Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications.The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As  Mdk-deficient mice display an increased bone formation rate [Mdk is an estrogen-responsive gene and that Mdk expression is enhanced in the murine postmenopausal diabetic kidney [Osteoporosis affects 200 million women worldwide; one major risk factor is the decline in estrogen after menopause , 2. The ion rate . Bone foion rate . The negion rate , a cruciion rate . We receion rate . We alsoc kidney , we hypoAll animal experiments were in compliance with international regulations for the care and use of laboratory animals  with the approval of the Local Ethical Committee .2) on a 14 h light and 10 h dark circadian rhythm with water and food ad libitum. Three-month-old mice underwent a bilateral sham-operation or OVX as described previously [Female C57BL/6J mice were provided by the University of Ulm and were maintained in groups of two to four animals per cage  as described in the manufacturer’s protocol, which was cross-reactive with murine Mdk.Biomechanical testing of the intact and fractured femurs of the mice sacrificed at day 23 was performed using a nondestructive 3-point-bending test as described previously . Briefly3 and 750 mg/cm3) within each scan. The BMD of the fracture callus was evaluated without a threshold, whereas the TMD of the cortical bone was determined using a global threshold of 642 mg hydroxyapatite/cm3. The TMD of the trabecular bone was evaluated using a global threshold of 395 mg hydroxyapatite/cm3 as described previously and in accordance with the American Society for Bone and Mineral Research (ASBMR) guidelines for μCT analysis [Femurs and vertebral bodies were analyzed using a μCT scanning device  operating at a voxel resolution of 8 μm . Four volumes of interest (VOIs) were determined for μCT analysis: VOI 1  covered the periosteal callus between the two inner pinholes. VOI 2  covered the area from 80 μm proximal to 80 μm distal from the middle of the diaphysis of the intact femur. The starting point for VOI 3 (intact trabecular bone) was 200 μm proximal to the metaphyseal growth plate of the intact femur and the endpoint was 280 μm proximal from the starting point. VOI 4 covered the trabecular region of the second lumbar vertebral body. Tissue and bone mineral density (TMD and BMD) were assessed using two phantoms with a defined hydroxyapatite density  for 10–12 days and embedded in paraffin after dehydration in an ascending ethanol series. Longitudinal sections of 7 μm thickness were prepared and stained using Safranin O for tissue quantification. Immunohistochemical staining of total and active beta-catenin was performed using the following antibodies: polyclonal rabbit anti-mouse beta-catenin antibody , monoclonal rabbit anti-mouse active beta-catenin antibody , horse-radish peroxidase-conjugated streptavidin , and goat anti-rabbit IgG . Species-specific non-targeting immunoglobulins were used as isotype controls. 3-Amino-9-ethylcarbazol (Zytomed Systems) was used as the chromogen and the sections were counterstained using hematoxylin  as described previously .Sample size was calculated based on a previous fracture-healing study for the main outcome parameter flexural rigidity in the fractured femur  . The resAs expected, OVX delayed fracture healing as demonstrated by a significantly decreased relative flexural rigidity of the fractured femurs of the vehicle-treated mice . Callus Mdk-Ab treatment tended to increase the relative flexural rigidity in the sham-operated group and significantly in the OVX group . μCT anaWe determined Mdk serum levels 0, 3, 10 and 23 days after fracture . None ofWe were next interested in the molecular mechanism behind the increased bone formation in the fracture callus of Mdk-Ab-treated mice. Therefore, we stained callus sections for beta-catenin expression. Beta-catenin expression was mainly found in proliferative chondrocytes at the cartilaginous callus and in osteoblasts at the bony fracture callus, whereas hypertrophic chondrocytes were negative . OVX sigAntibody treatment increased the cortical TMD in the intact femur of OVX animals , whereasMdk-Ab treatment significantly increased trabecular TMD, BV/TV, trabecular thickness, osteoblast numbers and surface in OVX mice , Table 1OVX also led to a decreased trabecular BV/TV and number in the vertebral bodies of vehicle-treated mice . Mdk-Ab Because osteoporotic patients display an increased risk for fracture healing complications , there iMdk-deficiency were more pronounced in 12- and 18-month-old mice than in 4-month-old mice [As expected, OVX mice displayed a significantly lower relative flexural rigidity and bone content in the fracture callus compared to non-OVX mice , 22, 23.old mice . This inold mice –31. Wnt/old mice . TherefoMdk-deficient mice exhibited increased trabecular bone formation in the vertebral bodies and were protected from OVX-induced trabecular bone loss [Dmp1, Ank and Enpp1 [Ank and Enpp1 are essential in preventing ectopic tissue mineralization by raising the extracellular level of pyrophosphatase [Because one loss , we alsoone loss . On the nd Enpp1 . In partsphatase . TherefoIn conclusion, the findings of the present study indicate that there is a strong therapeutic potential for the Mdk-Ab to enhance fracture healing in patients with delayed bone healing resulting from postmenopausal osteoporosis.


Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) poses a worldwide and serious health threat. Although new antibiotics, such as daptomycin and linezolid, have been developed for the treatment of infections of Gram-positive pathogens, the emergence of daptomycin-resistant and linezolid-resistant strains during therapy has now increased clinical treatment failures. In the past few years, studies using quantitative proteomic methods have provided a considerable progress in understanding antibiotic resistance mechanisms. In this review, to understand the resistance mechanisms to four clinically important antibiotics  used in the treatment of Gram-positive pathogens, we summarize recent advances in studies on resistance mechanisms using quantitative proteomic methods, and also examine proteins playing an important role in the bacterial mechanisms of resistance to the four antibiotics. Proteomic researches can identify proteins whose expression levels are changed in the resistance mechanism to only one antibiotic, such as LiaH in daptomycin resistance and PrsA in vancomycin resistance, and many proteins simultaneously involved in resistance mechanisms to various antibiotics. Most of resistance-related proteins, which are simultaneously associated with resistance mechanisms to several antibiotics, play important roles in regulating bacterial envelope biogenesis, or compensating for the fitness cost of antibiotic resistance. Therefore, proteomic data confirm that antibiotic resistance requires the fitness cost and the bacterial envelope is an important factor in antibiotic resistance.The increase of methicillin-resistant  Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), multidrug-resistant (MDR) Pseudomonas aeruginosa, imipenem-resistant Acinetobacter baumannii, and third-generation cephalosporin-resistant Escherichia coli and Klebsiella pneumonia, has become an increasingly severe problem in many hospitals worldwide ] that crosslinks the linear peptidoglycan polymer chains  regulatory elements, and fem factors  that is not inhibited by classical β-lactam antibiotics including methicillin  involved in pathogenesis can enhance the fitness of methicillin-resistant pathogens  or D-Ala-D-Ser peptide  in peptidoglycan synthesis. VanH protein (α-keto acid reductase) reduces pyruvate to D-Lac, and the D,D-dipeptidase VanX selectively removes the D-Ala-D-Ala produced by the native ligase to enhance the incorporation of the D-Ala-D-Lac or D-Ala-D-Ser into the peptidoglycan precursor. VanR and VanS constitute a two-component regulatory system that activates the transcription of the van gene cluster , to which vancomycin binds Table . The D-ALinezolid is a first synthetic oxazolidinone antibiotic used to treat infections caused by VRE and MRSA. Although the mechanism of action of linezolid is not fully understood, it seems to bind to the 50S subunit of the bacterial ribosome through interaction with the central loop of the 23S rRNA and block the formation of protein synthesis initiation complexes  that methylates G2445 of the 23S rRNA, and mutations causing increased expression of ABC transporter genes (patA and patB).Most Gram-negative bacteria have an intrinsic resistance to linezolid due to the high activity of efflux pumps, which actively pump linezolid out of the cell   and transfers the lysyl-PG (LPG) to the outer leaflet of the