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Systemic lupus erythematosus (SLE) is a chronic, remitting, and relapsing, inflammatory disease involving multiple organs, which exhibits abnormalities of both the innate and adaptive immune responses. A limited number of transcriptomic studies have characterized the gene pathways involved in SLE in an attempt to identify the key pathogenic drivers of the disease. In order to further advance our understanding of the pathogenesis of SLE, we used a novel Bayesian network algorithm to hybridize knowledge- and data-driven methods, and then applied the algorithm to build an SLE gene network using transcriptomic data from 1,760 SLE patients' RNA from the two tabalumab Phase III trials (ILLUMINATE-I & -II), the largest SLE RNA dataset to date. Further, based on the gene network, we carried out hub- and key driver-gene analyses for gene prioritization. Our analyses identified that the JAK-STAT pathway genes, including JAK2, STAT1, and STAT2, played essential roles in SLE pathogenesis, and reaffirmed the recent discovery of pathogenic relevance of JAK-STAT signaling in SLE. Additionally, we showed that other genes, such as IRF1, IRF7, PDIA4, FAM72C, TNFSF10, DHX58, SIGLEC1, and PML, may be also important in SLE and serve as potential therapeutic targets for SLE. In summary, using a hybridized network construction approach, we systematically investigated gene-gene interactions based on their transcriptomic profiles, prioritized genes based on their importance in the network structure, and revealed new insights into SLE activity.

Abstract Background Activation of microglia, the resident immune cells of the central nervous system, is a prominent pathological hallmark of Alzheimer’s disease (AD). However, the gene expression changes underlying microglia activation in response to tau pathology remain elusive. Furthermore, it is not clear how murine gene expression changes relate to human gene expression networks. Methods Microglia cells were isolated from rTg4510 tau transgenic mice and gene expression was profiled using RNA sequencing. Four age groups of mice (2-, 4-, 6-, and 8-months) were analyzed to capture longitudinal gene expression changes that correspond to varying levels of pathology, from minimal tau accumulation to massive neuronal loss. Statistical and system biology approaches were used to analyze the genes and pathways that underlie microglia activation. Differentially expressed genes were compared to human brain co-expression networks. Results Statistical analysis of RNAseq data indicated that more than 4000 genes were differentially expressed in rTg4510 microglia compared to wild type microglia, with the majority of gene expression changes occurring between 2- and 4-months of age. These genes belong to four major clusters based on their temporal expression pattern. Genes involved in innate immunity were continuously up-regulated, whereas genes involved in the glutamatergic synapse were down-regulated. Up-regulated innate inflammatory pathways included NF-κB signaling, cytokine-cytokine receptor interaction, lysosome, oxidative phosphorylation, and phagosome. NF-κB and cytokine signaling were among the earliest pathways activated, likely driven by the RELA, STAT1 and STAT6 transcription factors. The expression of many AD associated genes such as APOE and TREM2 was also altered in rTg4510 microglia cells. Differentially expressed genes in rTg4510 microglia were enriched in human neurodegenerative disease associated pathways, including Alzheimer’s, Parkinson’s, and Huntington’s diseases, and highly overlapped with the microglia and endothelial modules of human brain transcriptional co-expression networks. Conclusion This study revealed temporal transcriptome alterations in microglia cells in response to pathological tau perturbation and provides insight into the molecular changes underlying microglia activation during tau mediated neurodegeneration.

Abstract Introduction It has been proposed that the signal distribution on tau positron emission tomography (PET) images could be used to define pathologic stages similar to those seen in neuropathology. Methods Three topographic staging schemes for tau PET, two sampling the temporal and occipital subregions only and one sampling cortical gray matter across the major brain lobes, were evaluated on flortaucipir F 18 PET images in a test‐retest scenario and from Alzheimer's Disease Neuroimaging Initiative 2. Results All three schemes estimated stages that were significantly associated with amyloid status and when dichotomized to tau positive or negative were 90% to 94% concordant in the populations identified. However, the schemes with fewer regions and simpler decision rules yielded more robust performance in terms of fewer unclassified scans and increased test‐retest reproducibility of assigned stage. Discussion Tau PET staging schemes could be useful tools to concisely index the regional involvement of tau pathology in living subjects. Simpler schemes may be more robust.

