A post-translational modification (PTM) is a chemical modification of a protein that occurs naturally. Many of these modifications, such as phosphorylation, are known to play pivotal roles in the regulation of protein function. Henceforth, PTM perturbations have been linked to diverse diseases like Parkinson's, Alzheimer's, diabetes and cancer. To discover PTMs on a genome-wide scale, there is a recent surge of interest in analyzing tandem mass spectrometry data, and several unrestrictive (so-called 'blind') PTM search methods have been reported. However, these approaches are subject to noise in mass measurements and in the predicted modification site (amino acid position) within peptides, which can result in false PTM assignments.

Although the medial subdivision of the central nucleus of the amygdala (CeM) and serotonin-1A (5-HT1A) receptors are involved in the regulation of anxiety, their roles in Parkinson's disease (PD)-associated anxiety are still unknown. Here we assessed the importance of CeM 5-HT1A receptors for anxiety in rats with unilateral 6-hydroxydopamine (6-OHDA) lesion of the medial forebrain bundle (MFB). The lesion induced anxiety-like behaviors, increased the firing rate and burst-firing pattern of CeM γ-aminobutyric acid (GABA) neurons, as well as decreased dopamine (DA) levels in the striatum, medial prefrontal cortex (mPFC), amygdala and ventral part of hippocampus (vHip). Intra-CeM injection of the selective 5-HT1A receptor agonist 8-OH-DPAT produced anxiolytic effects in the lesioned rats, and decreased the firing rate of CeM GABAergic neurons in two groups of rats. Compared to sham-operated rats, the duration of the inhibitory effect on the firing rate of GABAergic neurons was shortened in the lesioned rats. The injection increased DA levels in the mPFC and amygdala in two groups of rats and the vHip in the lesioned rats, and increased 5-HT level in the lesioned rats, whereas it decreased NA levels in the mPFC in two groups of rats and the vHip in the lesioned rats. Moreover, the mean density of 5-HT1A receptor and GABA double-labeled neurons in the CeM was reduced after the lesioning. These results suggest that activation of CeM 5-HT1A receptor produces anxiolytic effects in the 6-OHDA-lesioned rats, which involves decreased firing rate of the GABAergic neurons, and changed monoamine levels in the limbic and limbic-related brain regions.

Studies have shown that exercise interventions can improve functional recovery after spinal cord injury, but the mechanism of action remains unclear. To investigate the mechanism, we established a unilateral corticospinal tract injury model in rats by pyramidotomy, and used a single pellet reaching task and horizontal ladder walking task as exercise interventions postoperatively. Functional recovery of forelimbs and forepaws in the rat models was noticeably enhanced after the exercises. Furthermore, TUNEL staining revealed significantly fewer apoptotic cells in the spinal cord of exercised rats, and western blot analysis showed that spinal cord expression of the apoptosis-related protein caspase-3 was significantly lower, and the expression of Bcl-2 was significantly higher, while the expression of Bax was not signifiantly changed after exercise, compared with the non-exercised group. Expression of these proteins decreased with time after injury, towards the levels observed in sham-operated rats, however at 4 weeks postoperatively, caspase-3 expression remained significantly greater than in sham-operated rats. The present findings indicate that a reduction in apoptosis is one of the mechanisms underlying the improvement of functional recovery by exercise interventions after corticospinal tract injury.

In order to explore the efficient utilization of plant resources from constructed wetlands, the potential anti-metastatic effects of flavonoids from Potamogeton crispus L. were investigated in human ovarian cancer cells (ES-2). Two major flavonoids, luteolin-3'-O-β-D-glucopyranoside and flavone-6-C-β-D-glucopyranoside, were isolated from P. crispus and identified. The effects of these flavonoids on cell proliferation, cell morphology, cell cycle, apoptosis, and cell migration and invasion were then investigated. Furthermore, reverse transcriptase polymerase chain reaction assays and western blotting analysis were conducted to examine the expression level of mRNA and protein. Results indicated that Luteolin-3'-O-β-D-glucopyranoside inhibited ES-2 cell migration and invasion and suppressed the expression of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, and Flavone-6-C-β-D-glucopyranoside had no significant inhibitory effects on ES-2 cells. Thus, this study demonstrated the potential anti-metastatic properties of a P. crispus flavonoid, and provided a scientific approach for the screening of promising natural resources from constructed wetlands to identify useful products for use in the pharmaceutical and healthcare industries.

