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1.  Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice 
PLoS ONE  2014;9(3):e90363.
Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5−/− embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5−/− brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.
doi:10.1371/journal.pone.0090363
PMCID: PMC3962345  PMID: 24658276
2.  Activation of cyclin-dependent kinase 5 mediates orofacial mechanical hyperalgesia 
Molecular Pain  2013;9:66.
Background
Cyclin-dependent kinase 5 (Cdk5) is a unique member of the serine/threonine kinase family. This kinase plays an important role in neuronal development, and deregulation of its activity leads to neurodegenerative disorders. Cdk5 also serves an important function in the regulation of nociceptive signaling. Our previous studies revealed that the expression of Cdk5 and its activator, p35, is upregulated in nociceptive neurons during peripheral inflammation. The aim of the present study was to characterize the involvement of Cdk5 in orofacial pain. Since mechanical hyperalgesia is the distinctive sign of many orofacial pain conditions, we adapted an existing orofacial stimulation test to assess the behavioral responses to mechanical stimulation in the trigeminal region of the transgenic mice with either reduced or increased Cdk5 activity.
Results
Mice overexpressing or lacking p35, an activator of Cdk5, showed altered phenotype in response to noxious mechanical stimulation in the trigeminal area. Mice with increased Cdk5 activity displayed aversive behavior to mechanical stimulation as indicated by a significant decrease in reward licking events and licking time. The number of reward licking/facial contact events was significantly decreased in these mice as the mechanical intensity increased. By contrast, mice deficient in Cdk5 activity displayed mechanical hypoalgesia.
Conclusions
Collectively, our findings demonstrate for the first time the important role of Cdk5 in orofacial mechanical nociception. Modulation of Cdk5 activity in primary sensory neurons makes it an attractive potential target for the development of novel analgesics that could be used to treat multiple orofacial pain conditions.
doi:10.1186/1744-8069-9-66
PMCID: PMC3882292  PMID: 24359609
Cdk5; p35; Trigeminal ganglia; Orofacial pain; Mouse model
3.  Differential expression of the FMRF gene in adult and hatchling stellate ganglia of the squid Loligo pealei 
Biology Open  2013;3(1):50-58.
Summary
The giant fiber system of the squid Loligo pealei mediates the escape response and is an important neurobiological model. Here, we identified an abundant transcript in the stellate ganglion (SG) that encodes a FMRFamide precursor, and characterized FMRFamide and FI/LRF-amide peptides. To determine whether FMRFamide plays a role in the adult and hatchling giant fiber system, we studied the expression of the Fmrf gene and FMRFamide peptides. In stage 29 embryos and stage 30 hatchlings, Ffmr transcripts and FMRFamide peptide were low to undetectable in the SG, in contrast to groups of neurons intensely expressing the Fmrf gene in several brain lobes, including those that innervate the SG. In the adult SG the Fmrf gene was highly expressed, but the FMRFamide peptide was in low abundance. Intense staining for FMRFamide in the adult SG was confined to microneurons and fibers in the neuropil and to small fibers surrounding giant axons in stellar nerves. This shows that the Fmrf gene in the SG is strongly regulated post-hatching, and suggests that the FMRFamide precursor is incompletely processed in the adult SG. The data suggest that the SG only employs the Fmrf gene post-hatching and restricts the biosynthesis of FMRFamide, demonstrating that this peptide is not a major transmitter of the giant fiber system. This contrasts with brain lobes that engage FMRFamide embryonically as a regulatory peptide in multiple neuronal systems, including the afferent fibers that innervate the SG. The biological significance of these mechanisms may be to generate diversity within Fmrf-expressing systems in cephalopods.
doi:10.1242/bio.20136890
PMCID: PMC3892160  PMID: 24326188
FMRFamide; Cephalopods; Mollusk; Stellate ganglion; Giant axon; Prohormone processing
4.  Deregulated Cdk5 Activity Is Involved in Inducing Alzheimer’s Disease 
Archives of medical research  2012;43(8):655-662.