Myeloid cell leukemia-1 (MCL-1) is a member of the antiapoptotic BCL-2 proteins family and a key regulator of mitochondrial homeostasis. Overexpression of MCL-1 is found in many cancer cells and contributes to tumor progression, which makes it an attractive therapeutic target. Pursuing our previous study of macrocyclic indoles for the inhibition of MCL-1, we report herein the impact of both pyrazole and indole isomerism on the potency and overall properties of this family of compounds. We demonstrated that the incorporation of a fluorine atom on the naphthalene moiety was a necessary step to improve cellular potency and that, combined with the introduction of various side chains on the pyrazole, it enhanced solubility significantly. This exploration culminated in the discovery of compounds ( R a )- 10 and ( R a )- 15 , possessing remarkable cellular potency and properties., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Avoidance of apoptosis is critical for the development and sustained growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins which is overexpressed in many cancers. Upregulation of Mcl-1 in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Therefore, pharmacological inhibition of Mcl-1 is regarded as an attractive approach to treating relapsed or refractory malignancies. Herein, we disclose the design, synthesis, optimization, and early preclinical evaluation of a potent and selective small-molecule inhibitor of Mcl-1. Our exploratory design tactics focused on structural modifications which improve the potency and physicochemical properties of the inhibitor while minimizing the risk of functional cardiotoxicity. Despite being in the "non-Lipinski" beyond-Rule-of-Five property space, the developed compound benefits from exquisite oral bioavailability in vivo and induces potent pharmacodynamic inhibition of Mcl-1 in a mouse xenograft model.

Introduction: Adrenoleukodystrophy (ALD) is an X-linked peroxisomal disorder with an incidence of 1 in 14-17,000 male births, caused by pathogenic variants within the ABCD1 gene. By adulthood, approximately 40% of the patients develop cerebral ALD, a severe, neuroinflammatory condition that is generally progressive and fatal without intervention., Areas Covered: Historically, only allogeneic hematopoietic stem cell transplantation (HSCT) has been shown to halt progression of cerebral ALD, with superior outcomes obtained when HSCT is performed early in the disease process. More recently, a lentiviral-based gene therapy approach has been investigated as therapy for cerebral ALD as an alternative to allogeneic transplantation. A focused literature review was performed using the terms 'hematopoietic stem cell transplantation,' 'gene therapy' and 'adrenoleukodystrophy' to include relevant literature, especially comparing the experience with gene therapy and HSCT outcomes. We review the history and experience with HSCT in cerebral ALD and its limitations, as well as the information currently available in association with the gene therapy trials for cerebral ALD., Expert Opinion: The data regarding this lentiviral-based gene therapy approach and its relative risks and benefits is still being evaluated. This information is explored in the context of the experience with allogeneic HSCT for cerebral ALD.

Pancreatic cancer (PC) is a deadly disease with a grim prognosis. Pancreatic tumor derived factors (TDF) contribute to the induction of an immunosuppressive tumor microenvironment (TME) that impedes the effectiveness of immunotherapy. PC-induced microRNA-155 (miRNA-155) represses expression of Src homology 2 (SH2) domain-containing Inositol 5'-phosphatase-1 (SHIP-1), a regulator of myeloid cell development and function, thus impacting anti-tumor immunity. We recently reported that the bioflavonoid apigenin (API) increased SHIP-1 expression which correlated with the expansion of tumoricidal macrophages (TAM) and improved anti-tumor immune responses in the TME of mice with PC. We now show that API transcriptionally regulates SHIP-1 expression via the suppression of miRNA-155, impacting anti-tumor immune responses in the bone marrow (BM) and TME of mice with PC. We discovered that API reduced miRNA-155 in the PC milieu, which induced SHIP-1 expression. This promoted the restoration of myelopoiesis and increased anti-tumor immune responses in the TME of heterotopic, orthotopic and transgenic SHIP-1 knockout preclinical mouse models of PC. Our results suggest that manipulating SHIP-1 through miR-155 may assist in augmenting anti-tumor immune responses and aid in the therapeutic intervention of PC.

