Revealing PAK2's Function in the Cell Division through MKLP1's Interactome.

Cell division-related proteins are essential for the normal development and differentiation of cells and may be related to the occurrence of cancer and the drug resistance mechanism of cancer cells. The mitotic kinesin-like protein 1 (MKLP1) is a kinesin protein that has been involved in the assembly of the midzone/midbody during mitosis and cytokinesis. In this study, we found that the tail domain of MKLP1 exhibited an autoinhibitory effect on its motor activity. Overexpression of the tail domain in HEK293 cells blocked cytokinesis and caused bi-/multinucleation. It is possible that protein binding to the MKLP1 tail relieves this autoinhibition and induces the motility of MKLP1. We used the GST pull-down assay followed by the LC-MS/MS analysis and identified 54 MKLP1 tail domain-specific binding proteins. Further, we confirmed the MS result by coimmunoprecipitation and FRET that a serine/threonine kinase, p21-activated kinase 2 (PAK2), binding to MKLP1. Endogenous PAK2 expression was found to be identical to that of MKLP1 in HEK293 cells during cytokinesis. Finally, functional studies indicated that when PAK2 expression was downregulated by siRNA, MKLP1 underwent a change in its localization away from the midbody, and cell cytokinesis was subsequently impeded. This study presents a novel regulatory mechanism that PAK2 promotes the activation of MKLP1 and contributes to complete cell cytokinesis.

Circ-camk4 involved in cerebral ischemia/reperfusion induced neuronal injury.

Stroke and subsequent cerebral ischemia/reperfusion (I/R) injury is a frequently occurring disease that can have serious consequences in the absence of timely intervention. Circular RNAs (circRNAs) in association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression. However, whether circRNAs have a role in cerebral I/R injury pathogenesis, especially soon after onset, is unclear. In this study, we used the SD rat middle cerebral artery occlusion (MCAO) model of stroke to examine the role of circRNAs in cerebral I/R injury. We used high-throughput sequencing (HTS) to compare the expression levels of circRNAs in cerebral cortex tissue from MCAO rats during the occlusion-reperfusion latency period 3 hours after I/R injury with those in control cerebral cortices. Our sequencing results revealed that expression levels of 44 circRNAs were significantly altered after I/R, with 16 and 28 circRNAs showing significant up- and down-regulation, respectively, relative to levels in control cortex. We extended these results in vitro in primary cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) using qRT-PCR to show that levels of circ-camk4 were increased in OGD/R neurons relative to control neurons. Bioinformatics analyses predicted that several miRNAs could be associated with circ-camk4 and this prediction was confirmed in a RNA pull-down assay. KEGG analysis to predict pathways that involve circ-camk4 included the glutamatergic synapse pathway, MAPK signaling pathway, and apoptosis signaling pathways, all of which are known to be involved in brain injury after I/R. Our results also demonstrate that levels of the human homolog to circ-camk4 (hsa-circ-camk4) are elevated in SH-SY5Y cells exposed to OGD/R treatment. Overexpression of hsa-circ-camk4 in SH-SY5Y cells significantly increased the rate of cell death after OGD/R, suggesting that circ-camk4 may play a key role in progression of cerebral I/R injury.

SIRT 1 binding with PKM and NSE and modulate their acetylation and activities.

SIRT1 (Silent mating type information regulation 2 homolog 1) play a neuroprotective effect through deacetylation target proteins in various neuronal diseases. However, the precise mechanisms remain elusive. In this study, we aim to identify those novel interacting partners of SIRT1 in rat brain tissue. By using a pre-clear GST-Pull down assay followed by the LC-MS/MS analysis, we've identified potential SIRT1's interacting partners, which function annotation by GO and KEGG analysis indicating some metabolic pathways are among the most enriched. Then we confirmed two candidates Enolase-1 (and NSE (Neuron-Specific Enolase) in brain) and PKM (Pyruvate Kinase Muscle) are associated with SIRT1 in brain tissue lysis by co-immunoprecipitation. Furthermore, increase or decrease the SIRT1 enzyme activity by its agonist SRT1720 or antagonist EX527 could significantly affect the acetylation level of endogenous NSE and PKM, SIRT1 overexpression or knock out expreiments also showed the same results as use SIRT1's agonist or antagonist. Moreover, the acetylation changes on NSE or PKM could finally lead to affection on their catalytic activity. Taken together, our findings suggest that the function of SIRT1 binding proteins is enriched in metabolic pathways. NSE and PKM are new SIRT1 binding molecules. SIRT1 may regulate acetylation level of NSE and PKM through deacetylation and further regulate their catalytic activity. Our study provides new evidence for the involvement of SIRT1 in the mechanisms of metabolic regulation in central nervous system.