Alzheimer’s disease (AD), the most devastating chronic neurodegenerative disease in adults, causes dementia and eventually, death of the affected individuals. Clinically, AD is characterized as late-onset, age-dependent cognitive decline due to loss of neurons in cortex and hippocampus. The pathologic corollary of these symptoms is the formation of senile plaques and neurofibrillary tangles. Senile plaques are formed due to accumulation of oligomeric amyloid beta (Aβ) forming fibrillary plaques. This occurs due to the amyloidogenic processing of the amyloid precursor protein (APP) by various secretases. On the other hand, neurofibrillary tangles are formed due to hyperphosphorylation of cytoskeleton proteins like tau and neurofilament. Both are hyperphosphorylated by cyclin-dependent kinase-5 (Cdk5) and are part of the paired helical filament (PHF), an integral part of neurofibrillary tangles. Unlike other cyclin-dependent kinases, Cdk5 plays a very important role in the neuronal development. Cdk5 gets activated by its neuronal activators p35 and p39. Upon stress, p35 and p39 are cleaved by calpain resulting in truncated products as p25 and p29. Association of Cdk5/p25 is longer and uncontrolled causing aberrant hyperphosphorylation of various substrates of Cdk5 like APP, tau and neurofilament, leading to neurodegenerative pathology like AD. Additionally recent evidence has shown increased levels of p25, Aβ, hyperactivity of Cdk5, phosphorylated tau and neurofilament in human AD brains. This review briefly describes the above-mentioned aspects of involvement of Cdk5 in the pathology of AD and at the end summarizes the advances in Cdk5 as a therapeutic target.
doi:10.1016/j.arcmed.2012.10.015
PMCID: PMC3532552  PMID: 23142263
Cyclin dependent kinase 5 (Cdk5); Hyperphosphorylation; Neurodegeneration; Alzheimer’s disease; Plaques; Tangles
5.  Cdk5 Inhibitory Peptide (CIP) Inhibits Cdk5/p25 Activity Induced by High Glucose in Pancreatic Beta Cells and Recovers Insulin Secretion from p25 Damage 
PLoS ONE  2013;8(9):e63332.
Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG) results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4–12 hrs), however was detectable in the long exposure in HG cells (24 hrs and 48 hrs). Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5) with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes.
doi:10.1371/journal.pone.0063332
PMCID: PMC3764143  PMID: 24039692
6.  Cyclin-dependent kinase 5 regulates E2F transcription factor through phosphorylation of Rb protein in neurons 
Cell Cycle  2012;11(8):1603-1610.
Recent studies have shown the involvement of cyclin-dependent kinase 5 (Cdk5) in cell cycle regulation in postmitotic neurons. In this study, we demonstrate that Cdk5 and its co-activator p35 were detected in the nuclear fraction in neurons and Cdk5/p35 phosphorylated retinoblastoma (Rb) protein, a key protein controlling cell cycle re-entry. Cdk5/p35 phosphorylates Rb at the sites similar to those phosphorylated by Cdk4 and Cdk2. Furthermore, increased Cdk5 activity elevates activity of E2F transcription factor, which can trigger cell cycle re-entry, leading to neuronal cell death. A normal Cdk5 activity in neurons did not induce E2F activation, suggesting that Cdk5 does not induce cell cycle re-entry under normal conditions. Taken together, these results indicate that Cdk5 can regulate cell cycle by its ability to phosphorylate Rb. Most importantly, increased Cdk5 activity induces cell cycle re-entry, which is especially detrimental for survival of postmitotic neurons.
doi:10.4161/cc.20009
PMCID: PMC3341229  PMID: 22456337
Cdk5; cell cycle; cell death; E2F transcription factor; neurons; phosphorylation; retinoblastoma protein
7.  Declining phosphatases underlie aging-related hyperphosphorylation of neurofilaments 
Neurobiology of aging  2009;32(11):2016-2029.
Cytoskeletal protein phosphorylation is frequently altered in neuropathologic states but little is known about changes during normal aging. Here we report that declining protein phosphatase activity, rather than activation of kinases, underlies aging-related neurofilament hyperphosphorylation. Purified PP2A or PP2B dephosphorylated the heavy neurofilament (NFH) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting either phosphatase induced NFH phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 phosphoepitope associated with late axon maturation, more than doubled during the 12 month period after NFH expression plateaued at p21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.
doi:10.1016/j.neurobiolaging.2009.12.001
PMCID: PMC2891331  PMID: 20031277
neurofilament; phosphorylation; dephosphorylation; kinases; phosphatases; maturation; aging; RT-97 epitope; immunoreactivity
8.  Achaete-scute homologue-1 (ASH1) stimulates migration of lung cancer cells through Cdk5/p35 pathway 
Molecular Biology of the Cell  2012;23(15):2856-2866.