Renal ischemia-reperfusion (IR) injury is one of the major causes of acute kidney injury and represents a significant risk factor for renal transplantation. The level of renal damage is influenced by the ischemic duration and is caused by excessive amounts of produced reactive oxygen species (ROS). Adaptor protein p66Shc is known to regulate cellular and organ's sensitivity to oxidative stress and to contribute significantly to mitochondrial production of hydrogen peroxide in stress conditions. Studies carried out in cultured renal cells suggest that p66Shc-mediated mitochondrial dysfunction and ROS production are responsible for renal ischemic injury. We used our genetically modified rats, which either lack p66Shc expression, or express p66Shc variant, which constitutively generates increased quantities of hydrogen peroxide, to evaluate potential contribution of p66Shc signaling to renal damage in ischemia reperfusion rat model. Analysis of outer medulla tubule damage revealed the lack of contribution of either p66Shc expression or its constitutive signaling to IR injury in rat model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cerebral blood flow and perfusion are tightly maintained through autoregulation despite changes in transmural pressure. Oxidative stress impairs cerebral blood flow, precipitating cerebrovascular events. Phosphorylation of the adaptor protein p66Shc increases mitochondrial-derived oxidative stress. The effect of p66Shc gain or loss of function in nonhypertensive rats is unclear. We hypothesized that p66Shc gain of function would impair autoregulation of cerebral microcirculation under physiological and pathological conditions. Three previously established transgenic [salt-sensitive (SS) background] p66Shc rats were used, p66-Del/SS (express p66Shc with a nine-amino acid deletion), p66Shc-knockout (KO)/SS (frameshift premature termination codon), and p66Shc signaling and knock-in substitution of Ser36Ala (p66Shc-S36A)/SS (substitution of Ser36Ala). The p66Shc-Del were also bred on Sprague-Dawley (SD) backgrounds (p66-Del/SD), and a subset was exposed to a hypertensive stimulus [ N G -nitro-l-arginine methyl ester (l-NAME)] for 4 wk. Active and passive diameters to increasing transmural pressure were measured and myogenic tone was calculated in all groups (SS and SD). Myogenic responses to increasing pressure were impaired in p66Shc-Del/SS rats relative to wild-type (WT)/SS and knock-in substitution of Ser36Ala (S36A; P < 0.05). p66-Del/SD rats did not demonstrate changes in active/passive diameters or myogenic tone relative to WT/SD but did demonstrate attenuated passive diameter responses to higher transmural pressure relative to p66-Del/SS. Four weeks of a hypertensive stimulus (l-NAME) did not alter active or passive diameter responses to increasing transmural pressure ( P = 0.86-0.99), but increased myogenic responses relative to p66-Del/SD ( P < 0.05). Collectively, we demonstrate the functional impact of p66Shc within the cerebral circulation and demonstrate that the genetic background of p66Shc rats largely drives changes in cerebrovascular function. NEW & NOTEWORTHY We demonstrate that the modulation of p66Shc signaling impairs cerebral artery myogenic tone in a low renin model of hypertension. This impairment is dependent upon the genetic background, as modulated p66Shc signaling in Sprague-Dawley rats does not impair cerebral artery myogenic tone.

Aims: Adaptor protein p66Shc, encoded by Shc1 gene, contributes to the pathogenesis of oxidative stress-related diseases. p66Shc ability to promote oxidative stress-related diseases requires phosphorylation of serine 36 residue (Ser36) and depends on translocation of p66Shc to the mitochondria. We tested the hypothesis that abnormal p66Shc-mediated reactive oxygen species (ROS) production could be critically involved in nephrons development during nephrogenesis., Main Methods: We have generated unique mutant rats (termed p66Shc-Del), which express endogenous p66Shc with a 9-amino acid deletion, and lack regulatory Ser36. H 2 O 2 renal production was measured by enzymatic microelectrode biosensors. Nephron numbers in 3-5 weeks old p66Shc-Del rats were quantified using the acid maceration method., Key Findings: p66Shc-Del rats, as wild type salt sensitive rats, display increased mean arterial blood pressure following chronic exposure to a high salt diet. In contrast to wild type rats, p66Shc-Del rats display increased H 2 O 2 renal production and are characterized by a reduction in renal function. The number of glomeruli is significantly reduced in adult p66Shc-Del rats., Significance: Since low nephron number is an established risk factor for kidney disease and hypertension in humans and rodents, our data suggest that H 2 O 2 renal production, caused by irregular signaling of p66Shc, could be critical in regulating nephrogenesis and that abnormal p66Shc signaling negatively impacts kidney development and renal function by increasing susceptibility to diabetic nephropathy and hypertension-induced nephropathy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The ubiquitously expressed adaptor protein Shc exists in three isoforms p46Shc, p52Shc, and p66Shc, which execute distinctly different actions in cells. The role of p46Shc is insufficiently studied, and the purpose of this study was to further investigate its functional significance. We developed unique rat mutants lacking p52Shc and p46Shc isoforms (p52Shc/46Shc-KO) and carried out histological analysis of skeletal and cardiac muscle of parental and genetically modified rats with impaired gait. p52Shc/46Shc-KO rats demonstrate severe functional abnormalities associated with impaired gait. Our analysis of p52Shc/46Shc-KO rat axons and myelin sheets in cross-sections of the sciatic nerve revealed the presence of significant anomalies. Based on the lack of skeletal muscle fiber atrophy and the presence of sciatic nerve abnormalities, we suggest that the impaired gait in p52Shc/46Shc-KO rats might be due to the sensory feedback from active muscle to the brain locomotor centers. The lack of dystrophin in some heart muscle fibers reflects damage due to dilated cardiomyopathy. Since rats with only p52Shc knockout do not display the phenotype of p52Shc/p46Shc-KO, abnormal locomotion is likely to be caused by p46Shc deletion. Our data suggest a previously unknown role of 46Shc actions and signaling in regulation of gait.

Trace elements analysis is a fundamental challenge in environmental sciences. Scientists measure trace elements in environmental media in order to assess the quality and safety of ecosystems and to quantify the burden of anthropogenic pollution. Among the available analytical techniques, X-ray based methods are particularly powerful, as they can quantify trace elements in situ . Chemical extraction is not required, as is the case for many other analytical techniques. In the last few years, the potential for X-ray techniques to be applied in the environmental sciences has dramatically increased due to developments in laboratory instruments and synchrotron radiation facilities with improved sensitivity and spatial resolution. In this report, we summarize the principles of the X-ray based analytical techniques most frequently employed to study trace elements in environmental samples. We report on the most recent developments in laboratory and synchrotron techniques, as well as advances in instrumentation, with a special attention on X-ray sources, detectors, and optics. Lastly, we inform readers on recent applications of X-ray based analysis to different environmental matrices, such as soil, sediments, waters, wastes, living organisms, geological samples, and atmospheric particulate, and we report examples of sample preparation.

