Density fractionations of yeast stationary phase cells have shown that after day 2, the cultures contain a mixture of quiescent cells, generated as daughter cells in the last cell division after the diauxic shift, and non-quisecent cells, which ultimately lose the ability to reproduce and become necrotic or apoptotic. The cell population after 3 days is thus heterogenous, which should be kept in mind when interpreting the data. Microtubule dynamics allow cells to rapidly assemble, remodel, or disassemble polarized arrays of microtubules. Pure tubulin in vitro shows intrinsic dynamic instability, whereby microtubules spontaneously nucleate, grow steadily, and then spontaneously and rapidly depolymerise. Dynamic instability is well described by four parameters: growth rate, shrinkage rate, catastrophe frequency and rescue frequency. In cells, these parameters are all heavily regulated. Amongst the regulators are members of the kinesin-13, -14 and -8 families, which modulate the catastrophe frequency. MCAK and related kinesins-13 can diffuse along the microtubule lattice to the growing microtubule tip and drive tubulin subunits to dissociate, either alone or in complex with the MCAK, driven by an ATPase cycle that is distinct from that of translocating kinesins. Kar3, a kinesin-14, is a minus end directed translocase that heterodimerises with Cik1, targets the plus ends of taxol-stabilised microtubules, and depolymerizes them at a rate dependent on the taxol concentration. The kinesins-8 are plus end-directed translocases, at least one of which, S. cerevisiae Kip3, can step processively along the lattice of GMPCPP microtubules to their plus ends, where it enhances the Rapamycin 53123-88-9 off-rate of GMPCPP tubulin heterodimers. The tail of Kip3 contains a microtubule and tubulin-heterodimer binding site that enhances its processivity and microtubule end binding. Although, like MCAK, Kip3 ATPase is stimulated by tubulin heterodimers the mechanism of depolymerisation is different. In that Kip3- tubulin complexes are displaced from the microtubule tip by the arrival of another Kip3. The cell biology of Kip3 is consistent with this type of length-dependent catastrophe mechanism operating in vivo. The velocity of Kip3 in vivo is 47–73 nm s21, similar to its single molecule velocity of 50 nm s21 on brain microtubules in vitro, and is sufficient to account for its accumulation at the ends of microtubules growing at 23 nm s21 in vivo. Deletion of kip3 leads to unusually long spindles, longer cytoplasmic microtubules, effects on chromosome congression and a decrease in microtubule catastrophe frequency consistent with microtubule depolymerase activity. However in vivo Kip3 also increases microtubule growth rate, rescue frequency and pause duration whilst decreasing shrinkage rate, suggesting Kip3 has a wide range of effects on dynamic microtubules. The tail of Kip3 is required in vivo for the increase in microtubule rescue frequency and reduction in microtubule shrinkage rate. These effects may result directly from binding of the Kip3 tail to microtubules since in vitro the tail reduces the shrinkage rate of GDP microtubules. The cell biology of other kinesins-8 is also only partially consistent with their having solely a microtubule depolymerase activity. Some observations are consistent with depolymerase activity.

Hsp27 is ubiquitously expressed throughout the human body. We have shown that Hsp27 is particularly vulnerable to MGO modification in kidney mesangial cells. Others have shown a similar vulnerability of Hsp27 in other cell types. Furthermore, we showed that the chaperone and anti-apoptotic functions of Hsp27 were improved after its modification by MGO. Thus, Hsp27 appears to be a prime target for MGO modification, and consequently, its function could be altered in cells. Altogether, it is clear now that MGO modification of the small heat shock proteins results in an improvement in their key functions. Whether the improvement in the chaperone function of small heat shock proteins occurs via modification of a conserved arginine residue and whether physiological levels of MGO could improve the chaperone function through a hydroimidazolone modification is not known. In this study, we modified human Hsp27 and aA- and aB-crystallin with 2–10 mM MGO and identified hydroimidazolone AGEs using mass spectrometry. Interestingly, the only conserved arginine residue that was modified to hydroimidazolone by MGO was R12 in all three proteins. To determine if the hydroimidazolone modification of this arginine residue is responsible for the improvement of the chaperone function, we replaced R12 with alanine and explored the effect of this mutation on the structure and chaperone function of Hsp27 and aA- and aB-crystallin. MGO is derived mostly from triose phosphate intermediates of glycolysis by non-enzymatic mechanisms in vivo. It is a major precursor of AGEs in tissue proteins. In previous studies, we have shown that MGO modifications of small heat shock proteins, such as aA-crystallin and Hsp27, enhanced their chaperone function. In this study, our primary goal was to determine whether a similar increase in the chaperone function occurred with physiological levels of MGO and to determine whether a modification of the conserved R12 to hydroimidazolone contributed to the increased chaperone function. We first LEE011 determined the “first hit” arginine residues for modification to hydroimidazolone. To accomplish this, we modified the proteins with 2, 5 and 10 mM of MGO. With 2 mM MGO, we found that 6, 6 and 8 arginine residues were modified to hydroimidazolone in aA- and aB-crystallin and Hsp27, respectively. With 10 mM of MGO, this modification reached 10, 8 and 10 arginine residues in the three respective proteins. R12 was the only common residues among the three proteins converted to hydroimidazolone with 2 mM MGO, which suggested that in small heat shock proteins, R12 is the most susceptible for modification to hydroimidazolone by MGO. Notably, a previous study detected a modification of R12 in human lens aA-crystallin that had a molecular weight identical to hydroimidazolone. The modification of arginine residues to hydroimidazolone converts the positive charge on arginine to a neutral charge. Previously, we reported that the substitution of MGO-modifiable arginine residues with neutral alanine residues enhanced the chaperone function of aA-crystallin, similarly to MGO-modification. Because R12 is the most susceptible arginine for MGO modification, we sought to determine if the chaperone function would be improved if it was replaced with alanine. Our results also demonstrated that TNS binding sites are different than the client protein binding sites in all three proteins.