Biochar produced by pyrolysis of biomass can be used to counter nitrogen (N) pollution. The present study investigated the effects of feedstock and temperature on characteristics of biochars and their adsorption ability for ammonium N (NH4(+)-N) and nitrate N (NO3(-)-N). Twelve biochars were produced from wheat-straw (W-BC), corn-straw (C-BC) and peanut-shell (P-BC) at pyrolysis temperatures of 400, 500, 600 and 700°C. Biochar physical and chemical properties were determined and the biochars were used for N sorption experiments. The results showed that biochar yield and contents of N, hydrogen and oxygen decreased as pyrolysis temperature increased from 400°C to 700°C, whereas contents of ash, pH and carbon increased with greater pyrolysis temperature. All biochars could sorb substantial amounts of NH4(+)-N, and the sorption characteristics were well fitted to the Freundlich isotherm model. The ability of biochars to adsorb NH4(+)-N followed: C-BC>P-BC>W-BC, and the adsorption amount decreased with higher pyrolysis temperature. The ability of C-BC to sorb NH4(+)-N was the highest because it had the largest cation exchange capacity (CEC) among all biochars (e.g., C-BC400 with a CEC of 38.3 cmol kg(-1) adsorbed 2.3 mg NH4(+)-N g(-1) in solutions with 50 mg NH4(+) L(-1)). Compared with NH4(+)-N, none of NO3(-)-N was adsorbed to biochars at different NO3(-) concentrations. Instead, some NO3(-)-N was even released from the biochar materials. We conclude that biochars can be used under conditions where NH4(+)-N (or NH3) pollution is a concern, but further research is needed in terms of applying biochars to reduce NO3(-)-N pollution.

New Zealand rabbits were randomly divided into an ischemia group (occlusion of the abdominal aorta for 60 minutes), an ischemia-reperfusion group (occlusion of the abdominal aorta for 60 minutes followed by 48 hours of reperfusion) and a sham-surgery group. Two-dimensional gel electrophoresis detected 49 differentially expressed proteins in spinal cord tissue from the ischemia and ischemia/ reperfusion groups and 23 of them were identified by mass spectrometry. In the ischemia group, the expression of eight proteins was up regulated, and that of the remaining four proteins was down regulated. In the ischemia/reperfusion group, the expression of four proteins was up regulated, and that of two proteins was down regulated. In the sham-surgery group, only one protein was detected. In the ischemia and ischemia/reperfusion groups, four proteins overlapped between groups with the same differential expression, including three that were up regulated and one down regulated. These proteins were related to energy metabolism, cell defense, inflammatory mechanism and cell signaling.

Excessive reactive oxygen species (ROS) play an important role in the development of osteoporosis. Ophiopogonin D (OP-D), isolated from the traditional Chinese herbal agent Radix Ophiopogon japonicus, is a potent anti-oxidative agent. We hypothesized that OP-D demonstrates anti-osteoporosis effects via decreasing ROS generation in mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells and a macrophage cell line RAW264.7 cells. We investigated OP-D on osteogenic and osteoclastic differentiation under oxidative status. Hydrogen peroxide (H2O2) was used to establish an oxidative damage model. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Then, we searched the molecular mechanism of OP-D against osteoporosis. Our results revealed that OP-D significantly promoted the proliferation of MC3T3-E1 cells and improved some osteogenic markers. Moreover, OP-D reduced TRAP activity and the mRNA expressions of osteoclastic genes in RAW264.7 cells. OP-D suppressed ROS generation in both MC3T3-E1 and RAW264.7 cells. OP-D treatment reduced the activity of serum bone degradation markers, including CTX-1 and TRAP. Further research showed that OP-D displayed anti-osteoporosis effects via reducing ROS through the FoxO3a-β-catenin signaling pathway. In summary, our results indicated that the protective effects of OP-D against osteoporosis are linked to a reduction in oxidative stress via the FoxO3a-β-catenin signaling pathway, suggesting that OP-D may be a beneficial herbal agent in bone-related disorders, such as osteoporosis.