In microvessels of patients with coronary artery disease (CAD), flow-mediated dilation (FMD) is largely dependent upon the endothelium-derived hyperpolarizing factor H 2 O 2 . The goal of this study is to examine the influence of age and presence or absence of disease on the mechanism of FMD. Human coronary or adipose arterioles (~150 µm diameter) were prepared for videomicroscopy. The effect of inhibiting COX [indomethacin (Indo) or NOS (L-NAME), eliminating H 2 O 2 (polyethylene glycol-catalase (PEG-CAT)] or targeting a reduction in mitochondrial ROS with scavengers/inhibitors [Vitamin E ( mt Vitamin E); phenylboronic acid ( mt PBA)] was determined in children aged 0-18 years; young adults 19-55 years; older adults >55 years without CAD, and similarly aged adults with CAD. Indo eliminated FMD in children and reduced FMD in younger adults. This response was mediated mainly by PGI 2 , as the prostacyclin-synthase-inhibitor trans-2-phenyl cyclopropylamine reduced FMD in children and young adults. L-NAME attenuated dilation in children and younger adults and eliminated FMD in older adults without CAD, but had no effect on vessels from those with CAD, where mitochondria-derived H 2 O 2 was the primary mediator. The magnitude of dilation was reduced in older compared to younger adults independent of CAD. Exogenous treatment with a sub-dilator dose of NO blocked FMD in vessels from subjects with CAD, while prolonged inhibition of NOS in young adults resulted in a phenotype similar to that observed in disease. The mediator of coronary arteriolar FMD evolves throughout life from prostacyclin in youth, to NO in adulthood. With the onset of CAD, NO-inhibitable release of H 2 O 2 emerges as the exclusive mediator of FMD. These findings have implications for use of pharmacological agents, such as nonsteroidal anti-inflammatory agents in children and the role of microvascular endothelium in cardiovascular health.

Aims: Endothelin-1 (ET-1) is a potent vasoconstrictor which regulates the physiology of cardiorenal system. The aim of this study was to evaluate ET-1-mediated elevation of intracellular Ca(2+) in smooth muscle cells (SMC) of renal resistance arteries., Main Methods: In in vitro studies of primary SMC, which were isolated from rat renal microvessels, the levels of intracellular Ca(2+) were calculated from the ratio of emissions at 340 and 380nm after loading cells with Fura 2-AM dye. In ex vivo studies we used two-photon imaging of renal resistance arteries excised from rat kidneys and loaded with fluorescent Ca(2+) indicator Fluo-4 AM., Key Findings: The two-photon imaging demonstrates that treatment of isolated rat renal resistance arteries with ET-1 causes a rapid increase of intracellular Ca(2+) concentration in smooth muscle vasculature of these vessels. These ex vivo observations are in accordance with in vitro findings indicating that ET-1 mediates activation of TRPC channels and increases the level of intracellular Ca(2+) in cultured SMC to 510±83nM., Significance: ET-1-mediated elevation of intracellular Ca(2+) is strongly linked to renal microvascular contraction and is crucial for ET-1-induced contraction of SMC. The two-photon imaging of intracellular Ca(2+) in intact SMC of rat renal resistance arteries is a powerful technique which allows the detailed ex vivo analysis of intracellular Ca(2+) handling by ET-1, an important player in hypertension-related kidney diseases., Competing Interests: statement The authors declare that there are no conflicts of interest., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Renal preglomerular arterioles regulate vascular tone to ensure a large pressure gradient over short distances, a function that is extremely important for maintaining renal microcirculation. Regulation of renal microvascular tone is impaired in salt-sensitive (SS) hypertension-induced nephropathy, but the molecular mechanisms contributing to this impairment remain elusive. Here, we assessed the contribution of the SH2 adaptor protein p66Shc (encoded by Shc1) in regulating renal vascular tone and the development of renal vascular dysfunction associated with hypertension-induced nephropathy. We generated a panel of mutant rat strains in which specific modifications of Shc1 were introduced into the Dahl SS rats. In SS rats, overexpression of p66Shc was linked to increased renal damage. Conversely, deletion of p66Shc from these rats restored the myogenic responsiveness of renal preglomerular arterioles ex vivo and promoted cellular contraction in primary vascular smooth muscle cells (SMCs) that were isolated from renal vessels. In primary SMCs, p66Shc restricted the activation of transient receptor potential cation channels to attenuate cytosolic Ca2+ influx, implicating a mechanism by which overexpression of p66Shc impairs renal vascular reactivity. These results establish the adaptor protein p66Shc as a regulator of renal vascular tone and a driver of impaired renal vascular function in hypertension-induced nephropathy.

