon have more job opportunities after the city passed a new tax exemption Tuesday night.
The initiative proposed giving exemptions to new and expanding businesses that would bring in more jobs.
The measure passed with 81% support.
Mayor Matt Surrency said, while Hawthorne has the ability to give tax incentives to these businesses, the exemptions will need to be approved on a case-by-case basis.


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From now on, students and researchers from the Computer Science Department of FCT NOVA will be able to develop projects related to OutSystems' low-code technology, with the support of a team of specialists.
The NOVA LINCS / OutSystems Collaborative Research Laboratory, launched on February 26 at FCT NOVA's Department of Informatics (DI) facilities, opens a new phase in the 10-year partnership for research, training and innovation among these institutions.
The event was attended by Minister of Science, Technology and Higher Education, Manuel Heitor, who provided the success of this collaborative research initiative, and Paulo Rosado, CEO of OutSystems, who highlighted the unique success of this partnership with FCT NOVA. to the future of building IT systems.
"The Laboratory, fully equipped by OutSystems, will allow researchers and students of the DI / NOVA LINCS (Integrated Master in Computer Engineering, PhD Program in Computer Science) to develop projects related to OutSystems' programming technologies and systems, in collaboration with experts OutSystems R & D team, NOVA LINCS researchers and students" said Luís Caires, President of the Department and Director of NOVA LINCS.
"Over the ten years of the partnership, OutSystems and ID have deepened an atmosphere of remarkable alignment of innovation activities, fundamental research and advanced training, which strengthens and strengthens all partners around issues of high technological potential," he said. João Costa Seco, researcher at NOVA LINCS.
Also present at the session were the Rector of the NOVA University in Lisbon, João SàÁgua, the FCT NOVA Director, Virgílio Machado, teachers, students, researchers and staff from DI / NOVA LINCS and OutSystems.


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EVER fancy a game of bowling after a drink at the pub?
Now you can do just that, with the opening of the Lookout Bar Bowling Bites at Scarborough Beach.
Co-owner David Heaton took over the venue following Matisse Beach Club’s closure last year and said it was always his intention to reinvent the space.
He formed the idea after visiting a restaurant with bowling lanes while in Nashville and was already working on the concept for Northbridge’s old IMAX theatre when the Scarborough opportunity presented itself.
“It was following the concept of another bowling bar on the drawing board,” he said.
Mr Heaton said it was a bar first and bowling venue second, and aimed to give people something more to do at a pub.
Along with eight bowling lanes, sports games will be televised on two large screens, there are pool tables and an expanded menu with weekly specials.
“In this day and age with millenials’ expectations, the old stand around having a drink is boring,” he said.
“There’s a little bit of everything for everyone.
He wanted to offer something a bit different for Scarborough following the redevelopment.
“We’re hoping it will attract a lot more people,” he said.

industry_corpus2: Sports


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The Fire Protection Association (FPA) is holding the “Building a safer future” seminar on Wednesday, February 27.
The Fire Protection Association’s seminar will feature leading experts who will focus on the detail of the Government’s implementation plan of Hackitt’s recommendations and how safety standards and regulations are changing. Speakers will also address the implications for industry, stakeholders, and residents of tall buildings, and how different agencies can work together to improve and promote fire safety.
Nick Coombe – protection vice-chair of the National Fire Chief’s Council – How will the enforcement regulation landscape evolve?

industry_corpus2: Safety Management


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The lamps can be ordered by contacting SemperNovum.
You can then specify your requirements: what model you want with which material and from which monument.
Also material of your own "monument" is possible!
Other functional and technical requirements also can often be realized.
Each lamp becomes, therfore, custom made lamp: a lamp to which only your story belongs.
The prices of the lamps vary between €500,- and €3500,- and depend on the material, the monument and your specific needs. The more exclusive the monument and the scarcer the material, the more exclusive, but also the more expensive a lamp will be. The processability of the material and its condition also play a role.
E.g. a granite pedestal is more difficult to process than a foot of marlstone. The latter requires however more attention for good preservation.
So please feel free to contact us for prices.
If you would first like to see the lamps, you can.
To do this just contact us for the current showroom and gallery addresses.