In terms of making genes expression data more interpretable and comprehensible, there exists a significant superiority on sparse methods. Many sparse methods, such as penalized matrix decomposition (PMD) and sparse principal component analysis (SPCA), have been applied to extract plants core genes. Supervised algorithms, especially the support vector machine-recursive feature elimination (SVM-RFE) method, always have good performance in gene selection. In this paper, we draw into class information via the total scatter matrix and put forward a class-information-based penalized matrix decomposition (CIPMD) method to improve the gene identification performance of PMD-based method. Firstly, the total scatter matrix is obtained based on different samples of the gene expression data. Secondly, a new data matrix is constructed by decomposing the total scatter matrix. Thirdly, the new data matrix is decomposed by PMD to obtain the sparse eigensamples. Finally, the core genes are identified according to the nonzero entries in eigensamples. The results on simulation data show that CIPMD method can reach higher identification accuracies than the conventional gene identification methods. Moreover, the results on real gene expression data demonstrate that CIPMD method can identify more core genes closely related to the abiotic stresses than the other methods.

Recent progress suggests gene therapy may one day be an option for treating some forms of limb girdle muscular dystrophy (LGMD). Nevertheless, approaches targeting LGMD have so far focused on gene replacement strategies for recessive forms of the disease. In contrast, no attempts have been made to develop molecular therapies for any of the eight dominantly inherited forms of LGMD. Importantly, the emergence of RNA interference (RNAi) therapeutics in the last decade provided new tools to combat dominantly inherited LGMDs with molecular therapy. In this study, we describe the first RNAi-based, preclinical gene therapy approach for silencing a gene associated with dominant LGMD. To do this, we developed adeno-associated viral vectors (AAV6) carrying designed therapeutic microRNAs targeting mutant myotilin (MYOT), which is the underlying cause of LGMD type 1A (LGMD1A). Our best MYOT-targeted microRNA vector (called miMYOT) significantly reduced mutant myotilin mRNA and soluble protein expression in muscles of LGMD1A mice (the TgT57I model) both 3 and 9 months after delivery, demonstrating short- and long-term silencing effects. This MYOT gene silencing subsequently decreased deposition of MYOT-seeded intramuscular protein aggregates, which is the hallmark feature of LGMD1A. Histological improvements were accompanied by significant functional correction, as miMYOT-treated animals showed increased muscle weight and improved specific force in the gastrocnemius, which is one of the most severely affected muscles in TgT57I mice and patients with dominant myotilin mutations. These promising results in a preclinical model of LGMD1A support the further development of RNAi-based molecular therapy as a prospective treatment for LGMD1A. Furthermore, this study sets a foundation that may be refined and adapted to treat other dominant LGMD and related disorders.

This study is a meta-analysis comparing the efficacy, predictability, and safety of correcting myopia via implantation of two types of phakic intraocular lens (PIOLs): the implantable collamer lens (ICL) and iris-fixed PIOL. The Cochrane library, Pubmed, and EMBASE were searched. Study selection, data exclusion, and quality assessment were performed by two independent observers. The pooled relative risk (RR), pooled standardized mean difference (SMD), and their 95% confidence intervals (CIs) were used to compare lenses. Seven studies, involving 511 eyes, were included. The pooled SMD in postoperative uncorrected distance visual acuity (UDVA) comparing ICLs to iris-fixed PIOLs was -0.22 (95% CI, -0.58 to 0.13; P = .22). The pooled RR values of UDVA of 20/20 or better and of 20/40 or better comparing ICLs to iris-fixed PIOLs were 1.15 (95% CI, 0.89 to 1.47; P = .29) and 1.01 (95% CI, 0.95 to 1.08; P = .75), respectively. The pooled RR of loss of best spectacle-corrected visual acuity (BSCVA) and gain in BSCVA comparing ICLs to iris-fixed PIOLs were 1.20 (95% CI, 0.24 to 6.00; P = .82) and 1.14 (95% CI, 0.89 to 1.48; P = .31), respectively. The pooled RR comparing ICLs to iris-fixed PIOLs was 0.78 (95% CI, 0.29 to 2.12; P = .63) for all reported complications and 2.80 (95% CI, 1.04 to 7.52; P = .04) for severe complications. The pooled RR of achieving a result within ± 0.5 D (diopter) of the intended target comparing ICLs to iris-fixed PIOLs was 1.35 (95% CI, 1.04 to 1.77; P = .03). Overall, there is no significant difference in efficacy between the two types of PIOLs or in safety, except that the ICL is associated with a greater incidence of severe complications, especially anterior subcapsular cataract, primarily in the Version 2 and Version 3 groups. However, ICL has better predictability.

