Circadian time series proteomics reveals daily dynamics in cartilage physiology.

OBJECTIVE: Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue. METHODS: We investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis. RESULTS: Out of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing. CONCLUSIONS: Our circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis.

LncRNA HCP5 promotes the development of cervical cancer by regulating MACC1 via suppression of microRNA-15a.

OBJECTIVE: To explore the role of long non-coding RNA (lncRNA) HCP5 in the development of cervical cancer and its underlying mechanism. PATIENTS AND METHODS: Expression levels of HCP5, MACC1 and microRNA-15a in cervical cancer tissues and paracancerous tissues were detected. The relationship between HCP5 expression and prognosis of patients with cervical cancer was analyzed by Kaplan-Meier. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay after altering expressions of HCP5 and microRNA-15a by plasmids transfection. The binding condition of HCP5, MACC1 and microRNA-15a was evaluated by luciferase reporter gene assay. The regulatory effect of microRNA-15a on MACC1 expression was determined by Western blot. RESULTS: HCP5 and MACC1 were overexpressed in cervical cancer tissues than those of paracancerous tissues. The survival rate of patients with cervical cancer was negatively correlated to HCP5 expression, but positively correlated to microRNA-15a expression. Luciferase reporter gene assay showed that microRNA-15a was directly bound to HCP5 and MACC1. Besides, overexpression of microRNA-15a could remarkably inhibited MACC1 expression. In vitro experiments showed that HCP5 promoted proliferation of cervical cancer cells, which was reversed by microRNA-15a knockdown. CONCLUSIONS: Overexpressed HCP5 promoted the development of cervical cancer through increasing MACC1 expression by microRNA-15a adsorption.

Catabolic cytokines disrupt the circadian clock and the expression of clock-controlled genes in cartilage via an NFкB-dependent pathway.

OBJECTIVE: To define how the catabolic cytokines (Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFα)) affect the circadian clock mechanism and the expression of clock-controlled catabolic genes within cartilage, and to identify the downstream pathways linking the cytokines to the molecular clock within chondrocytes. METHODS: Ex vivo cartilage explants were isolated from the Cry1-luc or PER2::LUC clock reporter mice. Clock gene dynamics were monitored in real-time by bioluminescence photon counting. Gene expression changes were studied by qRT-PCR. Functional luc assays were used to study the function of the core Clock/BMAL1 complex in SW-1353 cells. NFкB pathway inhibitor and fluorescence live-imaging of cartilage were performed to study the underlying mechanisms. RESULTS: Exposure to IL-1ß severely disrupted circadian gene expression rhythms in cartilage. This effect was reversed by an anti-inflammatory drug dexamethasone, but not by other clock synchronizing agents. Circadian disruption mediated by IL-1ß was accompanied by disregulated expression of endogenous clock genes and clock-controlled catabolic pathways. Mechanistically, NFкB signalling was involved in the effect of IL-1ß on the cartilage clock in part through functional interference with the core Clock/BMAL1 complex. In contrast, TNFα had little impact on the circadian rhythm and clock gene expression in cartilage. CONCLUSION: In our experimental system (young healthy mouse cartilage), we demonstrate that IL-1ß (but not TNFα) abolishes circadian rhythms in Cry1-luc and PER2::LUC gene expression. These data implicate disruption of the chondrocyte clock as a novel aspect of the catabolic responses of cartilage to pro-inflammatory cytokines, and provide an additional mechanism for how chronic joint inflammation may contribute to osteoarthritis (OA).

Real-time analysis of gene regulation by glucocorticoid hormones.