Cyclin-dependent kinase 5 (Cdk5) activity is important for the migration and invasion of cancer cells. Our results indicate that in the lung one of the mechanisms that hASH1 regulates—migration—takes place through induction of Cdk5 activity. Our data suggest that Cdk5 and its activator p35 promote lung cancer cell migration through hASH1-mediated signaling.
Our previous data suggested that the human basic helix–loop–helix transcription factor achaete-scute homologue-1 (hASH1) may stimulate both proliferation and migration in the lung. In the CNS, cyclin-dependent kinase 5 (Cdk5) and its activator p35 are important for neuronal migration that is regulated by basic helix–loop–helix transcription factors. Cdk5/p35 may also play a role in carcinogenesis. In this study, we found that the neuronal activator p35 was commonly expressed in primary human lung cancers. Cdk5 and p35 were also expressed by several human lung cancer cell lines and coupled with migration and invasion. When the kinase activity was inhibited by the Cdk5 inhibitor roscovitine or dominant-negative (dn) Cdk5, the migration of lung cancer cells was reduced. In neuroendocrine cells expressing hASH1, such as a pulmonary carcinoid cell line, knocking down the gene expression by short hairpin RNA reduced the levels of Cdk5/p35, nuclear p35 protein, and migration. Furthermore, expression of hASH1 in lung adenocarcinoma cells normally lacking hASH1 increased p35/Cdk5 activity and enhanced cellular migration. We were also able to show that p35 was a direct target for hASH1. In conclusion, induction of Cdk5 activity is a novel mechanism through which hASH1 may regulate migration in lung carcinogenesis.
doi:10.1091/mbc.E10-12-1010
PMCID: PMC3408413  PMID: 22696682
9.  Cysteinyl-tRNA Deacylation Can Be Uncoupled from Protein Synthesis 
PLoS ONE  2012;7(3):e33072.
Aminoacyl-tRNA synthetases (ARSs) are critical components of protein translation, providing ribosomes with aminoacyl-tRNAs. In return, ribosomes release uncharged tRNAs as ARS substrates. Here, we show that tRNA deacylation can be uncoupled from protein synthesis in an amino acid specific manner. While tRNAs coupled to radiolabeled Met, Leu Lys, or Ser are stable in cells following translation inhibition with arsenite, radiolabeled Cys is released from tRNA at a high rate. We discuss possible translation independent functions for tRNACys.
doi:10.1371/journal.pone.0033072
PMCID: PMC3302863  PMID: 22427952
10.  Mechanism of Acetylcholine Receptor Cluster Formation Induced by DC Electric Field 
PLoS ONE  2011;6(10):e26805.
Background
The formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.
Methodology/Principal Findings
To understand its molecular mechanism, quantum dots (QDs) were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering.
Conclusions
These results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.
doi:10.1371/journal.pone.0026805
PMCID: PMC3201969  PMID: 22046365
11.  Neuropathologic Correlates of Hippocampal Atrophy in the Elderly: A Clinical, Pathologic, Postmortem MRI Study 
PLoS ONE  2011;6(10):e26286.
The volume of the hippocampus measured with structural magnetic resonance imaging (MRI) is increasingly used as a biomarker for Alzheimer's disease (AD). However, the neuropathologic basis of structural MRI changes in the hippocampus in the elderly has not been directly assessed. Postmortem MRI of the aging human brain, combined with histopathology, could be an important tool to address this issue. Therefore, this study combined postmortem MRI and histopathology in 100 elderly subjects from the Rush Memory and Aging Project and the Religious Orders Study. First, to validate the information contained in postmortem MRI data, we tested the hypothesis that postmortem hippocampal volume is smaller in subjects with clinically diagnosed Alzheimer's disease compared to subjects with mild or no cognitive impairment, as observed in antemortem imaging studies. Subsequently, the relations of postmortem hippocampal volume to AD pathology, Lewy bodies, amyloid angiopathy, gross infarcts, microscopic infarcts, and hippocampal sclerosis were examined. It was demonstrated that hippocampal volume was smaller in persons with a clinical diagnosis of AD compared to those with no cognitive impairment (P = 2.6×10−7) or mild cognitive impairment (P = 9.6×10−7). Additionally, hippocampal volume was related to multiple cognitive abilities assessed proximate to death, with its strongest association with episodic memory. Among all pathologies investigated, the most significant factors related to lower hippocampal volume were shown to be AD pathology (P = 0.0018) and hippocampal sclerosis (P = 4.2×10−7). Shape analysis allowed for visualization of the hippocampal regions most associated with volume loss for each of these two pathologies. Overall, this investigation confirmed the relation of hippocampal volume measured postmortem to clinical diagnosis of AD and measures of cognition, and concluded that both AD pathology and hippocampal sclerosis affect hippocampal volume in old age, though the impacts of each pathology on the shape of the hippocampus may differ.