membrane. The LPG is less acidic than PG, and membranes lacking LPG are more acidic than those containing PG and LPG  reduces the binding of Ca2+-bound daptomycin to bacterial membranes by a less favorable electrostatic interaction. YycG is a membrane spanning sensor histidine kinase of a two-component signal transduction system that partners with the YycF response regulator. YycFG functions as a master regulatory system for cell wall metabolism and biofilm formation and is the only two-component system required for viability in many Gram-positive bacteria  and methicillin-resistant S. aureus (MRSA) in the absence of methicillin were reported  and 8325 (methicillin-susceptible) in the absence of methicillin , only FemA protein, which is known as a host-mediated factor essential for methicillin resistance in S. aureus . This study also showed that three proteins linked to the alternative sigma factor σB, Asp23, anti-anti- σB factor RsbV, and conserved hypothetical protein SA0772, were also present at significantly higher levels in methicillin-resistant cells  regulons are involved in methicillin resistance of S. aureus . This study also showed that the stage V sporulation protein G (SpoVG), originally identified in Bacillus subtilis as being involved in the formation of the spore cortex , indicating that SpoVG may be involved in resistance mechanisms to other antibiotics as well as methicillin and glycopeptide.Two studies exploring proteomic profiles of methicillin-susceptible S. aureus . In addition, elongation factor G (encoded by the fusA gene) was also increased in MRSA. Our analysis showed that PusA is one of the three proteins affected in all four antibiotic-resistant strains (Table 5). Although elongation factor G is a major target of fusidic acid which has been used as a topical agent for skin infection and for some systemic infections caused by S. aureus  and vancomycin-intermediate S. aureus (VISA) with a minimal inhibitory concentration (MIC) of 4–8 μg/ml, one study exploring proteomic profiles in VSSA and heterogeneous vancomycin-intermediate S. aureus (hVISA) with a vancomycin MIC of ≤2 μg/ml, one study exploring proteomic profiles in VISA and vancomycin-resistant S. aureus (VRSA) with MIC of ≥8 μg/ml, one study analyzing global proteomes of vancomycin stress in S. aureus, and two studies examining vancomycin-induced proteomes of Enterococcus faecalis under vancomycin treatment  and the vancomycin-intermediate S. aureus strain 14-4, and most proteins play a role in energy metabolism, cell envelope biosynthesis, protein turnover, amino acids transport, and metabolism, and inorganic ion transport. Genes or gene products known to be involved in resistance mechanisms to different antibiotics, such as PBP 2a (MecA), O-nucleotidyltransferase(9) [Ant(9)], UDP-N-acetylmuramyl tripeptide synthetase (MurE), and penicillin-binding methicillin resistant-related protein (FmtA), were up-regulated in the VISA strain , indicating the importance of this protein on peptidoglycan metabolism and antibiotic resistance.There were two studies exploring proteomic profiles in vancomycin-susceptible B. subtilis  regulating expression of a set of genes involved in the cell wall biosynthesis or degradation   pathway enzymes, which are under the control of the PurR regulator, strongly increased in protein abundance in the vancomycin-resistant Abundance changes were also found in proteins such as the single-stranded DNA binding protein (TraM), DNA gyrase subunit B (GyrB), and topoisomerase IV subunit B (ParE), which catalyze or influence the fidelity of DNA replication and repair Table . This resceD mRNA expression levels were compared between 25 VSSA and VISA clinical isolates by real-time RT-PCR   Table . Among tG) Table . In addiG) Table , and decG) Table , suggestG) Table . S. auren = 24) and VSSA stains (n = 30)   that play a role in vancomycin stress response. The cluster of proteins under positive σB control mainly increased, whereas negatively regulated proteins primarily decreased in amount after vancomycin addition  under 64 μg/ml vancomycin treatment  with linezolid MICs of 0.5–0.75 μg/ml and linezolid-resistant S. pneumonia strains (1974M2-LZD and R6M2-LZD) with MIC of 32 μg/ml, Feng et al. showed that the proteomic and transcriptomic approaches were poorly correlated with previously known resistance factors , as modulated proteins rarely had significant concomitant changes at the expression level , 3-ketoacyl-ACP reductase (FabG), acetyl-CoA carboxylase biotin carboxyl carrier protein subunit (AccB), acetyl-CoA carboxylase subunit alpha (AccA), and hydroxymyristoyl-ACP dehydratase (FabZ), were down-regulated in linezolid-resistant strains  under linezolid stresses   affecting cell membrane lipid homeostasis, cell wall metabolism and biofilm formation, and two-component regulation system genes  involved in pathogenesis of methicillin-resistant strains    Table , recent e) Table  and a sue) Table . These rB regulon, and hydroxymyristoyl-ACP dehydratase (FabZ). These proteins can be divided into two groups, proteins involved in bacterial envelope regulation and proteins compensating for a fitness cost of antibiotic resistance. Proteins such as LipA, VraSR, FmtA, SsaA, SceD, IsaA, Atl, and FabZ, are directly or indirectly involved in envelope regulation. In order to modify or thicken the bacterial cell wall for antibiotic resistance, cells require abundant energy, and proteins involved in stress adaptation are necessary to neutralize various damages by antibiotic. To sustain these fitness costs associated with resistance mechanisms, proteins involved in energy metabolism  and stress-related proteins  seem to be identified in resistance mechanisms to several antibiotics. Therefore, these proteomic results confirm that antibiotic resistance requires a fitness cost.Interestingly, comparative proteomic approaches in methicillin, linezolid, and daptomycin, except for vancomycin, were poorly correlated with known resistance-related factors found by non-proteomic approaches Table . This reDetailed studies on the mechanism by which these proteins affect antibiotic resistance are required. In particular, because these proteins can act as the global factor affecting resistance mechanisms to most antibiotics, it is necessary to examine whether they affect resistance mechanism of other antibiotics having different action modes. These studies will provide important clues for understanding and managing antibiotic resistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vivo validation of these pro-atherogenic and osteogenic genes also demonstrates a broad consistent differential expression pattern in mouse aorta vs. vena cava ECs, which cannot be explained by the difference in hemodynamic flow. These data reveal phenotypic modulation by different levels of COUP-TFII in arterial and venous ECs, and suggest COUP-TFII may play an important role in the different susceptibilities of arteries and veins to vascular diseases such as atherosclerosis and vascular calcification.Arteries and veins have very different susceptibility to certain vascular diseases such as atherosclerosis and vascular calcification. The molecular mechanisms of these differences are not fully understood. In this study, we discovered that COUP-TFII, a transcription factor critical for establishing the venous identity during embryonic vascular development, also regulates the pathophysiological functions of adult blood vessels, especially those directly related to vascular diseases. Specifically, we found that suppression of COUP-TFII in venous ECs switched its phenotype toward pro-atherogenic by up-regulating the expression of inflammatory genes and down-regulating anti-thrombotic genes. ECs with COUP-TFII knockdown also readily undergo endothelial-to-mesenchymal transition (EndoMT) and subsequent osteogenic differentiation with dramatically increased osteogenic transcriptional program and calcium deposition. Consistently, over-expression of COUP-TFII led to the completely opposite effects.  