Intelligent identification of multi-level nanopore signatures for accurate detection of cancer biomarkers.

To achieve accurate detection of cancer biomarkers with nanopore sensors, the precise recognition of multi-level current blockage events (signature) is a pivotal problem. However, it remains rather a challenge to identify the multi-level current blockages of target biomarkers in nanopore experiments, especially for the nanopore analysis of serum samples. In this work, we combined a modified DBSCAN (Density-Based Spatial Clustering of Applications with Noise) algorithm with the Viterbi training algorithm of the hidden Markov model (HMM) to achieve intelligent retrieval of multi-level current signatures from microRNA in serum samples. The results showed that the developed intelligent data analysis method is highly efficient for processing the large-scale nanopore data, which facilitates future application of nanopores to the clinical detection of cancer biomarkers.

Applying Side-chain Flexibility in Motifs for Protein Docking.

Conventional rigid docking algorithms have been unsatisfactory in their computational results, largely due to the fact that protein structures are flexible in live environments. In response, we propose to introduce the side-chain flexibility in protein motif into the docking. First, the Morse theory is applied to curvature labeling and surface region growing, for segmentation of the protein surface into smaller patches. Then, the protein is described by an ensemble of conformations that incorporate the flexibility of interface side chains and are sampled using rotamers. Next, a 3D rotation invariant shape descriptor is proposed to deal with the flexible motifs and surface patches; thus, pairwise complementarity matching is needed only between the convex patches of ligand and the concave patches of receptor. The iterative closest point (ICP) algorithm is implemented for geometric alignment of the two 3D protein surface patches. Compared with the fast Fourier transform-based global geometric matching algorithm and other methods, our FlexDock system generates much less false-positive docking results, which benefits identification of the complementary candidates. Our computational experiments show the advantages of the proposed flexible docking algorithm over its counterparts.

Ratiometric two-photon fluorescent probes for mitochondrial hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) is an important signaling molecule with diverse biological roles. Various fluorescent probes for H2S with biological application have been developed. However, two-photon ratiometric imaging of mitochondrial H2S is scarce. In this paper, we report two ratiometric two-photon probes, AcHS-1 and AcHS-2, which employ 4-amino-1,8-naphthalimide as the fluorophore and 4-azidobenzyl carbamate as the H2S response site. These probes exhibit high selectivity toward H2S over biothiols and other reactive species, low detection limits of 50-85 nM, low cytotoxicity, and high stability under physiological conditions. Furthermore, through cell imaging with one-photon and two-photon microscopy, MCF-7 cells incubated with two probes show a marked change in emission color from blue to green in response to H2S. Cell images costraining with a mitochondrial dye reveal that AcHS-2 is a mitochondria-specific two-photon probe for H2S. These results show that AcHS-2 may find useful applications in biological research such as tracking mitochondrial H2S in living biological specimens.

Regioselectivity and competition of the Paternò-Büchi reaction and triplet-triplet energy transfer between triplet benzophenones and pyrimidines: control by triplet energy levels.