While increased COX2 expression and prostaglandin levels are elevated in human cancers, the mechanisms of COX2 regulation at the post-translational level are unknown. Initial observation that COX2 forms adduct with non-receptor tyrosine kinase FYN, prompted us to study FYN-mediated post-translational regulation of COX2. We found that FYN increased COX2 activity in prostate cancer cells DU145, independent of changes in COX2 or COX1 protein expression levels. We report that FYN phosphorylates human COX2 on Tyr 446, and while corresponding phospho-mimetic COX2 mutation promotes COX2 activity, the phosphorylation blocking mutation prevents FYN-mediated increase in COX2 activity.

The protective mechanisms employed by arbuscular mycorrhizal fungi (AMF) to reduce the toxic effects of arsenic on host plants remain partially unknown. The goal of this research was identifying the in situ localization and speciation of arsenic (As) in the AM fungus Rhizophagus intraradices [formerly named Glomus intraradices] exposed to arsenate [As(V)]. By using a two-compartment in vitro fungal cultures of R. intraradices-transformed carrot roots, microspectroscopic X-ray fluorescence (μ-XRF), and microspectroscopic X-ray absorption near edge structure (μ-XANES), we observed that As(V) is absorbed after 1 h in the hyphae of AMF. Three hours after exposure a decrease in the concentration of As was noticed and after 24 and 72 h no detectable As concentrations were perceived suggesting that As taken up was pumped out from the hyphae. No As was detected within the roots or hyphae in the root compartment zone three or 45 h after exposure. This suggests a dual protective mechanism to the plant by rapidly excluding As from the fungus and preventing As translocation to the plant root. μ-XANES data showed that gradual As(V) reduction occurred in the AM hyphae between 1 and 3 h after arsenic exposure and was completed after 6 h. Principal component analysis (PCA) and linear combination fitting (LCF) of μ-XANES data showed that the dominant species after reduction of As(V) by R. intraradices extra-radical hyphal was As(III) complexed with a reduced iron(II) carbonate compound. The second most abundant As species present was As(V)-iron hydroxides. The remaining As(III) compounds identified by the LCF analyses suggested these molecules were made of reduced As and S. These results increase our knowledge on the mechanism of As transport in AMF and validate our hypotheses that R. intraradices directly participates in arsenic detoxification. These fungal mechanisms may help AMF colonized plants to increase their tolerance to As at contaminated sites., (Published by Elsevier Ltd.)

The study evaluates the impact of polyvinylpyrrolidone (PVP) coated silver nanoparticles (PVP-AgNPs) on the composting of municipal solid waste. The results suggest that there was no statistically significant difference in the leachate, gas, and solid quality parameters and overall composting performance between the treatments containing the AgNPs, Ag(+), and negative control. Nonetheless, taxonomical analyses of 25 Illumina 16S rDNA barcoded libraries containing 2 393 504 sequences indicated that the bacterial communities in composted samples were highly diverse and primarily dominated by Clostridia (48.5%), Bacilli (27.9%), and beta-Proteobacteria (13.4%). Bacterial diversity studies showed that the overall bacterial community structure in the composters changed in response to the Ag-based treatments. However, the data suggest that functional performance was not significantly affected due to potential bacterial functional redundancy within the compost samples. The data also indicate that while the surface transformation of AgNPs to AgCl and Ag2S can reduce the toxicity, complexation with organic matter may also play a major role. The results of this study further suggest that at relatively low concentrations, the organically rich waste management systems' functionality may not be influenced by the presence of AgNPs.

Inflammation is a major contributor to the development of atherosclerotic plaque, yet the involvement of liver and visceral adipose tissue inflammatory status in atherosclerotic lesion development has yet to be fully elucidated. We hypothesized that an atherogenic diet would increase inflammatory response and lipid accumulation in the liver and gonadal adipose tissue (GAT) and would correlate with systemic inflammation and aortic lesion formation in low-density lipoprotein (LDL) receptor null (LDLr-/-) mice. For 32 weeks, LDLr-/- mice (n = 10/group) were fed either an atherogenic (high saturated fat and cholesterol) or control (low fat and cholesterol) diet. Hepatic and GAT lipid content and expression of inflammatory factors were measured using standard procedures. Compared with the control diet, the atherogenic diet significantly increased hepatic triglyceride and total cholesterol (TC), primarily esterified cholesterol, and GAT triglyceride content. These changes were accompanied by increased expression of acyl-CoA synthetase long-chain family member 5, CD36, ATP-binding cassette, subfamily A, member 1 and scavenger receptor B class 1, and they decreased the expression of cytochrome P450, family 7 and subfamily a, polypeptide 1 in GAT. Aortic TC content was positively associated with hepatic TC, triglyceride, and GAT triglyceride contents as well as plasma interleukin 6 and monocyte chemoattractant protein-1 concentrations. Although when compared with the control diet, the atherogenic diet increased hepatic tumor necrosis factor α production, they were not associated with aortic TC content. These data suggest that the LDLr-/- mice responded to the atherogenic diet by increasing lipid accumulation in the liver and GAT, which may have increased inflammatory response. Aortic TC content was positively associated with systemic inflammation but not hepatic and GAT inflammatory status., (© 2013 Elsevier Inc. All rights reserved.)