COUP-TFII , also known as NR2F2 , is an orphan member of the steroid receptor superfamily. COUP-TFII is widely expressed in a variety of tissues in the body, and plays an important role in the function and homeostasis of many tissues and organs, such as stem cell differentiation, angiogenesis, lipid/glucose metabolism, and organ development123467vs. venous ECs, including extracellular matrix (ECM), inflammation, lipid/metabolism, growth factors, junction proteins, vasomotor factors and thrombosis. Specifically, we found that suppression of COUP-TFII in venous ECs switched its phenotype toward pro-atherogenic by up-regulating the expression of inflammatory genes and down-regulating anti-thrombotic genes. We also discovered that COUP-TFII is a strong regulator of the TGFβ/BMP pathway. ECs with COUP-TFII knockdown readily undergo endothelial-to-mesenchymal transition (EndMT) and subsequent osteogenic differentiation with dramatically increased osteogenic transcriptional program and calcium deposition. Consistently, over-expression of COUP-TFII suppressed all these effects, leading to an anti-atherogenic phenotype and less calcium deposition. These data suggest COUP-TFII may play multifaceted roles in defining the distinctive susceptibilities of adult arteries and veins to vascular diseases such as atherosclerosis and vascular calcification.In vascular endothelial cells (ECs), COUP-TFII is expressed at much higher level in venous than arterial endothelium, thus serves as a robust venous EC marker102 on 0.1% gelatin (Sigma) coated tissue culture plate. After 24 hours, the confluence of each EC monolayer was verified by phase contrast examination. To validate that COUP-TFII regulated gene patterns are also true in other endothelial cell types, human aortic endothelial cells  were cultured and selected genes were validated using RT-PCR  were cultured at 37 °C in 5% CO2 in Endothelial Cell Growth Medium-2 . The culture media was changed every two days. HSVECs were routinely passaged onto tissue culture flasks and discarded after 8 passages to ensure representation of key endothelial characteristics. For each experiment, EC were plated at an initial density of 50,000 cells/cmg RT-PCR .The human Nr2f2 (COUP-TFII) cDNA was amplified from pCMV6-XL5-Nr2f2 (OriGene) by PCR, then sub-cloned into pCDH-EF1-T2A-GFP lentiviral vector (System Biosciences) between EcoRI and NotI digestion sites. Sequences of the PCR primer pair are: Forward, GGAATTCGCCACCATGGCAATGGTAGTCAGCAC; Reverse, ATTTGCGGCCGCTTGAATTGCCATATACGGCCA. To package the lentivirus, the pCDH-EF1-Nr2f2-T2A-GFP or the pCDH-EF1-T2A-GFP constructs were co-transfected with pCMV-VSVG and pCMV-dvpr into 293FT cells using X-tremeGENE HP transfection reagent (Roche). Supernatant was harvested 2 and 3 days later and further concentrated by ultra-centrifugation. The virus pellet was re-suspended in PBS and aliquoted, then put into −80 C for storage. Lentiviruses were used at 10 MOI for cell transduction, and undergo puromycin (2 μg/ml) selection to establish stable ECs that permanently over-express COUP-TFII.Endothelium RNA from mouse aorta and vena cava was isolated using protocol previously described with minor modificationEn face immunofluorescence microscopy of mouse aortic arch was performed as described previouslyen face preparation were obtained using a Zeiss LSM META 510 confocal microscope system with Zeiss Zen software, or Zeiss Axio Observer Z1 inverted microscope with the Apotome module and Zeiss AxioVision software.To identify nuclei, DAPI  was added during the incubation of secondary antibodies. For the aorta, the lesser curvature of the aortic arch and a portion of thoracic aorta were cut and mounted with the endothelium facing up. Images of the To assay the osteogenic potential of cultured HSVECs, the culture medium was switched from EGM-2 to StemPro osteogenesis differentiation medium (Life Technologies). After 7 days of culture, RNA was isolated and Taqman RT-PCR was performed to assess the gene expression involved in osteogenic pathway: RUNX2, SP7, DLX5, ALPL, COL1A1, IBSP, BGLAP, SPP1 and BMP4. To further validate the functional consequences of the osteogenic potential, Alkaline Phosphatase (ALP) and Alizarin Red S staining were performed on cells following 7 days of culture under osteogenic differentiation medium. Briefly, for Alkaline Phosphatase staining, cells were rinsed twice by PBS and then fixed using 1 mL 10% formaldehyde in each well for 1 min . After the fixation, the fixative was aspirated and the cells were rinsed again using PBS. ALP activities were detected by SigmaFast BCIP/NBT (Sigma-Aldrich) following the manufacturer protocol. For Alizarin Red S staining, cells were rinsed twice using calcium and magnesium free PBS and then fixed using 1 mL 10% formaldehyde in each well for 10 min. Cells were then washed twice using PBS and stained using an Alizarin Red S kit (American MasterTech) to evaluated the calcium deposit following the manufacturer protocol.Additional materials and methods are described in detail in Supporting Information.We first applied RNAi to knockdown COUP-TFII expression in cultured human saphenous vein endothelial cells (HSVEC), and performed transcriptional profiling of the genes that are differentially expressed. As shown in Importantly, we found large number of chemokine/inflammatory genes are up-regulated as well as several genes involved in thrombosis . To valiThe overall pattern of pro-/anti-atherogenic gene expression suggests that the ECs with COUP-TFII knockdown switch to a more pro-atherogenic phenotype. To further validate this, we assessed inflammatory cytokine induced VCAM-1, the adhesion molecule involved in leukocyte-endothelial interaction in early atherosclerosis and is essential for disease progressionTo further understand the functions of COUP-TFII in adult ECs, we made lentivirus over-expressing COUP-TFII and used it to generate confluent EC monolayer that stably expresses COUP-TFII. The transcriptional profiling analysis  revealed1719e.g., VCAM-1, von Willebrand factor, thrombospondin, multimerin), and increased anti-thrombotic gene expression , SPP1 (secreted phosphoprotein 1 or osteopontin) and BMP4. The effect is so significant that some of them have more than 10 or 100 fold of induction, suggesting that COUP-TFII in ECs acts as a strong repressor of its osteogenic potential in normal condition, and upon removal, osteogenic programs are greatly increased. Consistent with this, over-expression of COUP-TFII suppressed these genes: ALPL, COL1A1, IBSP, SPP1, BMP4  are not differentially expressed in adult arteries vs. veins, although they have been used extensively in the past as the proof of arterial differentiation in many developmental biology studies. It is likely that they appear as early arterial marker during vascular development, and then the difference disappears after maturation into adult blood vessel. Interestingly, although COUP-TFII controls many arterial venous markers, EphB4 seems not influenced by COUP-TFII at all. This suggests that other additional unknown endogenous programs are involved in venous specification besides COUP-TFII.We have also identified many new molecular markers that are highly expressed in adult artery or vein. Some of these markers show more than 10–100 fold of differential expression, and therefore, can be used as a more robust marker to identify artery en face VCAM-1 staining in blood vessels also confirmed that arterial ECs have a much stronger response to inflammatory stimuli by turn on VCAM-1 expression whereas venous ECs do not express VCAM-1 at all. VCAM-1 was reported to express at the arteries and post-capillary venules after cytokine stimulationIn arteries, atherosclerosis often develops in regions of disturbed flow, which promotes a pro-inflammatory phenotype of ECs23404243in vitroin vivo is also striking, indicating a stronger osteogenic potential of arteries. EndMT has been described in different pathologies, and is implicated in many disease situations such as cancer progressionVascular calcification is a frequent complication of vascular disease, such as diabetes mellitus, renal disease, and atherosclerosis. It is a regulated process with strong similarities to bone formation driven by osteo-progenitor cells in the media layer of blood vessel. Vascular EC represents a distinct cell lineage from osteo-progenitor cells. In healthy blood vessels and also in normal EC culture, the osteogenic program is usually absent and thus ECs do not contribute to the calcium deposition in healthy blood vessels. However, recent studies have demonstrated a role for the endothelium in vascular calcificationIn summary, COUP-TFII, a transcription factor expressed higher in venous EC, plays multifaceted roles in determining the functional difference between arteries and veins. Multiple important gene pathways involved in vascular diseases are regulated by COUP-TFII. ECs with higher COUP-TFII expression confer a more anti-atherogenic phenotype and lower osteogenic potential, while lower COUP-TFII expression elicits a more pro-atherogenic phenotype and greater sensitivity to EndMT and osteogenic stimulation. Our findings provide new insights into the mechanism of arterial diseases and suggest potential roles of COUP-TFII in determining the distinctive susceptibilities of arteries and veins to vascular diseases.How to cite this article: Cui, X. et al. Venous Endothelial Marker COUP-TFII Regulates the Distinct Pathologic Potentials of Adult Arteries and Veins. Sci. Rep.5, 16193; doi: 10.1038/srep16193 (2015).


Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain GrollT together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes. Desulfotomaculum gibsoniae strain GrollT (DSM 7213) is a mesophilic sulfate-reducing bacterium isolated from a freshwater ditch in Bremen, Northern Germany  and three [NiFe] hydrogenases, suggesting a lower redundancy in the case of [FeFe] enzymes than other members of the genus. The [FeFe] hydrogenases include one membrane-associated protein (Desgi_0926-0928) that contains a tat motif in the alpha subunit (Desgi_0926), suggesting an extracellular localization; one monomeric hydrogenase (Desgi_0935) encoded close to the membrane-bound enzyme, which suggests the possibility of co-regulation; two copies of trimeric NAD(P)-dependent bifurcating hydrogenases (Desgi_4669-4667 and Desgi_3197-3195); one enzyme (Desgi_0771) that is part of a multi-gene cluster encoding two flavin-dependent oxidoreductases that is also present in other Desulfotomaculum spp., and one HsfB-type hydrogenase (Desgi_3194) encoding a PAS-sensing domain that is likely involved in sensing and regulation, and possibly with the bifurcating Desgi_3195 hydrogenase.The [NiFe] hydrogenases include one enzyme (Desgi_1398 – 1397) that may also be bound to the membrane by a cytochrome b (Desgi_1402); one simple dimeric enzyme (Desgi_1231-1230); and one trimeric group 3 hydrogenase (Desgi_1166-1164), similar to methyl-viologen reducing hydrogenases from methanogens, and which is encoded next to a HdrA-like protein (Desgi_1163).NifE, nitrogenase molybdenum-iron protein, alpha and beta chains, nitrogenase cofactor biosynthesis protein NifB; ferredoxin, iron only nitrogenase protein AnfO (AnfO_nitrog) (Desgi_2428-2419) were detected within the annotated genome sequence. Thus, D. gibsoniae probably has the capacity for nitrogen fixation. However, the fixation of molecular nitrogen has not been analyzed in this species so far.A cluster of nitrogenase genes, specifically genes encoding nitrogenase iron protein, nitrogen regulatory protein PII, nitrogenase molybdenum-iron protein alpha chain, nitrogenase molybdenum-iron protein beta chain, nitrogenase molybdenum-iron cofactor biosynthesis protein


Cortical projection neurons are classified by hodology in corticocortical, commissural and corticofugal subtypes. Although cortical projection neurons had been regarded as only glutamatergic neurons, recently corticocortical GABAergic projection neurons has been also reported in several species. Here, we demonstrate corticofugal GABAergic projection neurons in the mouse frontal cortex. We employed viral-vector-mediated anterograde tracing, classical retrograde tracing, and immunohistochemistry to characterize neocortical GABAergic projection neurons. Injections of the Cre-dependent adeno-associated virus into glutamate decarboxylase 67 (GAD67)-Cre knock-in mice revealed neocortical GABAergic projections widely to the forebrain, including the cerebral cortices, caudate putamen (CPu), ventral pallidum (VP), lateral globus pallidus (LGP), nucleus accumbens, and olfactory tubercle (Tu). Minor GABAergic projections were also found in the mediodorsal thalamic nucleus, diagonal band of Broca, medial globus pallidus, substantial nigra, and dorsal raphe nucleus. Retrograde tracing studies also demonstrated corticofugal GABAergic projection neurons in the mouse frontal cortex. Further immunohistochemical screening with neurochemical markers revealed the majority of corticostriatal GABAergic projection neurons were positive for somatostatin (SS)-immunoreactivity. In contrast, corticothalamic GABAergic projection neurons were not identified by representative neurochemical markers for GABAergic neurons. These findings suggest that corticofugal GABAergic projection neurons are heterogeneous in terms of their neurochemical properties and target nuclei, and provide axonal innervations mainly to the nuclei in the basal ganglia. The overwhelming majority of long-range corticocortical connections in the cerebral cortex originate from glutamatergic pyramidal neurons. Recently, several investigations in rodents, carnivores, and monkeys have provided evidence that a small number of inhibitory nonpyramidal neurons have long-range corticocortical connections -Cre knock-in mice, classical retrograde tracing in GAD67-green fluorescent protein (GFP) knock-in mice. Improved detection of GABAergic neurons could provide more in depth characterization of GABAergic projections neurons in the mouse frontal cortex.All experiments were approved by the Committee for Animal Experiments of Kumamoto University, and were performed in accordance with the Guidelines for Use of Animals in Experiments of Kumamoto University. All efforts were made to minimize animal suffering and the number of animals used. Two lines of transgenic mice targeting GABAergic neurons via GAD67 promoter were used in this study: GAD67-Cre knock-in mice  were used in this study. One of AAVs, AAV-Ef1a-DIO-hChR2-mCherry, drives Cre-dependent channel rhodopsin-fluorescent protein fusion  were anesthetized by intraperitoneal injection of chloral hydrate and placed in a stereotaxic apparatus. Less than 100 nl of 1% Fast Blue solution  dissolved in distilled water was injected into the ventral part of CPu , mediodorsal thalamus , or dorsal raphe nucleus  by pressure through a glass micropipette. The FB-injected mice survived for 5–7 days after the injection.The mice were deeply anesthetized by intraperitoneal injection of chloral hydrate (100 mg/100 g body weight), and perfused transcardially with 5 ml of PBS , followed by 40 ml of PBS containing 4% (w/v) formaldehyde. After the mice were left for 2 h, the brains were removed and immersed in 30% sucrose in PBS overnight for cryoprotection. The brains were cut into 40 μm-thick frontal sections on a freezing microtome.2O2 for 10 min at room temperature. After washing in PBS, the sections were blocked in PBS with 0.3% Triton X-100 (PBS-T) and 1% normal donkey serum for 1 h at room temperature. This was followed by overnight incubation with the rabbit anti-mCherry antibody or the rabbit anti-EGFP antibody in PBS-T containing 1% normal donkey serum and 0.02% sodium azide (PBS-TSS) at room temperature. After washing in PBS-T, the sections were incubated with the biotinylated secondary antibody against rabbit  in PBS-TSS for 2 h at room temperature. After washing in PBS-T, the sections were incubated with an ABC  in PBS at room temperature for 1 h. After washing in 0.1 M phosphate buffer , the sections were incubated for 30 min in the biotinylated tyramine-glucose oxidase reaction mixture containing 1.25 μM biotinylated tyramine, 2 mg/ml of glucose oxidase , 2 mg/ml of beta-D-glucose, and 1% BSA in 0.1 M PB. After washing in PBS, the sections were again incubated for 1 h with an ABC in PBS. Finally, the bound peroxidase was developed black by staining with nickel-enhanced coloring solution .In order to enhance the immunoreactivity of anterograde labeling, we employed the method of combining the avidin biotinylated peroxidase complex (ABC) method with the biotinylated tyramine-glucose oxidase amplification in this study . Secondly, we processed the sections for immunofluorescent staining with the following antibodies: anti-calretinin (CR) rabbit serum , anti-neuronal nitric oxide synthase (nNOS) rabbit IgG , anti-neuropeptide Y (NPY) rabbit serum , anti-PV mouse IgG , anti-SS rabbit affinity purified polyclonal antibody , and anti-VIP rabbit polyclonal antibody . The sections were incubated with one of the primary antibodies overnight at room temperature in PBS-TSS. After washing with PBS-T, the sections were further incubated with Alexa594-conjugated secondary antibody against rabbit or mouse IgG  for 2 h at room temperature. The sections were mounted onto non-coated glass slides in PBS. When the FB- and GFP-double-labeled cells were negative for one of neurochemical markers, the sections were re-incubated with other neurochemical marker, and then developed with an Alexa647-conjugated secondary antibody (Molecular Probes). After immunofluorescent procedures, the sections were incubated with Hoechst33342  to identify laminar structures of neocortex (Cx).For double or triple immunofluorescent staining, some sections from the AAV injected materials were incubated with several primary antibodies in PBS-TSS Table , followeImmunofluorescence was observed under either an epifluorescence microscope BZ-9000 or an confocal laser scanning microscope FV1200  with appropriate filter sets for FB , GFP , Alexa594  Alexa647 . In the anterograde labeling experiments, we obtained the integrated images of wide area of the sections with a ×10 objective using BZ-9000, according to the manufacturer’s instruction. We took higher magnification images of individual axons with