Age-related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Loss of retinal pigment epithelium (RPE) is a major pathological hallmark in AMD with or without pathological neovascularization. Although activation of the immune system is implicated in disease progression, pathological pathways remain diverse and unclear. Here, we report an unexpected protective role of a pro-inflammatory cytokine, interleukin-33 (IL-33), in ocular angiogenesis. IL-33 and its receptor (ST2) are expressed constitutively in human and murine retina and choroid. When RPE was activated, IL-33 expression was markedly elevated in vitro. We found that IL-33 regulated tissue remodelling by attenuating wound-healing responses, including reduction in the migration of choroidal fibroblasts and retinal microvascular endothelial cells, and inhibition of collagen gel contraction. In vivo, local administration of recombinant IL-33 inhibited murine choroidal neovascularization (CNV) formation, a surrogate of human neovascular AMD, and this effect was ST2-dependent. Collectively, these data demonstrate IL-33 as a potential immunotherapy and distinguishes pathways for subverting AMD pathology. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Carbonic anhydrase I (CA1) is the cytosolic isoform of mammalian α-CA family members which are responsible for maintaining pH homeostasis in the physiology and pathology of organisms. A subset of CA isoforms are known to be expressed and function in the central nervous system (CNS). CA1 has not been extensively characterized in the CNS. In this study, we demonstrate that CA1 is expressed in the motor neurons in human spinal cord. Unexpectedly, a subpopulation of CA1 appears to be associated with endoplasmic reticulum (ER) membranes. In addition, the membrane-associated CA1s are preferentially upregulated in amyotrophic lateral sclerosis (ALS) and exhibit altered distribution in motor neurons. Furthermore, long-term expression of CA1 in mammalian cells activates apoptosis. Our results suggest a previously unknown role for CA1 function in the CNS and its potential involvement in motor neuron degeneration in ALS.

BACKGROUND This study aimed to explore whether 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) restrains pathological hyperplasia of fibroblasts from hyperplastic scar tissues, and to investigate the potential mechanism. MATERIAL AND METHODS We used MTT assay, flow cytometry, and terminal-deoxynucleotidyl transferase mediated nick-end labeling (TUNEL) to examine the effects of ALA-PDT on proliferation, cell cycle, and apoptosis of fibroblasts isolated from hyperplastic scar tissues. The growth-promoting effect of fibroblasts on vascular endothelial cells was measured by cell co-culture. Real-time PCR and Western blot analysis were performed to detect the expression levels of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (a-SMA), Collagen I, Collagen III, vascular endothelial growth factor-A (VEGFA), and basic fibroblast growth factor (bFGF). RESULTS ALA-PDT inhibited proliferation delayed cell cycle progress, promoted apoptosis of fibroblasts, and suppressed its growth-promoting effect on vascular endothelial cells, and decreased expression of TGF-β1, α-SMA, Collagen I, Collagen III, VEGFA, and bFGF. CONCLUSIONS ALA-PDT effectively restrained pathological hyperplasia of fibroblasts from hyperplastic scar tissues, which may provide a research basis for clinical therapy of hyperplastic scars.