Background: Corticobasal degeneration (CBD) is a neurodegenerative, sporadic disorder of unknown cause. Few familial cases have been described., Objective: We aim to characterize the clinical, imaging, pathological and genetic features of two familial cases of CBD., Methods: We describe two first cousins with CBD associated with atypical MRI findings. We performed exome sequencing in both subjects and in an unaffected first cousin of similar age., Results: The cases include a 79-year-old woman and a 72-year-old man of Native American and British origin. The onset of the neurological manifestations was 74 and 68 years respectively. Both patients presented with a combination of asymmetric parkinsonism, apraxia, myoclonic tremor, cortical sensory syndrome, and gait disturbance. The female subject developed left side fixed dystonia. The manifestations were unresponsive to high doses of levodopa in both cases. Extensive bilateral T1-W hyperintensities and T2-W hypointensities in basal ganglia and thalamus were observed in the female patient; whereas these findings were more subtle in the male subject. Postmortem examination of both patients was consistent with corticobasal degeneration; the female patient had additional findings consistent with mild Alzheimer's disease. No Lewy bodies were found in either case. Exome sequencing showed mutations leading to possible structural changes in MRS2 and ZHX2 genes, which appear to have the same upstream regulator miR-4277., Conclusions: Corticobasal degeneration can have a familial presentation; the role of MRS2 and ZHX2 gene products in CBD should be further investigated., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: 20-Hydroxyeicosatetraenoic acid (20-HETE), a metabolite of arachidonic acid (AA) produced by the CYP4A and CYP4F enzyme families has been reported to induce mitogenic and angiogenic responses both in vitro and in vivo, and inhibitors of this pathway reduced growth of brain and kidney tumors., Materials and Methods: Real-Time PCR, western blot and immunohistochemistry were used to compare the expression of CYP4A/F mRNA and protein levels in human cancer tissue samples versus normal controls. Liquid chromatography/mass spectrometry analysis (LC-MS/MS) was performed to measure 20-HETE formation in tumor homogenates. Activation of Ras in human proximal tubule epithelial cells (HRPTEC) treated with stable agonist of 20-HETE was measured using a Ras pull-down detection kit., Results: The expression of CYP4A/4F genes was markedly elevated in thyroid, breast, colon, and ovarian cancer samples in comparison to matched normal tissues. Furthermore, the levels of the CYP4F2 protein and of 20-HETE were higher in ovarian cancer samples compared to normal control tissues. A stable 20-HETE agonist induced activation of the small-GTPase Ras in HRPTEC cells., Conclusion: The present finding of elevated expression of CYP4A/F enzymes in human cancer tissue suggests that 20-HETE inhibitors and antagonists may be useful in the treatment of cancer.

Diseases of the central nervous system (CNS) pose a significant health challenge, but despite their diversity, they share many common features and mechanisms. For example, endothelial dysfunction has been implicated as a crucial event in the development of several CNS disorders, such as Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, human immunodeficiency virus (HIV)-1-associated neurocognitive disorder and traumatic brain injury. Breakdown of the blood-brain barrier (BBB) as a result of disruption of tight junctions and transporters, leads to increased leukocyte transmigration and is an early event in the pathology of these disorders. The brain endothelium is highly reactive because it serves as both a source of, and a target for, inflammatory proteins and reactive oxygen species. BBB breakdown thus leads to neuroinflammation and oxidative stress, which are implicated in the pathogenesis of CNS disease. Furthermore, the physiology and pathophysiology of endothelial cells are closely linked to the functioning of their mitochondria, and mitochondrial dysfunction is another important mediator of disease pathology in the brain. The high concentration of mitochondria in cerebrovascular endothelial cells might account for the sensitivity of the BBB to oxidant stressors. Here, we discuss how greater understanding of the role of BBB function could lead to new therapeutic approaches for diseases of the CNS that target the dynamic properties of brain endothelial cells.