Indeed, it clearly shows the importance of ligand acyl chains to bind and induce heterodimerization of the receptors and provides a rationale to tentatively understand the ligand structure-function relationships, although the presence of binding sites other than that of lipopeptides cannot be excluded. For instance, LTA, that bears two acyl chains, has been unambiguously proved, using chemically synthesized analogs, to stimulate TLR2 and recently demonstrated to bind TLR2. However, its role as a physiological TLR2 ligand is still under debate. Indeed, a set of studies focusing on Staphylococcus aureus and using cell wall-derived compounds as well as a mutant lacking acylated lipoproteins, demonstrates that LTA is much less active than Vorinostat lipoproteins and suggests that not LTA but lipoproteins are the dominant immunobiologically active compounds in this Gram-positive bacterium. As a consequence, in a recent review, Za¨ hringer et al propose that lipoproteins/lipopeptides are the only compounds of microorganisms sensed at physiological concentrations by TLR2. Lipoglycans are surface-exposed molecules of mycobacteria that have been described by other and us to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, their real nature as MAMPs has never been validated by isogenic mycobacterial mutants in the context of a bacterium infection. Their structure is based on a mannosylphosphatidyl-myo-inositol anchor, which, although very similar to the GPI anchors found in eukaryotic cells, is specific of these microorganisms. The biosynthesis of the mannosyl-phosphatidyl-myo-inositol anchor is essential in mycobacteria. The most active lipoglycan, lipomannan, is sensed by TLR2 at concentrations similar to that of mycobacterial lipoproteins and we have shown recently that it can compete for lipopeptide binding to the receptor, suggesting that it shares at least in part the same binding site. Assuming that it is the case, straightforward structure-function relationships can account for the observed TLR2-stimulatory capacity of the various purified LM acyl-forms. Nevertheless, a contamination of lipoglycan fractions by highly active lipopeptides is formally difficult to rule out. Moreover, a Mycobacterium tuberculosis mutant deficient for lipoprotein processing is dramatically altered in its capacity to stimulate TLR2, suggesting, as for S. aureus, a predominant role of lipoproteins in mycobacteria sensing by TLR2. In order to determine whether lipoglycans are i) bona fide MAMPs and most particularly TLR2 ligands and ii) sensed at physiological concentrations in the context of the whole bacterium, we used here the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we attempted to construct some strains with either an increased or a reduced production of lipoglycans and we compared their ability to induce innate immune signaling relatively to control strains in reporter cells, macrophage cell line or dendritic cells. Finally, to compare the relative contribution of lipoglycans and lipoproteins in mycobacteria sensing by TLR2, we constructed a mutant deficient for lipoprotein processing.