doi:10.1371/journal.pone.0026286
PMCID: PMC3197137  PMID: 22043314
12.  Amyotrophic Lateral Sclerosis Multiprotein Biomarkers in Peripheral Blood Mononuclear Cells 
PLoS ONE  2011;6(10):e25545.
Background
Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.
Methodology/Principal Findings
We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.
Conclusions/Significance
Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.
doi:10.1371/journal.pone.0025545
PMCID: PMC3187793  PMID: 21998667
13.  Oxidative Stress in Neurodegeneration 
It has been demonstrated that oxidative stress has a ubiquitous role in neurodegenerative diseases. Major source of oxidative stress due to reactive oxygen species (ROS) is related to mitochondria as an endogenous source. Although there is ample evidence from tissues of patients with neurodegenerative disorders of morphological, biochemical, and molecular abnormalities in mitochondria, it is still not very clear whether the oxidative stress itself contributes to the onset of neurodegeneration or it is part of the neurodegenerative process as secondary manifestation. This paper begins with an overview of how oxidative stress occurs, discussing various oxidants and antioxidants, and role of oxidative stress in diseases in general. It highlights the role of oxidative stress in neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis. The last part of the paper describes the role of oxidative stress causing deregulation of cyclin-dependent kinase 5 (Cdk5) hyperactivity associated with neurodegeneration.
doi:10.1155/2011/572634
PMCID: PMC3177364  PMID: 21941533
14.  Identification of Novel Biomarkers in Seasonal Allergic Rhinitis by Combining Proteomic, Multivariate and Pathway Analysis 
PLoS ONE  2011;6(8):e23563.
Background
Glucocorticoids (GCs) play a key role in the treatment of seasonal allergic rhinitis (SAR). However, some patients show a low response to GC treatment. We hypothesized that proteins that correlated to discrimination between symptomatic high and low responders (HR and LR) to GC treatment might be regulated by GCs and therefore suitable as biomarkers for GC treatment.
Methodology/Principal Findings
We identified 953 nasal fluid proteins in symptomatic HR and LR with a LC MS/MS based-quantitative proteomics analysis and performed multivariate analysis to identify a combination of proteins that best separated symptomatic HR and LR. Pathway analysis showed that those proteins were most enriched in the acute phase response pathway. We prioritized candidate biomarkers for GC treatment based on the multivariate and pathway analysis. Next, we tested if those candidate biomarkers differed before and after GC treatment in nasal fluids from 40 patients with SAR using ELISA. Several proteins including ORM (P<0.0001), APOH (P<0.0001), FGA (P<0.01), CTSD (P<0.05) and SERPINB3 (P<0.05) differed significantly before and after GC treatment. Particularly, ORM (P<0.01), FGA (P<0.05) and APOH (P<0.01) that belonged to the acute phase response pathway decreased significantly in HR but not LR before and after GC treatment.
Conclusions/Significance
We identified several novel biomarkers for GC treatment response in SAR with combined proteomics, multivariate and pathway analysis. The analytical principles may be generally applicable to identify biomarkers in clinical studies of complex diseases.
doi:10.1371/journal.pone.0023563
PMCID: PMC3160968  PMID: 21887273
15.  P38 Mitogen-Activated Protein Kinase Inhibitor, FR167653, Inhibits Parathyroid Hormone Related Protein-Induced Osteoclastogenesis and Bone Resorption 
PLoS ONE  2011;6(8):e23199.
p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages(BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption.
doi:10.1371/journal.pone.0023199
PMCID: PMC3160289  PMID: 21886782
16.  Neurodegenerative Properties of Chronic Pain: Cognitive Decline in Patients with Chronic Pancreatitis 
PLoS ONE  2011;6(8):e23363.