a ×60 oil immersion objective using FV1200. In the retrograde labeling experiments, we scanned sections to find FB- and GFP-double-labeled neurons with a ×20 objective using BZ-9000. We took higher magnification images of individual neurons with a ×40 objective using FV1200. The FB fluorescence was often weakened after the immunohistochemical procedures, although some FB-labeled granules can be detected in the perikarya. When the FB-fluorescence was completely quenched, we identified FB- and GFP-double-labeled neurons by carefully comparing each GFP-labeled cell and its surrounding structures with those structures in the photomicrographs taken before immunostaining.Several previous reports have demonstrated highly specific expression of Cre recombinase in GAD67-Cre knock-in mice . We confirmed the injection sites were relatively larger (0.6–1.0 mm) and were confined only in frontal cortex . We confirmed the injection sites were mainly confined in the ventral part of CPu and LGP. Injections of FB into the ventral part of CPu resulted in numerous FB-labeled neurons mainly in layers 5 and 2/3 of the frontal cortex, consistent with the previous report in Gabbott et al. . We confirmed the injection sites were mainly confined in the mediodorsal thalamic nucleus (n = 8). We chose three brain samples for further analysis after scanning sections under an epifluorescence microscope, because we could find few FB- and GFP-double labeled neurons in most samples. Injection of FB into the mediodorsal thalamic nucleus resulted in numerous FB-labeled neurons in layer 6 of the frontal cortex, consistent with the previous report in Gabbott et al. . We confirmed the injection sites were mainly in the dorsal raphe nucleus and superior colliculus. Injection of FB resulted in numerous FB-labeled neurons in upper layer 5 and lower layer 3 of the frontal cortex, and occasionally in layer 2 and 6, consistent with the previous report in Gabbott et al. . Interestingly, mCherry-immunoreactive fibers were found mainly in the brain regions where a few GFP-immunoreactive fibers were found. For example, at the level of the crossing of the anterior commissure, abundant bundles of GFP-immunoreactive fibers were found in the ventromedial CPu, while mCherry-immunoreactive fibers were found mainly in further lateral part of main GFP-immunoreactive area Figure . TogetheWe report that a subset of GABAergic neurons in the mouse frontal cortex project widely to cortical areas and subcortical nuclei throughout the forebrain and midbrain. They project primarily to the basal ganglia including the CPu, VP, LGP, nucleus accumbens, and Tu Table . In someAlthough Cre-dependent expression of hChR2-mCherry in GAD67-Cre knock-in mice labeled nonpyramidal neurons, a natural concern is whether the apparent labeling could be specific. We think this concern is unlikely for several reasons. First, mCherry-immunoreactivity by Cre-dependent excision were found in nonpyramidal cells, but not in pyramidal and glia cells. In addition, we confirmed almost all mCherry-immunoreactive cells were positive for GABA-immunoreactivity. Second, retrograde tracing experiments were strongly consistent with the anterograde labeling by Cre-dependent excision. Third, our results are partly consistent with the previous report in Lee et al. , which hThe volume of viral injection is the primary factor to obtain strong labeling, especially when the distribution of axonal fibers is analyzed. In this study, the maximal volume of viral injection is limited to less than 50 nl, which size is roughly equal with a 350 μm cube. Considering that an injection solution is infused into extracellular space, a diameter of injection would be much larger than 350 μm. Indeed, the diameters of injection sites are 400–1000 μm in this study, although we carefully injected a small volume of viral solution several times for over 30 min in order to deal with this issue. This technical limitation by a pressure injection could be improved by a more sophisticated method iontophoresis (Wang et al., In the present study, we demonstrated corticofugal GABAergic projection neurons qualitatively, since the labeling efficiency of the viral-vector-mediated anterograde tracer were variably across animals. This technical limitation would be general among neuroanatomical tracers. Indeed we carefully injected the small volume of isosmotic viral solution into the frontal cortex to avoid any alternation such as tissue damage, but it was hard to control quality of injection. Recently single cell labeling techniques Pinault,  have sucSeveral studies have demonstrated glutamatergic projection fibers from the rodent frontal cortex to the nuclei throughout the forebrain and brainstem. In this study, the distribution of projection fibers, which are non-selective GFP-labeled Figure , are maiOur experiments confirm and extend the recent work describing GABAergic projection neurons in the medial prefrontal cortex using Dlxi12b–Cre transgenic mice (Lee et al., In order to examine the neurochemical properties of GABAergic projection neurons in the prefrontal cortex, Lee et al.  injectedSeveral studies have demonstrated corticocortical GABAergic projection neurons in rodents, carnivores, and monkeys (Peters et al., Corticocortical GABAergic projection neurons may account for 0.5% of the neocortical GABAergic neurons (Tamamaki and Tomioka, One puzzling observation is that the highly topographical overlap between corticothalamic GABAergic and glutamatergic projection fibers was demonstrated in retrograde labeling experiments Figure , whereasThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after  Like ubiquitination, SUMO conjugation occurs through an enzyme cascade. SUMO precursors are processed by SUMO-specific proteases (SENPs). Mature SUMO is activated by the SUMO E1 activating enzyme (SAE1/SAE2), transferred to the SUMO E2 conjugating enzyme (Ubc9), and then ligated to a lysine residue within SUMO consensus sequence inside the target protein . Althoughe cells  (RevieweSimilar to the Src homology 2 (SH2) domains and phosphotyrosine binding (PTB) domains that mediate selective protein-protein interaction with phosphorylated tyrosine, the SUMO-interacting motif (SIM) mediates the recognition of SUMO modification in both a paralog-specific and -non-specific manner . Via SIMDue to the involvement of SUMO in almost all cellular aspects, it is not surprising that viruses have evolved many different pathways to exploit the host SUMO system. In most cases, viruses target the SUMO pathway by encoding proteins that interact with various components of the pathway in order to modulate their enzyme activities. For instance, the avian adenovirus, chicken embryo lethal orphan (CELO), encodes Gam1 that inactivates the SUMO E1 activating enzyme . Human aKSHV, also known as human herpesvirus type 8 (HHV-8), is a γ-herpesvirus associated with Kaposi’s sarcoma (KS), primary effusion lymphomas (PEL) and multicentric Castlemen’s disease (MCD) . It primIn addition to chromatin remodeling, SUMO plays another important role in the herpesvirus life cycle, namely in the assembly and disassembly of PML-NBs or ND-10 (nuclear domain-10). PML-NBs are discrete subnuclear structures that contain multiple cellular proteins, including PML, Sp100, hDaxx, and ATRX. It has long been postulated that PML-NBs function as an intrinsic antiviral defense by repressing viral transcription and replication . DisruptThe PML-NB is a SUMO storage warehouse for the nucleus and it is here that a large number of sumoylated proteins congregate. Sumoylation of PML is a prerequisite for recruitment of Daxx, Sp100, and SUMO paralogs during PML-NB formation . De-sumoMoreover, KSHV targets SUMO because SUMO is capable of modulating interferon-related antiviral responses. Invading viruses are recognized by Toll-like receptors (TLRs), a type of pathogen recognition receptor (PRRs), and this then results in the production of type I interferon (IFN) and the subsequent activation of the host innate immune response. Transcriptional induction of the type I IFN genes is mainly regulated by the IFN regulatory factor-3 (IRF-3) and IFN regulatory factor-7 (IRF-7). Sumoylation of IRF-3 and IRF-7 brings about the negative regulation of type I IFN expression . As a rede novo infection or during the reactivation process. It is a potent transcription factor that is necessary and sufficient to trigger the full lytic phase of KSHV. Concomitant with its ability to induce lytic replication and break the virus latency, K-Rta expression remodels the viral chromatin and disassembles PML-NBs [K-Rta is the first KSHV protein expressed after  PML-NBs ,51. K-Rt PML-NBs . K-Rta h PML-NBs ,53 and h PML-NBs . Recentl PML-NBs   within the N-terminal domain and these preferentially bind SUMO-2 ,51 FiguC. Cai