The guanine nucleotide exchange factor C3G is one of the mediators of endothelin-1 (ET-1) intracellular signaling cascades and is vital for kidney development and homeostasis. The aim of the current study was to analyze the specificity of ET-1-induced signaling via C3G in rat glomerular mesangial cells (GMC) and to investigate the biological significance of C3G during mesangioproliferative glomerulonephritis. In GMC, C3G expression was increased (1) in vivo after induction of the anti-Thy1 model of glomerulonephritis and (2) in cell culture experiments after fetal bovine serum incubation. To examine the consequences of C3G up-regulation, adenovirus-mediated gene transfer of C3G into cultured glomerular cells was done, and the GTP loading of the small G proteins Rap1 and R-Ras was analyzed. Overexpression of C3G in mesangial cells resulted in enhanced activation of Rap1, but failed to affect the GTP-bound status of R-Ras in ET-1-stimulated cells. C3G overexpression led to significant changes in GMC spreading and migration patterns in response to ET-1 stimulation and increased stress fiber formation, which was mimicked by Rap1A overexpression. Together, these findings suggest (1) the existence of regulatory mechanisms resulting in disease-related up-regulation of C3G in GMC and (2) that an increase in the C3G protein level may contribute to the resolution stage of mesangioproliferative glomerulonephritis by reducing GMC sensitivity to ET-1, modulating cellular motility, and actin dynamics.

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have gained enormous interest as potential sources for regenerative biomedical therapies and model systems for studying early development. Traditionally, mouse embryonic fibroblasts have been used as a supportive feeder layer for the sustained propagation of hPSCs. However, the use of nonhuman-derived feeders presents concerns about the possibility of xenogenic contamination, labor intensiveness, and variability in experimental results in hPSC cultures. Toward addressing some of these concerns, we report the propagation of three different hPSCs on feeder-free extracellular matrix (ECM)-based substrates derived from human fibroblasts. hPSCs propagated in this setting were indistinguishable by multiple criteria, including colony morphology, expression of pluripotency protein markers, trilineage in vitro differentiation, and gene expression patterns, from hPSCs cultured directly on a fibroblast feeder layer. Further, hPSCs maintained a normal karyotype when analyzed after 15 passages in this setting. Development of this ECM-based culture system is a significant advance in hPSC propagation methods as it could serve as a critical component in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications., ((c) 2010 American Institute of Chemical Engineers)

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols., (2010 Elsevier Inc. All rights reserved.)

Background: We examined the ability of inhibitors of the synthesis or actions of 20-HETE, metabolite of arachidonic acid, to inhibit proliferation of human renal carcinoma cell lines., Materials and Methods: 786-O and 769-P cells were exposed to either 10 microM HET0016 (selective inhibitor of 20-HETE synthesis), 10 microM WIT002 (20-HETE antagonist), or vehicle. Subsequently, we assessed the effect of WIT002 on tumor growth in vivo using an ectopic mouse model of clear-cell renal carcinoma., Results: Addition of HET0016 and WIT002 inhibited the proliferation of 786-O and 769-P human renal cell carcinoma lines. HET0016 and WIT002 had little effect on the proliferation of primary cultures of normal human proximal tubule epithelial cells. WIT002 (10 mg/kg, s.c.) administered daily to athymic nude mice implanted subcutaneously with 786-O cells reduced the growth of the tumors by 84 % compared to vehicle (p<0.001)., Conclusion: 20-HETE is required for proliferation of human renal epithelial cancer.

Introduction: Open pelvic lymph node dissection (PLND) remains the gold standard in patients with intermediate and high-risk prostate cancer undergoing radical retropubic prostatectomy (RRP). Recently, our institution has adopted robotic assistance for performing radical prostatectomy. We sought to determine whether robot-assisted laparoscopic PLND yields comparable numbers of lymph nodes compared to open PLND., Methods: The medical records of patients undergoing open or robot-assisted laparoscopic radical prostatectomy (RALRP) with concurrent pelvic lymph node dissection (PLND) between 2003 and 2008 were reviewed. Demographic factors including age, PSA, and Gleason score were recorded. Pathology reports were reviewed to determine the number of pelvic lymph nodes obtained during PLND. Lymph node yield was further evaluated based on surgeon. Student's t-test was used to compare the number of lymph nodes obtained with each method., Results: A total of 61 patients undergoing open RRP with PLND and 62 patients undergoing RALRP with PLND were included. The mean number of lymph nodes obtained via open PLND was 7.3 while the mean number obtained via robotic PLND was 3.3. These means were significantly different with a p value < 0.001. One patient in the open cohort (1.6%) and two patients in the robotic cohort (3.2%) had micrometastatic disease on PLND., Conclusion: Robot-assisted laparoscopic PLND yielded fewer lymph nodes compared to open PLND at the time of radical prostatectomy for organ confined disease. Patients with higher risk disease may benefit from open prostatectomy with PLND early in a program's robotics experience. These findings may be related to the relative youth of our robotics program and further comparisons as our data mature will be revealing.

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1., ((c) 2006 Wiley-Liss, Inc.)