Chronic pain has been associated with impaired cognitive function. We examined cognitive performance in patients with severe chronic pancreatitis pain. We explored the following factors for their contribution to observed cognitive deficits: pain duration, comorbidity (depression, sleep disturbance), use of opioids, and premorbid alcohol abuse. The cognitive profiles of 16 patients with severe pain due to chronic pancreatitis were determined using an extensive neuropsychological test battery. Data from three cognitive domains (psychomotor performance, memory, executive functions) were compared to data from healthy controls matched for age, gender and education. Multivariate multilevel analysis of the data showed decreased test scores in patients with chronic pancreatitis pain in different cognitive domains. Psychomotor performance and executive functions showed the most prominent decline. Interestingly, pain duration appeared to be the strongest predictor for observed cognitive decline. Depressive symptoms, sleep disturbance, opioid use and history of alcohol abuse provided additional explanations for the observed cognitive decline in some of the tests, but to a lesser extent than pain duration. The negative effect of pain duration on cognitive performance is compatible with the theory of neurodegenerative properties of chronic pain. Therefore, early and effective therapeutic interventions might reduce or prevent decline in cognitive performance, thereby improving outcomes and quality of life in these patients.
doi:10.1371/journal.pone.0023363
PMCID: PMC3158076  PMID: 21876748
17.  Oxytocin and Vasopressin Involved in Restraint Water-Immersion Stress Mediated by Oxytocin Receptor and Vasopressin 1b Receptor in Rat Brain 
PLoS ONE  2011;6(8):e23362.
Aims
Vasopressin (AVP) and oxytocin (OT) are considered to be related to gastric functions and the regulation of stress response. The present study was to study the role of vasopressinergic and oxytocinergic neurons during the restraint water-immersion stress.
Methods
Ten male Wistar rats were divided into two groups, control and RWIS for 1h. The brain sections were treated with a dual immunohistochemistry of Fos and oxytocin (OT) or vasopressin (AVP) or OT receptor or AVP 1b receptor (V1bR).
Results
(1) Fos-immunoreactive (Fos-IR) neurons dramatically increased in the hypothalamic paraventricular nucleus (PVN), the supraoptic nucleus (SON), the neucleus of solitary tract (NTS) and motor nucleus of the vagus (DMV) in the RWIS rats; (2) OT-immunoreactive (OT-IR) neurons were mainly observed in the medial magnocellular part of the PVN and the dorsal portion of the SON, while AVP-immunoreactive (AVP-IR) neurons mainly distributed in the magnocellular part of the PVN and the ventral portion of the SON. In the RWIS rats, Fos-IR neurons were indentified in 31% of OT-IR neurons and 40% of AVP-IR neurons in the PVN, while in the SON it represented 28%, 53% respectively; (3) V1bR-IR and OTR-IR neurons occupied all portions of the NTS and DMV. In the RWIS rats, more than 10% of OTR-IR and V1bR-IR neurons were activated in the DMV, while lower ratio in the NTS.
Conclusion
RWIS activates both oxytocinergic and vasopressinergic neurons in the PVN and SON, which may project to the NTS or DMV mediating the activity of the neurons by OTR and V1bR.
doi:10.1371/journal.pone.0023362
PMCID: PMC3157380  PMID: 21858088
18.  Effects of Clinically Relevant MPL Mutations in the Transmembrane Domain Revealed at the Atomic Level through Computational Modeling 
PLoS ONE  2011;6(8):e23396.
Background
Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations.
Results
Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2.
Conclusions
Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems.
doi:10.1371/journal.pone.0023396
PMCID: PMC3157383  PMID: 21858098
19.  Influence of Stochastic Gene Expression on the Cell Survival Rheostat after Traumatic Brain Injury 
PLoS ONE  2011;6(8):e23111.