etng sites B and sumng sites A. Taken ng sites .vIRF-3 is another KSHV latent cycle gene that is encoded by K10.5. This protein is expressed exclusively in KSHV-infected B cells and is required for the survival of KSHV-infected primary effusion B-cell lymphoma (PEL) . vIRF-3 vPK is an early lytic gene encoded by Orf36. It is a serine/threonine protein kinase that is localized in the nucleus and can be autophosphorylated . K-bZIP Genome-wide profiling has shown that there is distinct dynamic to the chromatin-binding patterns of SUMO paralogs during viral reactivation. The SUMO-1 and SUMO-2/3 binding patterns are very similar in non-reactivated control cells. However, during KSHV reactivation, SUMO-2/3 binding, but not SUMO-1, is significantly increased in the human genome. This enrichment is focused on the promoters region that contain primary regulatory elements such as TFBSs. Annotation of SUMO sites with putative TFBSs identified potential SUMOylated TFs. It shows that potential SUMO-1 target TFs are decreased, while SUMO-2/3 targeting TFs are significantly increased during viral reactivation. Interestingly, three IRFs have been identified as top 10 most potent SUMO-2/3 target TFs after KSHV reactivation. Knockdown of SUMO-2/3 results in a higher induction of most those IRFs-regulated genes analyzed during viral reactivation. These findings suggest that one (or a few) of the KSHV lytic proteins are able to catalyze the sumoylation of these IRFs and attenuate their activity. Expression profiling revealed that these SUMO-2/3 targeted highly transcribed genes show no expression changes during viral reactivation. Gene ontology (GO) analysis showed that these genes are involved in cellular immune responses. Both TF identification and GO analysis indicates that SUMO-2/3 preferentially targets immune-related genes in order to make sure that the expression of these genes remains unaltered during viral reactivation .Sumoylation is a post-translational modification that is both rapid and reversible. Modulation of sumoylation has a profound effect on protein-protein interactions and chromatin assembly. A common property of herpesviruses is that these processes are involved in the biphasic euchromatin-to-heterochromatin transition and this helps to bring about the rapid lytic/latency switch. Most studies have indicated that many chromatin-remodeling proteins are SUMO modified, and such SUMO modified proteins can recruit more SIM containing chromatin-remodeling enzymes that help to regulate chromatin structure. The rapid and reversible character of SUMO modification is able to efficiently affect the local dynamic of euchromatization and heterochromatization. Therefore, this makes sumoylation an interesting process whereby herpesvirus is able to regulate their rapid entry into latency and their rapid reactivation as part of their life cycle.Sumoylation, like phosphorylation, is involved in nearly all cellular processes and viruses have evolved different strategies to manipulate the SUMO process to their own advantage. For herpesviruses, sumoylation is important to at least three steps of viral replication and pathogenesis. These are: firstly, modulation of the viral chromatin during the latent and lytic states; secondly, the disassembly of PML-NBs; and thirdly, the attenuation of interferon responses. To these ends, KSHV encodes at least five proteins that are involved in these processes. They are as follows. Firstly, K-Rta, a lytic gene product with STUbL activity, which preferentially catalyzes the degradation of SUMO-2/3 and sumoylated proteins including PML . Secondl


Pathogens, especially viruses, evolve abilities to utilize cellular machineries to facilitate their survival and propagation. Post-translational modifications (PTMs), especially phosphorylation and SUMOylation, that reversibly modulate the function and interactions of target proteins are among the most important features in cell signaling pathways. PTM-dependent events also serve as one of the favorite targets for viruses. Among the seven unambiguous human oncogenic viruses, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), human papillomavirus (HPV), Human T lymphotrophic virus-1 (HTLV-1), and Merkel cell polyomavirus (MCPyV), two are herpesviruses. The life cycle of herpesviruses consists of latent and lytic phases and the rapid switch between these states includes global remodeling of the viral genome from heterochromatin-to-euchromatin. The balance between lytic replication and latency is essential for herpesvirus to maintain a persistent infection through a combination of viral propagation and evasion of the host immune response, which consequently may contribute to tumorigenesis. It is no surprise that the swift reversibility of PTMs, especially SUMOylation, a modification that epigenetically regulates chromatin structure, is a major hijack target of the host for oncogenic herpesviruses. In this brief review, we summarize the varied ways in which herpesviruses engage the host immune components through PTMs, focusing on phosphorylation and SUMOylation. Post-translational modifications are reversible events that hold the key for the determination of protein function and increase the functional diversity of proteins. Phosphorylation, one of the most well-studied PTMs, regulates signal transduction through recruitment of different functional partners. Tyrosine phosphorylation recruits Src homology 2 (SH2) or phosphotyrosine binding (PTB) domain-containing proteins to propagate signaling events. Phospho-serine/threonine can be recognized by protein modules including 14-3-3 proteins, WW domains, WD40 repeats and the BRCT motif. Small ubiquitin-like modifier (SUMO) is another important PTM that was initially identified as an antagonist of ubiquitin (Ub). Ub is a small peptide that was the first identified protein in Ub-like (UBL) proteins family and labels protein through a conjugation system comprising E1 activation, E2 conjugation, and E3 ligation enzymes. Proteins carrying Lys-48 poly-Ub chains are targeted to the proteasome and degraded . ProteinMany of the important immunomodulatory proteins and chromatin remodeling molecules are functionally dependent on and regulated by PTMs, primarily phosphorylation and SUMOylation, respectively. Many cellular antiviral defense systems recognize DNA viruses and limit their replication and spread. It is not surprising that oncogenic herpesvirus, including Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), encode proteins that control cellular PTM pathways to help their life cycle and evade host immune responses [reviewed in ]. The obOncogenic herpesviruses include EBV and KSHV. EBV mainly infects B cells and causes lymphomas. KSHV infection is associated with Kaposi’s sarcoma (KS), primary effusion lymphomas (PEL), and multicentric Castlemen’s disease . HerpesvFigure 1-I).In response to DNA viruses, endosome-bound TLR9 induces a MyD88-dependent signaling with MyD88 forming complexes with IRAK-1, IRAK-4 and interferon regulatory factor-7 (IRF-7), which mediate the activation of type I interferon (IFN) pathway. Similar to other transcription factors (TFs), IRF-7 undergoes different PTMs, including phosphorylation, ubiquitylation, and SUMOylation. There are several examples of herpesvirus proteins targeting host IRFs. Open reading frame 45 (Orf45) of KSHV competitively inhibits IRF-7 phosphorylation, which is essential for IRF-7 transactivation activity . KSHV reFigure 1-II). Other inhibitory mechanisms employed by EBV which affect innate immunity via phosphorylation includes latent membrane protein 1 (LMP-1) interaction with non-receptor tyrosine kinase 2 (Tyk2); an interaction that inhibits Tyk2 phosphorylation and consequently prevents activation of IFN-α signaling , there is currently no evidence to indicate that IFI16 acetylation is targeted by herpesviral proteins. In addition to viral DNA, DNA virus encoded RNA can also be recognized by RNA sensors such as RIG-I, which mediates the type I IFN pathway against viral infection. RIG-I-mediated signaling is controlled by ubiquitylation of RIG-I. Orf64 encoded by KSHV is a tegument protein with deubiquitinase (DUB) activity. Orf64 suppresses RIG-I-mediated IFN signaling by reducing the ubiquitylation of RIG-I . This regulation is also observed in non-oncogenic herpesvirus. For example, Orf63 encoded by α-herpesvirus varicella zoster virus (VZV), the causative agent of chickenpox, inhibits type I IFN signaling through proteasome-dependent IRF-9 degradation (Cytosolic exogenous double-stranded DNA (dsDNA) is recognized by and triggers both Z-DNA binding protein 1/DNA-dependent activator of IFN-regulatory factors (ZBP1/DAI) and cGAMP synthase (cGAS) signaling that ultimately activates IRF-3-dependent type I IFN response. ZBP1, the first cytosolic