Phytoplasmas are a group of insect-vectored bacteria responsible for disease in many plant species worldwide. Among the crop species affected is the economically valuable forage species lucerne. Here we provide comprehensive molecular evidence for infection in multiple lucerne plants by a phytoplasma not previously known from this plant species. This phytoplasma had a >99% genetic similarity to an unclassified 16S rRNA subgroup previously reported as Stylosanthes little leaf from Stylosanthes spp. and was genetically and symptomatically distinct from a co-occurring but less common 16SrIIA group phytoplasma. Neighbour-joining analyses with publicly available sequence data confirmed the presence of two distinct phytoplasma lineages in the plant population. No PCR detections were made among 38 individuals of 12 co-occurring weed species. Sequence analysis revealed that all nine PCR detections from among 106 individuals of five Hemiptera insect species from the site, three of which had previously been reported as likely vectors, were false positives. This study demonstrates the importance of sequencing to complement PCR detection and avoid potentially inaccurate conclusions regarding vectors, highlights that sampling over a wide spatio-temporal scale is important for vector and alternative host studies, and extends to eight the number of phytoplasma 16 Sr groups known from lucerne.

Statins exert pleiotropic effects on endothelial cells in addition to lowering cholesterol. 15-Lipoxygenase-1 (ALOX15) has been implicated in vascular inflammation and disease. The relationship between atorvastatin and ALOX15 was investigated using a rat carotid artery balloon-injury model. Hematoxylin and eosin (HE) staining showed that ALOX15 overexpression increased the thickness of the intima-media (IMT). Immunohistochemistry and western blotting showed that atorvastatin increased the expression of cellular adhesion molecules (CAMs) but decreased the expression of endothelial nitric oxide synthase (eNOS); these effects of atorvastatin were blocked by ALOX15 overexpression. In human umbilical venous endothelial cells (HUVECs), silencing of ALOX15 enhanced the effects of atorvastatin on endothelial function. Expression levels of CAMs and Akt/eNOS/NO under oxidized low-density lipoprotein (ox-LDL) stimulation were modulated by ALOX15 inhibitor and ALOX15 small interfering RNA (siRNA). Atorvastatin abolished the activation of nuclear factor-kappa B (NF-κB) induced by ox-LDL. Exposure to ox-LDL induced upregulation of ALOX15 in HUVECs, but this effect was partially abolished by atorvastatin or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). These results demonstrate that regulation of ALOX15 expression might be involved in the effects of atorvastatin on endothelial dysfunction.

Previous studies demonstrated that dysfunction of the hypothalamus-pituitary-adrenal (HPA) axis played an important role in morphine dependence. Nonetheless, the molecular mechanism underlying morphine-induced HPA axis dysfunction and morphine dependence remains unclear. In the current study, 5'-aza-2'-deoxycytidine (5-aza), an inhibitor of DNA methyltransferases (DNMTs), was used to examine the effects of glucocorticoid receptor (GR) promoter 17 methylation on chronic morphine-induced HPA axis dysfunction and behavioral changes in rats and the underlying mechanism. Our results showed that chronic but not acute morphine downregulated the expression of nuclear GR protein and GR exon 17 variant mRNA, and upregulated the methylation of GR 17 exon promoter in the hippocampus of rats. Meanwhile, 5-aza per se had no effect on observed molecular and behavior change. In contrast, pretreatment of 5-aza into rat hippocampus reversed chronic morphine-induced hypermethylation of GR 17 promoter and decrease in GR expression. Moreover, pretreatment of 5-aza attenuated chronic morphine-enhanced HPA axis reactivity and the naloxone-precipitated somatic signs in morphine-dependent rats. Our results suggest that chronic morphine induced hypermethylation of GR 17 promoter, which then downregulated the expression of hippocampal GR, and was thus involved in chronic morphine-induced dysfunction of the HPA axis and the modulation of morphine dependence. Moreover, chronic morphine-induced hypermethylation of GR 17 promoter may be at least partially due to the increase in hippocampal DNMT 1 expression and its binding at GR 17 promoter in the rat hippocampus. © 2016 Wiley Periodicals, Inc.