Experimental evidence suggests that random, spontaneous (stochastic) fluctuations in gene expression have important biological consequences, including determination of cell fate and phenotypic variation within isogenic populations. We propose that fluctuations in gene expression represent a valuable tool to explore therapeutic strategies for patients who have suffered traumatic brain injury (TBI), for which there is no effective drug therapy. We have studied the effects of TBI on the hippocampus because TBI survivors commonly suffer cognitive problems that are associated with hippocampal damage. In our previous studies we separated dying and surviving hippocampal neurons by laser capture microdissection and observed unexplainable variations in post-TBI gene expression, even though dying and surviving neurons were adjacent and morphologically identical. We hypothesized that, in hippocampal neurons that subsequently are subjected to TBI, randomly increased pre-TBI expression of genes that are associated with neuroprotection predisposes neurons to survival; conversely, randomly decreased expression of these genes predisposes neurons to death. Thus, to identify genes that are associated with endogenous neuroprotection, we performed a comparative, high-resolution transcriptome analysis of dying and surviving hippocampal neurons in rats subjected to TBI. We found that surviving hippocampal neurons express a distinct molecular signature — increased expression of networks of genes that are associated with regeneration, cellular reprogramming, development, and synaptic plasticity. In dying neurons we found decreased expression of genes in those networks. Based on these data, we propose a hypothetical model in which hippocampal neuronal survival is determined by a rheostat that adds injury-induced genomic signals to expression of pro-survival genes, which pre-TBI varies randomly and spontaneously from neuron to neuron. We suggest that pharmacotherapeutic strategies that co-activate multiple survival signals and enhance self-repair mechanisms have the potential to shift the cell survival rheostat to favor survival and therefore improve functional outcome after TBI.
doi:10.1371/journal.pone.0023111
PMCID: PMC3154935  PMID: 21853077
20.  Evaluating Experimental Bias and Completeness in Comparative Phosphoproteomics Analysis 
PLoS ONE  2011;6(8):e23276.
Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically generated for this purpose, but also to extract insights through comparative analysis of the ever-increasing wealth of publically available phosphorylation data.
doi:10.1371/journal.pone.0023276
PMCID: PMC3154393  PMID: 21853102
21.  Cloning of a Novel Protein Interacting with BRS-3 and Its Effects in Wound Repair of Bronchial Epithelial Cells 
PLoS ONE  2011;6(8):e23072.
Bombesin receptor subtype 3 (BRS-3), the orphan bombesin receptor, may play a role in the regulation of stress responses in lung and airway epithelia. Bombesin receptor activated protein (BRAP )is a novel protein we found in our previous study which interacts with BRS-3. This study was designed to observe the subcellular location and wound repair function of BRAP in human bronchial epithelial cells (HBECs). BRAP ORF was amplified by RT-PCR and ligated to pEGFP-C1 vector, and then the recombinant plasmid pEGFP-C1-BRAP was transfected into Hela cells. The location of BRAP protein was observed by laser confocal microscope, and the expression of it was analyzed by Western-blot. At the same time,we built the recombinant plasmid pcDNA3.1(+)-BRAP, transfected it into HBECs and observed its impact on cell cycle and wound repair of HBECs. The results showed that BRAP locates in membrane and cytoplasm and increases significantly in transfected cells. Flow cytometry results demonstrated that the recombinant plasmid increases S phase plus G2 phase of cell cycle by 25%. Microscopic video analysis system showed that the repair index of wounded HBECs increases by 20% through stable expression of BRAP. The present study demonstrated that BRAP locates in the membrane and cytoplasm, suggesting that this protein is a cytoplasm protein, which promotes cell cycle and wound repair of HBECs.
doi:10.1371/journal.pone.0023072
PMCID: PMC3153462  PMID: 21857995
22.  Diagnosis of Alzheimer's Disease Based on Disease-Specific Autoantibody Profiles in Human Sera 
PLoS ONE  2011;6(8):e23112.
After decades of Alzheimer's disease (AD) research, the development of a definitive diagnostic test for this disease has remained elusive. The discovery of blood-borne biomarkers yielding an accurate and relatively non-invasive test has been a primary goal. Using human protein microarrays to characterize the differential expression of serum autoantibodies in AD and non-demented control (NDC) groups, we identified potential diagnostic biomarkers for AD. The differential significance of each biomarker was evaluated, resulting in the selection of only 10 autoantibody biomarkers that can effectively differentiate AD sera from NDC sera with a sensitivity of 96.0% and specificity of 92.5%. AD sera were also distinguishable from sera obtained from patients with Parkinson's disease and breast cancer with accuracies of 86% and 92%, respectively. Results demonstrate that serum autoantibodies can be used effectively as highly-specific and accurate biomarkers to diagnose AD throughout the course of the disease.
doi:10.1371/journal.pone.0023112
PMCID: PMC3149629  PMID: 21826230
23.  Influence of FADS Polymorphisms on Tracking of Serum Glycerophospholipid Fatty Acid Concentrations and Percentage Composition in Children 
PLoS ONE  2011;6(7):e21933.