dsDNA sensor, recruits TANK-binding kinase 1 (TBK1) and IFN regulatory factor-3 (IRF-3), with the consequent induction of type I IFN genes . cGAS, aignaling . Cytosolignaling  . EBV EBNA1, one of the nuclear proteins of EBV that is expressed during both latent and lytic phases, interacts with protein kinase CK2 and consequently increases PML phosphorylation, and subsequent polyubiquitylation and degradation . KSHV immediate-early protein K-Rta has recently been identified as a SUMO-Targeting Ubiquitin Ligase (STUbL) that degraded SUMOylated PML and dispersed PML-NBs during the viral lytic cycle . Emerging evidence suggests that, in addition to silencing viral gene transcription, PML functions as a co-activator of the type I IFN pathway  or PML oncogenic domains (PODs), target herpesviruses by inhibiting viral transcription and replication. PML-NB is a highly SUMO-modified nuclear compartment that is permanently composed of PML, hDaxx, and Sp100. Other factors can only be identified at PML-NB under certain unique conditions. Soon after viral DNA enters the nucleus, PML-NB rapidly associate with viral genomes in a SUMO-dependent manner. It is thus not surprising that herpesviruses have evolved mechanisms to target the SUMO machinery as a means to counteract the PML-NB-mediated antiviral response. Here, we focus on reviewing permanent PML-NB components but also include few important PML-NB variable proteins, such as ATRX that are targeted by oncogenic herpesviruses. Modification of PML by SUMO is a prerequisite for proper PML-NB formation. EBV BZLF1, also known as Zta, is SUMO modified and competitively reduces the SUMOylation of PML and consequently induces PML-NB disruption  . Phosphorylation of KAP-1 by vPK reduces KAP-1 SUMOylation and subsequently decreases the binding ability of KAP-1 on chromatin . MoreoveCurrent knowledge of the mechanisms involved in viral infection and tumor progression is still largely unknown. Emerging evidence shows the importance of immune response in cancer. Here, we summarize how the host cells use PTMs, especially phosphorylation and SUMOylation, to regulate many important immunomodulatory proteins. To survive in the host, viruses express different proteins that target PTMs and consequently repress host immune defenses. The potential mechanisms used by oncogenic herpesvirus, including EBV and KSHV, are depicted. Studying the interplay between virus and host immune system will provide novel insight for development of future therapeutic intervention in cancer.PCC and MC were involved in writing and editing of the review. ER was involved in paper construction and writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation. During gastrulation in amniote organisms, epiblast cells undergo an epithelial-to-mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm cell layers. EMT is driven by large-scale changes in gene expression involving the downregulation of the epithelial phenotype, including apical basal polarity, in epiblast cells and upregulation of genes that confer migratory behavior, and front-back polarity, to the emerging mesoderm , 2. CellAlthough diverse signaling pathways can regulate EMT depending on the cellular context , activatDrosophila embryos lacking Snail fail to form a ventral furrow and are deficient in mesoderm [shg) or N-cad , or both, has any effect on formation of the ventral furrow, germ layer formation or mesoderm spreading [Although E-cad downregulation and the accompanying reduction in adhesion have often been considered a prerequisite for EMT –14, recemesoderm , neitherpreading . Similarpreading . In factCDH3 gene that is adjacent to the gene coding for E-cad (CDH1). P-cad and E-cad share identical cytoplasmic domains, and so both are recognized by the commercially available E-cad antibodies used in published studies. The high sequence similarity between E-cad and P-cad suggest some functional redundancy, though this has not been experimentally tested.Protein localization studies in gastrulating chicken embryos also suggest that downregulation of epithelial cadherins is not a prerequisite for EMT. Although E-cad mRNAs expressed in the epiblast are lost as cells approach the primitive streak, immunofluorescence analyses using antibodies recognizing the intracellular domain of E-cad have reported robust protein levels on the surface of cells as they undergo EMT , 2. HoweHere we investigate the relationship between E-cad and P-cad expression, Snail function and EMT during gastrulation in chicken embryos. We find that P-cad is retained on the surface of cells undergoing EMT in the primitive streak and after they emerge in the mesoderm, while evidence suggests that E-cad is downregulated prior to the onset of EMT. Overexpression of E-cad in chicken embryos did not alter the ability of epiblast cells to undergo EMT. Furthermore, gain and loss of function studies suggest that Slug (SNAI2) is neither required for gastrulation EMT nor sufficient to induce EMT in the epiblast.Gallus gallus) eggs  were incubated at 37°C with high humidity until embryos reached Hamburger-Hamilton (HH) stage 3 to 4 [Fertile chicken ; rabbit anti-FLAG ; mouse anti-E-cadherin ; mouse anti-N-cadherin ; rabbit anti-laminin ; rabbit monoclonal anti-Slug ; rabbit ZO1 ; rabbit anti-Myc monoclonal ; mouse anti-c-Myc ; mouse anti-GM130 ; mouse anti-fluorescein ; and anti-p120-catenin . The E-cadherin antibody was prepared against the intracellular domain that is 100% identical to the intracellular domain of P-cadherin, and so recognizes both proteins. An E-cadherin-specific antibody could not be identified, and attempts to produce one were not successful (see below). The following secondary antibodies were obtained from Invitrogen and used at 1:500 dilution: Alexa fluor 488 conjugated goat anti-rabbit; Alexa fluor 488 conjugated goat anti-mouse IgG1; Alexa fluor 488 conjugated goat anti-mouse IgG2a; Alexa fluor 594 conjugated goat anti-rabbit; Alexa fluor 594 conjugated goat anti-mouse IgG1; Alexa fluor 594 conjugated goat anti-mouse IgG2a; Alexa fluor 647 conjugated goat anti-rabbit; Alexa fluor 647 conjugated goat anti-mouse IgG1; Alexa fluor 647 conjugated goat anti-mouse IgG2a.GRVESCAQKPRVDTGVP corresponding to amino acids 541–557 of the extracellular domain that shows only 29% identity to the corresponding sequence in E-cad. E-cadherin-specific antibodies were prepared using the peptides LKKELEPGEYNIFVKL and YETKSRYDLVVTVENKV, corresponding to amino acids 659 to 673 and 450 to 467, respectively, of the extracellular domain. Although the resulting affinity purified antibodies recognized the corresponding E-cad antigen peptides, attempts to use these antibodies to detect E-cad protein by immunocytochemistry were unsuccessful.For the detection of P-cadherin protein, an affinity-purified antibody was generated in rabbit using the peptide TGGCATTTTGAAGGCAGGCTTTCTC) targeting the initiation of translation of the SNAI2 mRNA, or a five base pair mismatch control morpholino (GeneTools), were electroporated at a concentration of 0.5uM along with 0.5ug/ml of carrier plasmid. Following electroporation, embryos were incubated for 3–16 hours before fixation and processing for immunohistochemistry and/or in situ hybridization. For immunofluorescence, embryos were fixed for 2 hours at R.T. in freshly prepared 4% paraformaldehyde, dehydrated through methanol and stored at -20°C overnight. After rehydration, embryos were blocked in 5% goat serum in PBST for 1 hour at room temperature before incubation overnight at 4°C with primary antibody diluted in blocking solution. Embryos were then washed extensively and incubated overnight at 4°C with the appropriate secondary antibody conjugated to Alexa fluor 488, Alexa fluor 594 or Alexa fluor 647. For In situ Hybridization (ISH), embryo processing, antisense RNA probe preparation and whole-mount ISHs were performed as described [Electroporation and subsequent immunohistochemistry were carried out essentially as described , 20 usinCell localization in the epiblast, primitive streak, and mesoderm was analyzed as previously described . SignifiCDH1) and the appearance of N-cad (CDH2) [SNAI2; CDH3 gene, which is adjacent to and shows high sequence similarity to CDH1. The intracellular domains of P-cad and E-cad are identical, while the extracellular domains have diverged. P-cad transcripts are also detected in the epiblast, but additionally are evident in the primitive streak and in newly formed mesoderm cells (The movement of cells from the epiblast to the mesoderm via an EMT has been correlated with the loss of E-cad CDH and the d (CDH2) . In chicrm cells .All available E-cad antibodies have been prepared using sequences from the cytoplasmic domain as immunogens, and so recognize both E-cad and P-cad. To examine the relative localization of