There is increasing evidence that temporal factors are important in allowing cells to gain additional information from external factors, such as hormones and cytokines. We sought to discover how cell responses to glucocorticoids develop over time, and how the response kinetics vary according to ligand structure and concentration, and hence have developed a continuous gene transcription measurement system, based on an interleukin-6 (IL-6) luciferase reporter gene. We measured the time to maximal response, maximal response and integrated response, and have compared these results with a conventional, end point glucocorticoid bioassay. We studied natural glucocorticoids (corticosterone and cortisol), synthetic glucocorticoids (dexamethasone) and glucocorticoid precursors with weak, or absent bioactivity. We found a close correlation between half maximal effective concentration (EC50) for maximal response, and for integrated response, but with consistently higher EC50 for the latter. There was no relation between the concentration of ligand and the time to maximal response. A comparison between conventional end point assays and real-time measurement showed similar effects for dexamethasone and hydrocortisone, with a less effective inhibition of IL-6 seen with corticosterone. We profiled the activity of precursor steroids, and found pregnenolone, progesterone, 21-hydroxyprogesterone and 17-hydroxyprogesterone all to be ineffective in the real-time assay, but in contrast, progesterone and 21-hydroxyprogesterone showed an IL-6 inhibitory activity in the end point assay. Taken together, our data show how ligand concentration can alter the amplitude of glucocorticoid response, and also that a comparison between real-time and end point assays reveals an unexpected diversity of the function of glucocorticoid precursor steroids, with implications for human disorders associated with their overproduction.

The biology of the circadian Ck1epsilon tau mutation in mice and Syrian hamsters: a tale of two species.

The tau mutation in the Syrian hamster resides in the enzyme casein kinase 1 epsilon (CK1epsilon), resulting in a dramatic acceleration of wheel-running activity cycles to about 20 hours. tau also impacts growth, energy, metabolism, feeding behavior, and circadian mechanisms underpinning seasonal timing, causing accelerated reproductive and neuroendocrine responses to photoperiodic changes. Modeling and experimental studies suggest that tau acts as a gain of function on specific residues of PER, consistent with hamster studies showing accelerated degradation of PER in the suprachiasmatic nucleus in the early circadian night. We have created null and tau mutants of Ck1epsilon in mice. Circadian period lengthens in CK1epsilon(/), whereas CK1epsilon(tau/tau) shortens circadian period of behavior in vivo in a manner nearly identical to that of the Syrian hamster. CK1epsilon(tau/tau) also accelerates molecular oscillations in peripheral tissues, demonstrating its global circadian role. CK1epsilon(tau) acts by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Our studies reveal that tau acts as a gain-of-function mutation, to accelerate degradation of PERIOD proteins. tau has consistent effects in both hamsters and mice on the circadian organization of behavior and metabolism, highlighting the global impact of this mutation on mammalian clockwork in brain and periphery.

Quasi-one-dimensional molecular magnets based on derivatives of (fluorobenzyl)pyridinium with the [M(mnt)2] monoanion (M = Ni, Pd or Pt; mnt2- = maleonitriledithiolate): syntheses, crystal structures and magnetic properties.

The syntheses, structural characterizations and magnetic behaviors of three new complexes, 1-(3',4',5'-trifluorobenzyl)pyridinium [M(mnt)2]- [M = Ni (1), Pd (2) or Pt (3)], are reported. These complexes are isomorphous and their prominent structural character is that the [M(mnt)2]- anions form columnar stacks, in which the dimerization was observed. Complexes 2 and 3 are diamagnetic, while 1 possesses an energy gap of 2474 K. For crystal 4, 1-(4'-fluorobenzyl)pyridinium [Ni(mnt)2] (its structure and magnetic susceptibility were briefly reported earlier), the magnetic behavior can be divided into two regimes, namely, weakly ferromagnetic coupling above 93 K and strongly antiferromagnetic coupling below 93 K. A transition occurs at 93 K which switches the magnetic exchange nature from ferromagnetic to antiferromagnetic. A sharp thermal abnormality with lambda-shape, associated with the transition, appears from its heat capacity measurement to indicate that the transition is first order. The temperature dependences of the superlattice diffractions revealed the existence of the pretransitional phenomena up to at least 140 K. The unusual magnetic behavior of 4, such as the origin of the ferromagnetic interaction in the high temperature phase and what causes the spin transition, are discussed further.

Ionic pair complexes with well-separated columnar stack structure based on [Pt(mnt)2]- ions showing unusual magnetic transition: syntheses, crystal structures, and magnetic properties.