Background
Tracking of fatty acid (FA) contribution to plasma or serum lipids over time was shown in children and adults. However, the potential role of FADS gene variants has not been investigated.
Methods and Principal Findings
Serum GP FA composition of 331 children aged 2 and 6 years, participating in an ongoing birth cohort study, was analyzed. Correlation coefficients were estimated to describe FA tracking over 4 years and to assess the influence of FADS variants on tracking. We found low to moderate tracking (r = 0.12–0.49) of FA compositions and concentration between 2 and 6 years. Concentration changes of total monounsaturated FA and total saturated FA over time correlated closely (r = 0.79) but percentage values were unrelated (r = −0.02). Tracking for n-6 long chain polyunsaturated fatty acid (LC-PUFA) concentrations was lower in subjects homozygous for the major allele of FADS variants and higher in carriers of at least one minor allele, whereas for total n-3 LC-PUFA concentrations and compositions this was vice versa. For individual n-3 PUFA inconsistent results were found.
Conclusions and Significance
Serum GP FA composition shows low to moderate tracking over 4 years with a higher tracking for LC-PUFA metabolites than for their precursor FA. Serum PUFA levels and their tracking seem to be more influenced by lipid and lipoprotein metabolism than by FA specific pathways.
doi:10.1371/journal.pone.0021933
PMCID: PMC3144869  PMID: 21818279
24.  A Functional Proteomic Method for Biomarker Discovery 
PLoS ONE  2011;6(7):e22471.
The sequencing of the human genome holds out the hope for personalized medicine, but it is clear that analysis of DNA or RNA content alone is not sufficient to understand most disease processes. Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies. However, the enormity of the proteome and limitations in proteomic methods make it difficult to determine the targets that are particularly relevant to human disease. Methods are therefore needed that allow rational identification of targets based on function and relevance to disease. Screening methodologies such as phage display, SELEX, and small-molecule combinatorial chemistry have been widely used to discover specific ligands for cells or tissues of interest, such as tumors. Those ligands can be used in turn as affinity probes to identify their cognate molecular targets when they are not known in advance. Here we report an easy, robust and generally applicable approach in which phage particles bearing cell- or tissue-specific peptides serve directly as the affinity probes for their molecular targets. For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer.
doi:10.1371/journal.pone.0022471
PMCID: PMC3139652  PMID: 21811618
25.  PP1 Forms an Active Complex with TLRR (lrrc67), a Putative PP1 Regulatory Subunit, during the Early Stages of Spermiogenesis in Mice 
PLoS ONE  2011;6(6):e21767.
Mammalian spermatogenesis is a highly regulated developmental pathway that demands dramatic rearrangement of the cytoskeleton of the male germ cell. We have described previously a leucine rich repeat protein, TLRR (also known as lrrc67), which is associated with the spermatid cytoskeleton in mouse testis and is a binding partner of protein phosphatase-1 (PP1), an extremely well conserved signaling molecule. The activity of PP1 is modulated by numerous specific regulators of which TLRR is a candidate. In this study we measured the phosphatase activity of the TLRR-PP1 complex in the adult and the developing mouse testis, which contains varying populations of developing germ cell types, in order to determine whether TLRR acts as an activator or an inhibitor of PP1 and whether the phosphatase activity of this complex is developmentally regulated during spermatogenesis. Additionally, we assayed the ability of bacterially expressed TLRR to affect the enzymatic activity of PP1. Furthermore, we examined phosphorylation of TLRR, and elements of the spermatid cytoskeleton during the first wave of spermatogenesis in the developing testis. We demonstrate here that the TLRR complex is associated with a phosphatase activity in adult mouse testis. The relative phosphatase activity of this complex appears to reach a peak at about 21 days after birth, when pachytene spermatocytes and round spermatids are abundant in the seminiferous epithelium of the mouse testis. TLRR, in addition to tubulin and kinesin-1B, is phosphorylated during the first wave of spermatogenesis. These findings indicate that the TLRR-PP1 complex is active prior to translocation of TLRR toward the sperm flagella and that TLRR, and constituents of the spermatid cytoskeleton, may be subject to regulation by reversible phosphorylation during spermatogenesis in murine testis.
doi:10.1371/journal.pone.0021767
PMCID: PMC3128092  PMID: 21738792

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