Although mitochondrial dysfunction and increased oxidative stress are found in Alzheimer's disease (AD), the origin(s) of the mitochondrial dysfunction, its causal relationship to oxidative stress and the mechanisms of their downstream effects to yield synaptic dysfunction and neuronal death are not known with certainty. The discovery of "classic" mitochondrial diseases where bioenergetic deficiencies were associated with causal mutations or deletions in mitochondrial DNA (mtDNA) generated a search for similar abnormalities in AD samples. At least three-dozen studies since 1992 have failed to find consistent mutational abnormalities in AD mtDNA beyond those associated with aging, with most studies carried out in postmortem brain. Historically, the publication of a new mutation or deletion is followed by other studies that fail to confirm the initial finding. Promising recent findings include heteroplasmic mutations in the D-loop control region. AD brain mtDNA consistently has more oxidative damage beyond that due to aging, providing the potential for generation of mutations/deletions and postgenomic problems with transcriptional regulation. To date no AD brain studies have examined individual neurons to search for clonal expansions of deleted mtDNA's like two recent reports in Parkinson's disease substantia nigra. Cybrid (cytoplasmic hybrid) models, in which mitochondrial DNA (mtDNA) from accessible tissue (platelets) of living AD patients is expressed in replicating human neural cells initially devoid of their own endogenous mtDNA (rho(0) cells) revealed that decreased cytochrome oxidase (CO) activity, increased oxidative stress, increased beta amyloid production, activation of detrimental intracellular signaling and caspases, accelerated mtDNA proliferation, and abnormal mitochondrial morphology and transport can be transmitted through expression of mtDNA from living AD patients. Carrying these cybrid observations into AD brain is necessary to demonstrate any causality of brain mtDNA to contribute to pathogenesis. A novel protein transfection technology that allows transfer of mtDNA into mitochondria of cells ("protofection") will allow this question to be examined. The contribution of altered mtDNA to pathogenesis and progression of AD is suggestive, not proven, and likely very heterogenous.

Cyclooxygenase-2 (COX-2) is constitutively expressed in restricted subpopulations of kidney cells, where it presumably acts as an antiapoptotic factor. In conditions that are characterized by inflammation, COX-2 expression also has been described in glomerular mesangial cells (GMC), where COX-2 is not expressed constitutively. It was shown previously that adenovirus-mediated gene transfer of COX-2 into rat GMC led to increased expression and activity of multidrug resistance protein 1 (MDR-1), a membrane transporter that functions as an efflux pump for chemotherapeutic drugs, including Adriamycin (ADR). In ADR nephrotoxicity, a pathologic change in glomeruli could be partially explained by ADR-mediated changes in GMC. Here it is demonstrated that ADR (also known as doxorubicin; 1 microg/ml) induced apoptosis in 15.3 +/- 2.2% of GMC, whereas after adenovirus-mediated COX-2 expression, only 6.6 +/- 0.4% of ADR-treated cells underwent apoptosis. This protective effect was nullified by treatment with NS398, specific COX-2 inhibitor. ADR efflux is greater in COX-2-overexpressing cells, when compared with control, which is attributed to the increased MDR-1 expression. Addition of PSC833, the specific MDR-1 inhibitor, completely abolished the protective effect of COX-2 overexpression and increased the level of apoptosis in GMC that were exposed to ADR. These data suggest that COX-2 protects GMC from ADR-mediated apoptosis via transcriptional upregulation of MDR-1 and that induced COX-2 expression would lessen ADR nephrotoxicity.

p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42- and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G alpha(s) also stimulated Cdc42. In addition, overexpression of beta(1)Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of beta(1)Pix SH3m(W43K), which lacks the ability to bind Pak, and beta(1)PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced beta(1)Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and beta(1)Pix translocation induced by ET-1, indicating the involvement of the PKA pathway. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assay, we have shown that beta(1)Pix is phosphorylated by PKA. Using purified recombinant beta(1)Pix(wt) and beta(1)Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. beta(1)Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks beta(1)Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by ET-1 or cAMP analog results in beta(1)Pix phosphorylation, which in turn controls beta(1)Pix translocation to focal complexes and Cdc42 activation.

We report a case of a thoracic intradural extramedullary capillary hemangioma. The MR imaging appearance was nonspecific for this lesion, and it could not be distinguished from more common spinal intradural extramedullary lesions. The presence of subtly enlarged abnormal vessels on contrast MR images should, however, lead one to consider a vascular mass and order preoperative spinal arteriography.

Cyclooxygenase 2 (COX-2) expressed in cultured neuronal PC12 cells under inducible promoter protects cells from trophic withdrawal apoptosis. Stimulation of SAPK is thought to play a significant role in initiation of PC12 cell death. We have therefore examined whether COX-2 expression inhibits trophic withdrawal-mediated activation of SAPK. SAPK activity increased during the first 6h after NGF removal in mock-transfected PC12 cells. COX-2 expression attenuated the increase of SAPK, as detected by Western blot analysis with phosphorylation state specific anti-SAPK antibodies and by SAPK activity assays. We propose that COX-2 attenuated SAPK activation by preventing activation of nNOS, which occurs, as we have shown before, via COX-2-mediated expression of dynein light chain (DLC). Activation of SAPK in neuronal cell death was attenuated by DLC expression. These observations support a role for NO production and SAPK activation in the neuronal death mechanisms.