Three ion pair complexes, [4-R-benzylpyridinium][bis(maleodinitriledithiolato)platinum(III)] (abbreviated as [RBzPy][Pt(mnt)(2)]; R = Cl (1), Br (2), or NO(2) (3)), have been synthesized. The cations and anions stack into well-separated columns in the solid state, and the Pt(III) ions form a 1-D zigzag chain within a [Pt(mnt)(2)](-) column through Pt...S, S...S, and Pt...S...Pt interactions. The chain is uniform in 1 and 2, while it alternates in 3. Unusual magnetic phase transitions from paramagnetism to diamagnetism were observed in these three complexes at approximately 275 K for 1, approximately 269 K for 2, and approximately 184 K for 3. These phase transitions were also found in DSC measurements for 1 and 2. The overall magnetic behaviors for 1-3 indicate the presence of antiferromagnetic exchange interactions in the high-temperature phase and spin-gapped systems in the low-temperature phase. Below 50 K, 2 exhibits weak ferromagnetism. The spontaneous moments are nearly repressed by a field of 1.0 T. The crystal structure of 2 at 173 K reveals that there are two crystallographically independent [Pt(mnt)(2)](-) entries in an asymmetric unit. These two crystallographically independent [Pt(mnt)(2)](-) entries satisfy the spin-canting condition, and the EPR spectra measured at room temperature exhibit anisotropic character. Therefore, the weak ferromagnetic behavior in the low-temperature region for 2 can be attributed to the spin-canting phenomenon.

2-Methyl-1-(4-nitrobenzyl)pyridinium bis(maleonitriledithiolato)nickelate(III).

In the title complex, 2-methyl-1-(4-nitrobenzyl)pyridinium bis(1,2-dicyanoethene-1,2-dithiolato)nickelate(III), (C(13)H(13)N(2)O(2))[Ni(C(4)N(2)S(2))(2)], the most prominent general structural feature of the complex is the completely segregated columnar stacks of anions and cations. Within the cation column, there may be stacking interactions between adjacent nitro groups and benzene rings.

The inclusion compound of a new ionizable derivative of beta-cyclodextrin with ferrocenium drug.

A new beta-cyclodextrin (beta-CD) derivative, mono[6-deoxy-6-(2-butenedinitrile-2,3-dimercapto sodium salt)]-beta-CD (6-mnt-beta-CD), and its inclusion compound with a ferrocenium drug, have been prepared and characterized by IR, UV, 13C-NMR spectroscopy, and mass spectrometry, elemental analysis, thermogravimetry, and cyclic voltammetry (CV). The interplay between the side-arm anion of beta-CD and the ferrocenium (guest) in the inclusion compound 6-mnt-beta-CD-/Fc+ has been investigated by 13C-NMR, UV, IR, and thermogravimetric methods. Charge transfer from the anion to the cation in 6-mnt-beta-CD-/Fc+ was then experimentally identified. The interaction between the guest and the host with side-arm in 6-mnt-beta-CD-/Fc+ resulted in smaller positive potential shifts compared to that in the inclusion compound [beta-CD/Fc+]BF4-.

The inclusion compounds of beta-cyclodextrin with 4-substituted benzoic acid and benzaldehyde drugs studied by proton nuclear magnetic resonance spectroscopy.

Inclusion compounds of some 4-substituted benzoic acid and benzaldehyde drugs with beta-cyclodextrin were prepared and characterized by IR spectroscopy, powder X-ray diffraction, thermogravimetry, and 1H-NMR spectroscopy. The thermal stability and chemical stability of these drugs were strikingly improved after inclusion. The effect of inclusion on the chemical-shifts of protons H-3 and H-5 in the NMR spectroscopy is discussed. Using the relative shift theory, the preferred inclusion mode was proposed. The center of the aromatic ring of the drug molecule was considered to be located in the cavity 1.2 A inside from the H-5 plane of beta-cyclodextrin.

[The advances of the research work on lens cells using patch clamp technique].

The study of lens cells by patch clamp technique began in the early 90's. It shows that the membrane of lens cells possess different types of ionic channels. These channels are responsible for the modulation of the hydration state and transparency of lens. Several patch clamp configurations have been used to study the activity of ionic channels. The resting potential and capacitance as well as resistance of different lens cells were also measured.