Although serum miR-122 was also elevated in HBV patients with HCC comparing with those without HCC, the difference was at the border line. Serum miR-122 yielded an AUC of 0.869 for discriminating HCC from healthy subjects and only 0.630 for discriminating HBV patients with HCC from those without HCC. At a cut-off value of 0.474, the sensitivity was 81.6% and the specificity was 83.3% in discriminating HCC from healthy subjects, and 77.6% sensitivity and 57.8% specificity in discriminating HBV patients with HCC from those without HCC at the cut-off value of 0.651. More importantly, it was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples, reaching levels comparable with healthy subjects, indicating that the elevation of serum miR-122 is likely derived from HCC. MiR-122 not only is evolutionary conserved across species and but also was identified as the most abundant liver specific miRNA constituting 70% of total hepatic miRNAs while cloning small RNAs from different tissues in mice. MiR-122 facilitates replication and translation of hepatitis C viral RNA and positively regulates cholesterol and triglyceride level. Significantly, the down-regulation of miR-122 was detected in more than 70% of HCC. It was shown that the level of miR122 expression increases in the mouse liver throughout development, to reach the maximum just Ibrutinib before birth. Thus, the loss of expression of miR-122 of HCC cells may represent either a differentiation reversion or a block to a less differentiated status of liver cells. In our study, it appears contrary and unexpected that the levels of miR-122 are elevated in serum of HCC patients. Our results showed that the elevated serum miR-122 is presented not only in HBV patients with HCC but also in HBV patients without HCC, suggesting that the elevated miR-122 in the serum of patients may also reflect liver injury. Hepatocytes contain abundant miR-122 and damage of hepatocytes caused by inflammation due to virus infection or cancer would be expected to release significant amount of this miRNA into the circulation. Because serum miRNAs have been shown to be very stable, miRNAs leaked from damaged hepatocytes would accumulate in blood to a high level. This might explain why miR-122 is downregulated in HCC tissues but elevated in serum of HBV patients without or with HCC. Interestingly, our data indicated that expression levels of miR-122 in serum were significantly higher in HCC patients than disease controls or healthy controls, while Xu et al. showed that expression levels of serum miR-122 were significantly higher in HBV patients than HCC or healthy controls. The reason may be that we and Xu et al. use the different normalization control. MiR-223 is one of the miRNAs that has been given much attention in the literature. In addition to this, a recent study observed that miR-223 was commonly repressed in HCC, suggesting a potential role of this miRNA in liver disease. In our study, levels of miR-223 were significantly elevated in HCC patients than in healthy controls, while no significant difference was observed for this miRNA between HBV subjects with and without HCC.

On the other hand, other growth factors such as VEGF, HGF and TGF-b1 were not changed, which suggests that the expression of IGF-1 was rather specific to RF radiation. The increased expression of IGF-1 was validated by Western blot against BCL-2, BAX, and pMAPK1, all of which seem to be targets of IGF-1. RF exposure activates IGF-1 mRNA expression to promote cell survival of hDPCs by Wortmannin increasing the ratio of BCL-2/BAX. CCND1 plays an important role in promoting G1-to-S phase progression. The signals downstream of MAPK related to mitogenesis contain the phosphorylation of transcription factors including JUN, ETS1, and ELK1, such as the cyclin D family. RF exposure could up-regulate the phosphorylation of MAPK1 and subsequently increase the expression of CCND1 as shown in Fig. 1. However, the overall cell cycle progression was not changed in RF-exposed cells, as shown in Fig. 2E. The increased levels of BCL-2 and CCND1 and the phosphorylation of MAPK1 may not be sufficient to induce the proliferation of hDPCs. The expression of BCL-2 and CCND1 and the phosphorylation of MAPK1 are important for cell survival by inhibiting apoptosis. Cell cycle progression requires additional changes in gatekeeper genes such as p53 and Rb for G1/S transition. We could not detect changes in cell cycle distribution in RF-irradiated hDPCs as other type of cells. RF exposure does not induce DNA strand breaks, chromosome aberrations, sister chromatid exchanges, DNA repair synthesis, phenotypic mutation, or transformation such as cancer-like changes. Exposure to RF radiation exerts no detectable effects on cell cycle distribution, cellular migration, or invasion at average SAR values of 2 or 10 W/kg. In contrast, another study noted that RF radiation induced basal cell proliferation and mild skin changes in rat skin cells. A diverse range of opinions exist with regard to whether RF exposure induces cellular change, and the results of these studies depend on the cell type, exposure time, frequency, or radiation dose used. We used higher SAR levels than previous reports, but we did not detect any evidence of oxidative stress. In this study, we demonstrated that RF exposure stimulated hair growth in an ex vivo system and an in vitro model using hDPCs. Our future studies will be focused on investigating the effect of RF radiation using various frequencies and doses to better understand the biological responses. In addition, we need to explore human hair growth on other types of HFs. Interestingly, 1,763 MHz RF radiation at a higher SAR, for example 60 W/kg, hardly showed linear responses in hDPCs and outer root sheath cells, which are keratinocytes of human scalp HFs. From these results, we propose that RF radiation at a specific dose and exposure time could be used to treat human hair disorders such as androgenetic alopecia. Fast and reliable identification of moulds would help manage the growing number of invasive mould infections, a leading cause of morbidity and lethality in immunocompromised patients. Currently, mould identification relies on the macroscopic and microscopic observation of colonies grown on mycological media. Adequate phenotypic identification of moulds requires highly skilled mycologists, who are found in a few reference laboratories.