OBJECTIVE: There is evidence for a disturbed intestinal barrier function in inflammatory bowel diseases [IBD] but the underlying mechanisms are unclear. Because mucins represent the major components of the mucus barrier and disturbed mucin expression is reported in the colon of IBD patients, we studied the association between mucin expression, inflammation and intestinal permeability in experimental colitis.
METHODS: We quantified 4-kDa FITC-dextran intestinal permeability and the expression of cytokines, mucins, junctional and polarity proteins at dedicated time points in the adoptive T cell transfer and dextran sodium sulfate [DSS]-induced colitis models. Mucin expression was also validated in biopsies from IBD patients.
RESULTS: In both animal models, the course of colitis was associated with increased interleukin-1β [IL-1β] and tumour necrosis factor-α [TNF-α] expression and increased Muc1 and Muc13 expression. In the T cell transfer model, a gradually increasing Muc1 expression coincided with gradually increasing 4-kDa FITC-dextran intestinal permeability and correlated with enhanced IL-1β expression. In the DSS model, Muc13 expression coincided with rapidly increased 4-kDa FITC-dextran intestinal permeability and correlated with TNF-α and Muc1 overexpression. Moreover, a significant association was observed between Muc1, Cldn1, Ocln, Par3 and aPKCζ expression in the T cell transfer model and between Muc13, Cldn1, Jam2, Tjp2, aPkcζ, Crb3 and Scrib expression in the DSS model. Additionally, MUC1 and MUC13 expression was upregulated in inflamed mucosa of IBD patients.
CONCLUSIONS: Aberrantly expressed MUC1 and MUC13 might be involved in intestinal barrier dysfunction upon inflammation by affecting junctional and cell polarity proteins, indicating their potential as therapeutic targets in IBD.

BACKGROUND: Survival benefit from surgical debulking of ovarian cancer (OC) is well established, but some women, despite total macroscopic clearance of disease, still have poor prognosis. We aimed to identify biomarkers to predict benefit from conventional surgery.
METHODS: Clinical data from women debulked for high-stage OC were analysed (Hammersmith Hospital, London, UK; 2001-2014). Infinium\'s HumanMethylation27 array interrogated tumour DNA for differentially methylated CpG sites, correlated to survival, in patients with the least residual disease (RD; Hammersmith Array). Validation was performed using bisulphite pyrosequencing (Charité Hospital, Berlin, Germany cohort) and The Cancer Genome Atlas\' (TCGA) methylation data set. Kaplan-Meier curves and Cox models tested survival.
RESULTS: Altogether 803 women with serous OC were studied. No RD was associated with significantly improved overall survival (OS; hazard ratio (HR) 1.25, 95% CI 1.06-1.47; P=0.0076) and progression-free survival (PFS; HR 1.23, 95% CI 1.05-1.43; P=0.012; Hammersmith database n=430). Differentially methylated loci within FGF4, FGF21, MYLK2, MYLK3, MYL7, and ITGAE associated with survival. Patients with the least RD had significantly better OS with higher methylation of MYLK3 (Hammersmith (HR 0.51, 95% CI 0.31-0.84; P=0.01), Charité (HR 0.46, 95% CI 0.21-1.01; P=0.05), and TCGA (HR 0.64, 95% CI 0.44-0.93; P=0.02)).
CONCLUSIONS: MYLK3 methylation is associated with improved OS in patients with the least RD, which could potentially be used to determine response to surgery.

BACKGROUND: To better understand the evolution of typical hypertrophic cardiomyopathy (HCM) to heart failure (HF), we investigated the relationship between serum biochemical abnormalities and changes in left ventricular (LV) remodeling.
RESULTS: Seventy-seven HCM patients were followed for 20 years. Creatine kinase (CK), CK-MB, lactate dehydrogenase (LDH), LDH-1, troponin T and myosin light chain-1 (MLC-1) were measured. Abnormal CK-MB elevation was observed in 64% of HCM patients. LDH-1 was not significantly different compared with the control subjects. Troponin T elevation was observed in 3 HCM patients and MLC-1 elevation was not observed. According to median CK-MB, HCM patients were divided into 2 groups: group H (CK-MB ≥2.5%, n=33) and group L (CK-MB <2.5%, n=44). During the follow-up period in group H, LV end-diastolic dimension increased (P<0.0001), fractional shortening decreased (P<0.0004), and left atrial dimension increased (P<0.0001). The markers reflecting LV hypertrophy were significantly decreased. In group L, LV end-diastolic dimension increased (P<0.02) and left atrial dimension increased (P<0.0001). HF was observed in 18 patients in group H and in 4 in group L. There were 14 HF deaths in group H and 2 in group L, and 3 sudden cardiac deaths in group H.
CONCLUSIONS: Persistent elevation of cardiac enzymes in HCM patients indicates ongoing myocardial injury, ultimately resulting in death by HF.

The present study was designed to identify changes in human serum caused by myosin light chain kinase (MLCK) in the context of Type 2 diabetes mellitus (T2DM), and to determine whether it had correlations with other biomarkers of T2DM. A total of 336 patients with T2DM and 90 sex- and age-matched, apparently healthy subjects were selected. The serum MLCK of all participants was detected by quantitative ELISA. Our results showed that the concentration of MLCK in the T2DM group was significantly higher than in control samples, and there were no correlations with age, FBG, TC, HDL-C, LDL-C or HbA1c. These findings suggest that serum MLCK is associated with T2DM, which gives some clues for identifying new biomarkers for the diagnosis of T2DM and its complications.