The photochemical reaction of a pyrimidine and a ketone occurs either as a Paternò-Büchi (PB) reaction or as energy transfer (ET) from the triplet ketone to the pyrimidine. It is rare for the two types of reactions to occur concurrently, and their competitive mechanism remains unknown. In this work, two classes of products, regioisomeric oxetane(s) (2, 3) from a PB reaction and three isomeric dimers of 5-fluoro-1,3-dimethyl uracil (FDMU) (4-6) from a photosensitized dimerization of FDMU, are obtained through the UV irradiation of FDMU with various benzophenones (BPs). The ratio of the two products (oxetanes to dimers) reveals that the two competitive reactions depend strongly on the triplet energy levels (ET ) of the BPs. The BPs with higher ET values lead to higher proportions of dimers, whereas those with lower ET values give higher proportions of oxetane(s), with the generation of just two regioisomeric oxetanes for the BP with the lowest ET of the eight BPs investigated. The ratio of the two oxetanes (2:3) decreases with the BP ET value. The competitive mechanism for the two types of photochemical reactions is demonstrated through quenching experiments and investigation of temperature effects. Kinetic analysis shows that the rate constants of the two [2+2] photocycloadditions are comparable. Furthermore, in combination with the results of previous studies, we have gained insight into the dependence of the photochemical type and the regioselectivity in the PB reaction on the triplet energy gaps (ΔE) between the pyrimidines and ketones. For ketones with higher ET values than the pyrimidines, the photochemical reaction is a photosensitized dimerization of the pyrimidine. In the opposite case, a PB reaction occurs, and the lower the ET of the ketones, the lower the ratio of oxetanes (2:3). When the ET of values of the ketones are close to those of the pyrimidines, the two reactions occur concurrently, and the higher the ET of the ketones, the higher the proportion of the dimers. The ratio of oxetanes (2:3) decreases with the ET value of the BPs.

Calcium sensing receptor absence delays postnatal brain development via direct and indirect mechanisms.

Calcium sensing receptor (CaSR) is implicated in the establishment of neural connections and myelin formation. However, its contribution to brain development remains unclear. We addressed this issue by analyzing brain phenotype in postnatal CaSR null mice, a model of human neonatal severe hyperparathyroidism. One- and 2-week-old CaSR null mice exhibited decreased brain weight and size with a developmental delay in expression of proliferating cell nuclear antigen. Neuronal and glial differentiation markers, neuronal specific nuclear protein, glial fibrillary acidic protein, and myelin basic protein, were also decreased compared with age-matched wild-type littermates. Moreover, deletion of the parathyroid hormone gene that corrects hyperparathyroidism, hypercalcemia, hypophosphatemia, and whole-body growth retardation normalized brain cell proliferation, but not differentiation, in CaSR null mice. Cultured neural stem cells (NSCs) derived from the subventricular zones of CaSR null neonatal mice exhibited normal proliferation capacity but decreased differentiation capacity, compared with wild-type controls. These results demonstrate that direct effects of CaSR absence impair NSC differentiation, while secondary effects of parathyroid hormone-related endocrine abnormalities impair NSC proliferation, both of which contribute to delayed brain development in CaSR null newborn mice.

Quinolinium-based fluorescent probes for the detection of thiophenols in environmental samples and living cells.

A new type of fluorescent probes for thiophenols, 6HQM-DNP and 7HQM-DNP, containing 6- or 7-hydroxy quinonlinium as fluorophore and 2,4-dinitrophenoxy (DNP) as nucleophilic recognition unit were constructed. As ethers, these non-fluorescent probe molecules can release the corresponding fluorescent quinolinium (6HQM and 7HQM) through aromatic nucleophilic substitution (S(N)Ar) by thiolate anions from thiophenols. The sensing reaction is highly sensitive (detection limit of 8 nM for 7HQM-DNP) and highly selective to thiophenols over aliphatic thiols and other nucleophiles under neutral conditions (pH 7.3). The probes respond rapidly to thiophenols, with second-order rate constants k=45 M(-1) s(-1) for 7HQM-DNP and 24 M(-1) s(-1) for 6HQM-DNP. Furthermore, the selective detection of thiophenols in living cells by 7HQM-DNP was demonstrated by confocal fluorescence imaging. In addition, these quinolinium salts show excellent chemical and thermal stability. In conclusion, this type of probes may find use in the detection of thiophenols in environmental samples and biosystems.