industry_corpus2: Others


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One Sunday evening, as I was browsing through the local newspaper, a story caught my eye. No more homework for learners in some schools, the headline shouted from the page. As a caregiver to a young child, this headline got me thinking and googling. I found many blog posts and newspaper articles from all over the world that claimed homework was bad for children’s happiness/stress levels/sleep patterns. Each article claimed that “recent research has found”, even for when the claims were contradictory. I felt overwhelmed and unsure of what to believe when I asked myself, What is the impact of homework on children’s wellbeing?
In a South African context, action against homework is already being taken in our schools. When it comes to education, we are told again and again how important it is for the future of our children (Vatterott, 2009, Galloway, Conner and Pope, 2013). The way we educate our children relates not only to the best we can afford as caregivers, but also our values as a family. We want the best for our children and we want our children to the best for their country. Their education is central to shaping them into the people they will be in the future. Since homework is an integral part of many schools’ current way of operating, it is essential to know what the evidence says about how homework impacts our children’s development.
Looking for evidence syntheses (check out our first blog in this series to understand why we avoid the danger of a single story), we found two evidence summaries that looked at reviews or meta-analyses on the impact of homework for primary or secondary school children on children’s academic achievement (see box ADD below for how we found this evidence). Both summaries are publicly available on the Education Endowment Fund’s Teaching and Learning Toolkit interface. These summaries focused on how homework impacted children’s academic achievement; they did not share findings on how children’s wellbeing was affected by homework. We think the findings are appropriate because the academic achievement of learners is one predictor of their future wellbeing.
Quality is more important than quantity. Detailed and timely feedback and how engaged caregivers are in the process of doing homework effects whether homework has a positive impact on learners’ academic achievement.
Shorter-term rather than routine. Homework given for specific shorter-term projects is more effective at enhancing academic achievement than homework given routinely.
Make the objective clear. Learners should know what the purpose of the homework is, and any homework given should be closely linked to what is being taught during the school day.
No much to say. There is weak evidence to suggest that homework is effective for academic achievement at a primary school level.
Homework is not punishment. Caregivers are cautioned to avoid using homework as reprimand for poor performance for secondary learners.
Use variety. Homework given at the secondary school level should include a variety of tasks with different levels of challenge.
So, what does this all mean in the South African and/or African context?
Whilst the evidence we found related to schools in developed nations, mainly Britain and the US, there are important differences and similarities between these contexts to take note of.
Similarities: The evidence presented here is applicable to schools in which homework policies are already taking place.
Differences: Schools in South Africa – and Africa more broadly – vary in standard because of constrained resources, teaching capacity, and infrastructure (Rakabe, 2014). This constraint extends to beyond the school day: resources to undertake homework may also differ. For example, characteristic of many households in South Africa are long commutes to school, malnutrition that might affect concentration, infrastructure challenges such as lack of electricity to complete homework by, and lack of support to complete homework in the case of child-headed households. These and other factors make for a vastly different context to the one the evidence was gathered from.
No matter which side of the homework debate you fall on, as caregivers we are united by a single value: we do what we believe is best for our children’s future. Whether I believe that the children in my care should be doing homework or not, there are definitely five things that evidence shows I can do to get the most out of the impact of homework on children’s academic achievement.
Question, encourage, and support our schools.
Ask our schools what they use to base their homework philosophies on.
Find out how our schools structure homework, how teachers provide feedback on homework, and how homework is related to what is taught in the classroom.
Share the results of evidence we know to be reliable and representative of the whole story – instead of one part of it – with our schools.
Understand the purpose of the homework your child has been given, and share with your children what the purpose is.
Engage with and participate in the homework lives of our children by sharing our feedback on the tasks they are completing as part of their homework.
In looking for reliable appropriate evidence, we searched the Campbell Collaboration’s working group of systematic reviews on education and the International Initiative for Impact Evaluation’s (3ie) education collection of evidence syntheses on their website, where we found no evidence syntheses on our exact topic.
We then revised our question to look at the impact of homework on children’s level of academic achievement. With this revised question, we reviewed the Education Endowment Fund’s Teaching and Learning Toolkit interface and found two summaries of the impact of homework on children’s academic achievement.
This talk by the Education Endowment Fund on how the evidence does not really support the use of homework at the primary level.
This systematic review (and brief) by the International Initiative for Impact Evaluation on the impact of education programmes in lower- and middle-income countries on learner’s school enrolment, attendance, completion and learning.
Rakabe, E. (2014). Equitable Resourcing of Schools for Better Outcomes (Vol. 16). Technical Report: Submission for the Division of Revenue 2015.