OBJECTIVE: To investigate the function of myosin light chain kinase (MLCK) in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing.
METHODS: Cross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response (ABR) to evaluate mice hearing function at different frequencies.
RESULTS: Mlck knockout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones (8 kHz, 16 kHz, 32 kHz), and the rise was over 20 dB at high-frequency(32 kHz). Further analyses of waveforms showed that wave-I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone (16 kHz) were less than control group(P < 0.05) on average, but the ratio of wave I/II and I/III were not difference (P > 0.05).
CONCLUSIONS: Mlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high-frequencies.

OBJECTIVE: To examine the content of phosphorylated myosin regulatory light chain (P-MLC20) and myosin light-chain kinase (MLCK) in the common bile duct of pediatric patients with pancreaticobiliary maljunction (PBM) accompanied by bile duct dilatation (BDD), and investigate their potential role in PBM accompanied by BDD.
METHODS: Twenty-one specimens of the common bile duct from pediatric patients with PBM accompanied by BDD were collected. P-MLC20 was examined with immunohistochemistry. The expression of P-MLC20 and MLCK was also examined with Western blot. Twenty-one specimens of the common bile duct from pediatric patients without PBM and BDD were used as controls.
RESULTS: The mean optical density (MOD), mean labeling intensity (MLI) and minimum qualifying scores (MQS) of P-MLC20 were 115.6856 ± 58.1634, 21.7125 % ± 9.6555 and 21.3531 ± 6.5255, respectively. In the control group, MOD, MLI and MQS were 96.5581 ± 9.7859, 11.1813 % ± 3.6208 and 10.7819 ± 3.5323, respectively. There was no significant difference in MOD between the two groups (P > 0.05), whereas there was a significant difference in MLI and MQS between the two groups (P < 0.05). The expression of P-MLC20 and MLCK, as determined with Western blot, was also significantly higher in the PBM group than in the control group (P < 0.05).
CONCLUSIONS: P-MLC20 is associated with increased contractile force of the smooth muscle of the common bile duct in pediatric patients with PBM accompanied by BDD. The enhanced expression of P-MLC20 in the common bile duct probably contributes to increased bile duct pressure in PBM via the MLCK pathway.

OBJECTIVE: To investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) on intestinal epithelial barrier function.
METHODS: The Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts.They were divided into control group (ordinary treatment), IFN-γ group (with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyanate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of variance and t test.
RESULTS: (1) There was no obvious difference in TER in control group at each time point (F = 0.86, P > 0.05). TER in IFN-γ group and TNF-α group were gradually decreased during PTH 6-48, but showed no statistical difference as compared with that at PTH 0 (with F value respectively 1.69, 2.47, P values all above 0.05). TER in IFN-γ plus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH 0 (t = 4.97, P < 0.05) and that in each of the other three groups (F = 11.54, P < 0.05). (2) The permeability of monolayers in IFN-γ plus TNF-α group [(1197 ± 215)pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [(303 ± 93), (328 ± 76), (797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups (F = 0.26, P > 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γ and TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-α group at PTH 48 was interrupted, with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ± 0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ± 0.12, 0.56 ± 0.07, 0.59 ± 0.10, respectively, F = 17.97, P < 0.01). The protein expression of MLCK in IFN-γ plus TNF-α group (1.57 ± 0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0.23, 1.00 ± 0.07, respectively, F = 9.05, P < 0.05).
CONCLUSIONS: Combination of IFN-γ and TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.

The transient and localized signaling events between invasive breast cancer cells and the underlying endothelial cells have remained poorly characterized. We report a novel approach integrating vascular engineering with three-dimensional time-lapse fluorescence resonance energy transfer (FRET) imaging to dissect how endothelial myosin light chain kinase (MLCK) is modulated during tumor intravasation. We show that tumor transendothelial migration occurs via both paracellular (i.e. through cell-cell junctions) and transcellular (i.e. through individual endothelial cells) routes. Endothelial MLCK is activated at the invasion site, leading to regional diphosphorylation of myosin-II regulatory light chain (RLC) and myosin contraction. Blocking endothelial RLC diphosphorylation blunts tumor transcellular, but not paracellular, invasion. Our results implicate an important role for endothelial myosin-II function in tumor intravasation.

Calmodulin is known to be a target for oxidation, which leads to conversion of methionine residues to methionine sulfoxides. Previously, we reported that both methionine sulfoxide reductases MsrA and MsrB were able to reduce methionine sulfoxide residues in oxidized calmodulin. In the present study, we have made use of the interaction between calmodulin and RS20, a peptide model for calmodulin targets, to probe the structural consequences of oxidation and mode of repair both by MsrA and MsrB. Isothermal titration calorimetry and differential scanning calorimetry showed that oxidized calmodulin interacts with RS20 via its C-terminal domain only, resulting in a non-productive complex. As shown by spectrofluorometry, oxidized calmodulin treated with MsrA exhibited native binding affinity for RS20. In contrast, MsrB-treatment of oxidized calmodulin resulted in 10-fold reduced affinity. Mass spectrometry revealed that the sulfoxide derivative of methionine residue 124 was differentially repaired by MsrA and MsrB. This provided a basis for rationalizing the difference in binding affinities of oxidized calmodulin reported above, since Met124 residue had been shown to be critical for interaction with some targets. This study provides the first evidence that in an oxidized polypeptide chain MetSO residues might be differentially repaired by the two Msr enzymes.

The signal transduction pathways within corporal smooth muscle cells are the intracellular molecular mechanisms of corporal smooth muscle tone regulation. Various neurotransmitters activate the membrane receptor proteins or intracellular enzyme pathways and result in the production of extracellular chemical signals. Second messenger molecules and ions transmit and amplify the signals, and subsequently induce the relaxation of smooth muscle cells and penile erection. Therefore, the study of signal transduction in cavernous smooth muscle play an important role in understanding the physiology of erection and the pathophysiology of erectile dysfunction as well as in developing new selective drugs for the treatment of erectile dysfunction.