3-Benzyl-1-methyl-imidazolium picrate.

In the title salt, C(11)H(13)N(2) (+)·C(6)H(2)N(3)O(7) (-), the dihedral angles between the benzene ring in the cation and the imidazolium ring and the benzene ring of the picrate anion are 113.7 (2) and 116.3 (2)°, respectively. The imidazolium ring is nearly parallel to the benzene ring of the picrate anion, the dihedral angle between the planes being 2.6 (1)°. The nitro groups in the picrate anions are disordered (occupancy ratio 0.54:0.46). The crystal packing is stabilized by weak C-H⋯O inter-actions between the cation-anion pairs.

[Effect of the ethyl acetate extract of zhi ju zi on serum makers and the expression of TGF-beta1 in rats with hepatic fibrosis].

OBJECTIVE: To study the effect of the Ethyl Acetate Extract of Zhi Ju Zi on hepatic fibrosis. METHODS: Model of liver fibrosis in rats was induced by CCl4. The level of hyaluronic acid (HA) and laminin (LN) was detected by RIN. The expression of TGF-beta1 of the rat liver was detected by RT-PCR. RESULTS: After treatment, the level of HA was apparently decreased (P < 0.05); the level of LN wsn't apparently different with the Colchicin group (P > 0.05); The expression of TGF-beta1, was apparently decreased (P < 0.05) CONCLUSION: The Ethyl Acetate Extract of Zhi Ju Zi can effectviely decrease the level of serum markers of hepatic fibrosis and the expression of TGF-beta1.

[Effect of Hovenia dulcis extract on expression of MMP-13 and TIMP-1 in hepatic tissue].

OBJECTIVE: To investigate the effects of Hovenia dulcis extract on mRNA expression of MMP-13 and TIMP-1 mRNA in hepatic tissue in experimental rats. METHOD: 48 male Sprague-Dawley rats were randomly divided into 2 groups: normal group (16) and model group (32), hepatic fibrosis was induced by CCl4 for 6 weeks in rats, 8 rats were sacrificed at the end of the 6th week from every group respectively, HE staining of hepatic tissue was performed; In the model group, rats randomly subdivided into 3 groups: spontaneous recovery group, control group and medication administration group, 8 rats were sacrificed at the end of the 12th week from every group respectively, the mRNA levels of MMP-13 and TIMP-1 in hepatic tissue were assayed by semi-quantitative RT-PCR. RESULT: The mRNA expression of MMP-13 among the 4 groups were not statistically significant, but the mRNA expression of TIMP-1 among the 4 groups were statiscally significant. The levels of TIMP-1 mRNA were significantly increased in control group and medication administration group compared, with those in the model group (P < 0.05), and reverse effects of medication administration groups were significantly high than those of control group (P < 0.05). CONCLUSION: Inhibition of the mRNA expression of TIMP-1 may be the mechanism of reversing hepatic fibrosis H. dulcis, for thus collogen degradation system was recoveried gradually.

High Efficient Expression in Escherichia coli of Chitinase Gene Cloned from Bacillus circulans C-2.

Subcloning analysis of a cloned DNA fragment from Bacillus circulans containing the chitinase gene Chi1 showed that the chitinase gene lies on a 1.7kb PstI-StyI fragment. The chitinase gene could be expressed in Escherichia coli strains JM107, DH5alpha, XL1-blue and TG-1 with various efficiencies. The expression level of chitinase gene was highest in JM107, which was almost the same as that in B. circulans C-2. The molecular weight of extracellular chitinase was 66 kD by SDS-PAGE analysis. Cell location determination of the expressed chitinase showed that the enzyme existed not only in cell periplasm and cytoplasm, but also in extracellular broth. When the expression of the enzyme was optimal, the distribution of enzyme activity in extracellular broth, periplasm and cytoplasm was 35.8%, 32.1% and 32.9%, respectively.