In articular chondrocytes, nitric oxide (NO) production triggers dedifferentiation and apoptotic cell death that is regulated by the converse functions of two mitogen-activated protein kinase subtypes, extracellular signal-regulated kinase (ERK) and p38 kinase. Since protein kinase C (PKC) transduces signals that influence differentiation, survival, and apoptosis of various cell types, we investigated the roles and underlying molecular mechanisms of action of PKC isoforms in NO-induced dedifferentiation and apoptosis of articular chondrocytes. We report here that among the expressed isoforms, activities of PKC␣ and -were reduced during NO-induced dedifferentiation and apoptosis. Inhibition of PKC␣ activity was independent of NO-induced activation of ERK or p38 kinase and occurred due to blockage of expression. On the other hand, PKC activity was inhibited as a result of NO-induced p38 kinase activation and was observed prior to proteolytic cleavage by a caspase-mediated process to generate enzymatically inactive fragments. Inhibition of PKC␣ or -activities potentiated NO-induced apoptosis, whereas ectopic expression of these isoforms significantly reduced the number of apoptotic cells and blocked dedifferentiation. Ectopic expression of PKC␣ or -did not affect p38 kinase or ERK but inhibited the p53 accumulation and caspase-3 activation that are required for NO-induced apoptosis of chondrocytes. Therefore, our results collectively indicate that p38 kinase-independent and -dependent inhibition of PKC␣ and -, respectively, regulates NO-induced apoptosis and dedifferentiation of articular chondrocytes.

The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

We have shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor that regulates chondrogenesis of mouse mesenchymal cells (Kim, J. S., Ryoo, Z. Y., and Chun, J. S. (2007) J. Biol. Chem. 282, 29359 -29367). In this previous work, we found that Cytl1 expression was very low in mesenchymal cells, increased dramatically during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. Moreover, exogenous addition or ectopic expression of Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells. In the current study, we generated a Cytl1 knockout (Cytl1 ؊/؊ ) mouse to investigate the in vivo role of Cytl1. Although a number of regulatory factors have been identified, the precise regulatory mechanisms underlying chondrogenesis and cartilage and bone development remain to be elucidated. In permanent cartilage tissue, cartilage homeostasis is maintained by chondrocytes, which are a cell type unique to cartilage tissue. Cartilage homeostasis is disrupted in osteoarthritis (OA), leading to eventual cartilage destruction. OA is a progressive and degenerative disorder of the joint primarily characterized by articular cartilage destruction. A variety of potential OA-causing mechanisms have been proposed (5-7). Biophysical and biochemical factors, such as mechanical stress and proinflammatory cytokines, respectively, are responsible for disruption of cartilage homeostasis and initiation of the catabolic pathway. This, in turn, activates intracellular pathways in chondrocytes that lead to the production of pro-inflammatory cytokines, inflammation, degradation of the ECM by matrixdegrading enzymes, and cessation of ECM synthesis via dedifferentiation and apoptosis of chondrocytes (5-7). Deletion of theWe have previously shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor involved in chondrogenesis of mouse mesenchymal cells in vitro (8). CYTL1 was originally cloned as a functionally unknown cytokine candidate from human bone marrow and cord blood mononuclear cells bearing the CD34 surface marker (9). The CYTL1 protein contains four ␣-helices and six conserved cysteine residues, which may form intra-disulfide bonds to yield a globular structure, a common structural characteristic of cytokines. CYTL1 also contains an N-terminal secretory signal peptide, and its secretion is associated with post-translational modifications, although the nature of these modifications remains to be established (8

The potential of spherical-harmonics beamforming (SHB) techniques for the auralization of target sound sources in a background noise was investigated and contrasted with traditional head-related transfer function (HRTF)-based binaural synthesis. A scaling of SHB was theoretically derived to estimate the free-field pressure at the center of a spherical microphone array and verified by comparing simulated frequency response functions with directly measured ones. The results show that there is good agreement in the frequency range of interest. A listening experiment was conducted to evaluate the auralization method subjectively. A set of ten environmental and product sounds were processed for headphone presentation in three different ways: (1) binaural synthesis using dummy head measurements, (2) the same with background noise, and (3) SHB of the noisy condition in combination with binaural synthesis. Two levels of background noise (62, 72 dB SPL) were used and two independent groups of subjects (N=14) evaluated either the loudness or annoyance of the processed sounds. The results indicate that SHB almost entirely restored the loudness (or annoyance) of the target sounds to unmasked levels, even when presented with background noise, and thus may be a useful tool to psychoacoustically analyze composite sources.

Multisensory integration assists us to identify objects by providing multiple cues with respect to object category and spatial location. We used a semantic audiovisual object matching task to determine the effect of spatial congruency on response behavior and fMRI brain activation. Fifteen subjects responded in a four-alternative response paradigm, which visual quadrant contained the object best matched to the sound presented. Realistic sounds based on head-related transfer functions were presented binaurally with the simulated sound source corresponding to one of the four quadrants. Following a random sequence, the location of the sound corresponded to the quadrant containing the semantically congruent target on half the trials, whereas on other trials the sound arose from an incongruent location. We examined the effects of spatial congruency on response latencies, hit-rates and fMRI responses. Preliminary behavioral results revealed a significant effect of spatial congruency on response latency or performance for stimuli with noise added. In the fMRI experiment, spatial congruency had a significant effect on the BOLD response. A cluster in the right middle and superior temporal gyrus was more activated when the auditory sound sources were spatially congruent with the semantically matching visual stimulus. In an exploratory post-hoc analysis, in which we correlated the BOLD signal with the subjects' ability to locate the sound sources, we found a significant cluster in the left inferior frontal cortex, where the BOLD response increased with sound-source localization performance. Thus spatial congruency appears to enhance the semantic integration of audiovisual object information in these brain regions.

Continuous wave and pulsed plasma polymerization coatings of allylamine were investigated for antibody immobilization as a function of plasma power, monomer pressure and treatment time, and duty cycle. Conditions were optimized by evaluating the surface amine density of plasma polymer coated samples before and after aging in dry ethanol for 3 hrs. In addition, plasma polymer coatings were characterized by contact angle analyzer, a-step, and FT-IR/ATR. The continuous wave plasma polymerization provided amine density of 4.8 molecules/nm 2 at the optimum condition of 20 W, 1 min, and 60 mTorr, while the pulsed plasma polymerization coating resulted in further increased amine density to 5.2 molecules/nm 2 at the duty cycle of 60/100.

SummaryThis study examined whether cell cycle regulatory proteins, such as cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors, regulate type II collagen expression and mediate interlukin-1 (IL1b)-induced suppression of type II collagen expression in articular chondrocytes. IL-1b inhibited type II collagen expression, but activated CDK6. Ectopic expression of CDK2 did not alter type II collagen expression. However, overexpression of CDK6 inhibited type II collagen expression, whereas inhibition of CDK6 activity blocked IL-1b-induced suppression of type II collagen expression. IL-1b upregulated the expression of cyclin D1, which is known to activate CDK6. In turn, overexpression of cyclin D1 suppressed type II collagen expression. In contrast to cyclin D1, IL-1b triggered down-regulation of the CDK inhibitor, p21. Overexpression of p21 blocked IL-1b-or CDK6-induced suppression of type II collagen expression. Our results collectively indicate that CDK6/ cyclin D1/p21 complex regulates type II collagen expression in articular chondrocytes. IUBMB Life, 59: 90-98, 2007

The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and offtarget cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less offtarget mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable 2 diseases caused by genetic mutation.