Mass spectrometry-based targeted proteomic assays are experiencing a surge in awareness due to the diverse possibilities arising from the re-application of traditional LC-SRM technology. The FDA-approved quantitative LC-SRM-pipeline in drug discovery motivates the use to quantitatively validate putative proteomic biomarkers. However, complexity of biological specimens bears a huge challenge to identify, in parallel, specific peptides and proteins of interest from large biomarker candidate lists. Methods have been devised to increase scan speeds, improve detection specificity and verify quantitative SRM-features. In contrast, high-resolution mass spectrometers could be used to improve reliability and precision of targeted proteomics assays. Here, we present a new method for identifying, quantifying and reporting peptides in high-resolution targeted proteomics experiments performed on an orbitrap hybrid instrument using stable isotope-labeled internal reference peptides. This high precision targeted peptide monitoring (TPM) method has unique advantages over existing techniques, including the need to only detect the most abundant product ion of a given target for confident peptide identification using a scoring function that evaluates assay performance based on 1) m/z-mass accuracy, 2) retention time accuracy of observed species relative to prediction, and 3) retention time accuracy relative to internal reference peptides. Further, we show management of multiplexed precision TPM-assays using sentinel peptide standards. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Spinal glial gap junctions may play an important role in dorsal horn neuronal sensitization and neuropathic pain. In rats after an L5 spinal nerve ligation (SNL), we examined the effects of intrathecal injection of carbenoxolone (CBX), a gap junction decoupler, on neuropathic pain manifestations and on wide-dynamic range (WDR) neuronal activity in vivo. Intrathecal injection of CBX dose-dependently (0.1-50 μg, 10 μl) inhibited mechanical hypersensitivity in rats at 2-3 weeks post-SNL. However, the same doses of glycyrrhizic acid (an analogue of CBX that does not affect gap junctions) and mefloquine hydrochloride (a selective neuronal gap junction decoupler) were ineffective. Intrathecal CBX (5μg) also attenuated heat hypersensitivity in SNL rats. Further, rats did not develop tachyphylaxis to CBX-induced inhibition of mechanical hypersensitivity after repetitive drug treatments (25 μg/day) during days 14-16 post-SNL. Electrophysiological study in SNL rats showed that spinal topical application of CBX (100 μg, 50 μl), which mimics intrathecal drug administration, attenuated WDR neuronal responses to mechanical stimuli and to repetitive intracutaneous electrical stimuli (0.5 Hz) that induce windup, a short-form of activity-dependent neuronal sensitization. The current findings suggest that the inhibition of neuropathic pain manifestations by intrathecal injection of CBX in SNL rats may involve an inhibition of glial gap junctions and an attenuation of WDR neuronal activity in the dorsal horn.

miR-126, the miRNA considered to be specially expressed in endothelial cells and hematopoietic progenitor cells, is strongly associated with angiogenesis. The purpose is to evaluate the role of miR-126 in hypoxia-induced angiogenesis and the possible mechanisms.

To examine the association between sleep disorders, obesity status, and the risk of diabetes in adults, a total of 3668 individuals aged 40+ years from the NHANES 2009-2010 without missing information on sleep-related questions, measurements related to diabetes, and BMI were included in this analysis. Subjects were categorized into three sleep groups based on two sleep questions: (a) no sleep problems; (b) sleep disturbance; and (c) sleep disorder. Diabetes was defined as having one of a diagnosis from a physician; an overnight fasting glucose > 125 mg/dL; Glycohemoglobin > 6.4%; or an oral glucose tolerance test > 199 mg/dL. Overall, 19% of subjects were diabetics, 37% were obese, and 32% had either sleep disturbance or sleep disorder. Using multiple logistic regression models adjusting for covariates without including BMI, the odds ratios (OR, (95% CI)) of diabetes were 1.40 (1.06, 1.84) and 2.04 (1.40, 2.95) for those with sleep disturbance and with sleep disorder, respectively. When further adjusting for BMI, the ORs were similar for those with sleep disturbance 1.36 (1.06, 1.73) but greatly attenuated for those with sleep disorders (1.38 [0.95, 2.00]). In conclusion, the impact of sleep disorders on diabetes may be explained through the individuals' obesity status.