P-cad and E-cad, antibodies were generated against extracellular domain peptides that showed low identity to the corresponding peptide in the other protein. While a P-cad specific antibody was obtained that is useful for immunocytochemistry, we were unable to generate an E-cad specific antibody that could visualize the protein in cells.Immunolocalization of cadherin proteins using the P-cad specific antibody and an intracellular domain antibody that recognizes P-cad and E-cad is shown in An antibody specific for N-cad was used to determine the relative localization of E-cad/P-cad (using the intracellular domain antibody that recognizes both proteins) and N-cad. N-cad is detected weakly in cells of the dorsal and mid streak and more robustly in mesoderm cells away from the primitive streak . The relTo determine whether over expression of E-cad could inhibit EMT, the epiblast was electroporated with an E-cad expression construct. Cells expressing high levels of E-cad were observed undergoing EMT and residing in the mesoderm .SNAI2) is present in the nuclei of cells in the primitive streak and the mesoderm of avian embryos . SNAI1 transcripts were not detected in cells containing SNAI2 morpholino (data not shown). Zeb1 and 2 are expressed in the avian primitive streak (http://geisha.arizona.edu), and so it is possible that in the absence of Slug protein (SNAI2), Zeb1 and/or Twist2 could function redundantly to enable EMT.Almost all cells containing the els Figs . Morpholels Figs , includiels Figs  and rounels Figs . SNAI2 mrpholino , suggestSNAI1 (6SAhSNAI1) that shows strong nuclear localization and an enhanced ability to induce EMT in a variety of cell types [Published studies have reported conflicting results regarding the ability of the Snail transcription factors to induce EMT in gastrula stage chicken epiblast , 26. To ll types , or wildll types , previouRegardless of embryo stage at the time of electroporation or the duration of the experiment, cells expressing 6SAhSNAI1 remained within the epiblast , Table 1CDH1 (E-cad) and CDH3 (P-cad), EMT, and Slug function during chick gastrulation. We find that P-cad is present at robust levels on the surface of cells in the epiblast, primitive streak and early mesoderm, diminishing only once mesoderm cells have migrated away from the streak. Although antibodies that can specifically recognize E-cad are not available, E-cad mRNAs are present in the epiblast but not in the primitive streak or mesoderm. Assuming that E-cad protein distribution roughly corresponds to its mRNA distribution, these findings are consistent with the possibility that E-cad protein is diminished or absent in the primitive streak and mesoderm, while P-cad protein persists as cells undergo EMT and emerge into the mesoderm. The persistence of E-cad and/or P-cad around the periphery of newly formed mesodermal cells is also observed in mouse embryos  was first identified in avian embryos as a regulator of cell behavior during gastrulation and neural crest formation [SNAI1) directly repress transcription of E-cad (CDH1) and other genes involved in maintaining the epithelial phenotype. A model therefore arose in which EMT was triggered by the transcriptional activation of SNAI1 and/or SNAI2, leading to the loss of E-cad followed by the onset of EMT. Additional transcriptional repressors, including Twist and Zeb, have been shown to combine with Snail proteins to repress the epithelial phenotype. In the SNAI1 knockout mouse, , epiblast cells undergo gastrulation to form a mesoderm cell layer, though the mesoderm cells retain some epithelial characteristics including a polarized phenotype and intercellular junctions [SNAI2 mRNA. Although Slug protein levels were undetectable in most morpholino-containing cells, the movement of cells from the epiblast to the mesoderm through the primitive streak was not inhibited. Mesoderm cells lacking detectable Slug protein showed reduced ability to migrate into the lateral mesoderm, though morphologically they appeared indistinguishable from control cells.The transcription factor Slug  to redistribute to the entire cell surface, reducing intercellular adhesion. Although mechanisms regulating the loss of adherens junctions are not fully defined, Nakaya et al have shoS1 Fig The same microscopic field showing lateral epiblast cells expressing GFP (green) and E-cad mRNA (purple).  Epiblast cells expressing C3 had migrated into the mesoderm and had downregulated E-cad mRNAs.  Epiblast cells expressing 6SAhSNAI1 remained in the epiblast (F) but showed greatly reduced levels of E-cad mRNAs (brackets in E).(TIF)Click here for additional data file.


Investigators from the Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California and a large study group utilized a combination of exome sequencing, targeted gene panels, and Sanger sequencing to identify thirty-one pathogenic variants in thirty-nine affected individuals with ALG1-CDG from 32 families. Investigators from the Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California and a large study group utilized a combination of exome sequencing, targeted gene panels, and Sanger sequencing to identify thirty-one pathogenic variants in thirty-nine affected individuals with ALG1-CDG from 32 families. Congenital disorders of glycosylation (CDGs) cause impaired synthesis of glycoconjugates and are genetically heterogeneous with pathogenic variants in over one hundred genes that can cause it. ALG1 mutations cause a rare autosomal recessive disorder called ALG1-CDG. Of the thirty-one pathogenic variants reported, twenty-two (65%) are novel variants. This study has tripled the number of known patients with ALG1-CDG.Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. ALG1-CDG patients are known to accumulate a novel N-linked xeno-tetrasaccharide. Since this tetrasaccharide does not normally occur in mammals and is primarily detected in ALG1-CDG cases, it serves as a biomarker for either detecting or confirming a diagnosis of ALG1-CDG. This biomarker was identified in all 27 patients tested.This study also expands the clinical phenotype of ALG1-CDG patients. All ALG1-CDG patients have substantial neurological involvement; however other systems are variably affected. Several factors may cause this variability, including how ALG1 mutations affect residual mannosyltransferase activity or GDP-Man binding efficiency, complex formations with other proteins, tissue specific expression, or even protein stability. [COMMENTARY. Congenital disorders of glycosylation are a group of predominantly autosomal recessive diseases with multi-system affects ranging from developmental delay and hypotonia with multiple organ system involvement to hypoglycemia and enteropathy . CongeniThe clinical features in this group of 39 patients included developmental delay, hypotonia, epilepsy, microcephaly, cerebral and cerebellar atrophy, strabismus, dysmorphic facial features, gastrointestinal abnormalities and hypoalbuminemia . Thus a


High n-6 PUFA intake decreased very low-density lipoprotein (VLDL) cholesterol and triglyceride concentrations by up-regulating VLDL lipolysis and uptake. In a study of healthy subjects, the intake of saturated fatty-acids with increased palmitic acid at the sn-2 position was associated with decreased postprandial lipemia. Low medium-chain triglyceride may not appreciably alter TRL metabolism. Replacing carbohydrate with monounsaturated fatty-acids increased TRL catabolism. Trans-fatty-acid decreased LDL and enhanced high-density lipoprotein catabolism. Interactions between APOE genotype and n-3 PUFA in regulating lipid responses were also described. The major advances in understanding the effect of dietary fatty-acids on lipoprotein metabolism has centered on n-3 PUFA. This knowledge emphasizes the importance of regulating lipoprotein metabolism as a mode to improve plasma lipids and potentially CVD risk. Additional studies are required to better characterize the cardiometabolic effects of other dietary fatty-acids.Dyslipidemia is a major risk factor for cardiovascular disease (CVD). Dietary fatty-acid composition regulates lipids and lipoprotein metabolism and may confer CVD benefit. This review updates understanding of the effect of dietary fatty-acids on human lipoprotein metabolism. In elderly participants with hyperlipidemia, high  Nutritional and lifestyle modifications are the frontline for the treatment of dyslipidemia to minimize and lower CVD risk. Current dietary recommendations focus on fatty acids and include reductions in saturated and ty acids . A betteRecent advances in understanding the impact and use of dietary fatty acids in cardiometabolic medicine have focused on polyunsaturated fatty acids (PUFA), and reviewed below.n-3 PUFA, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Compelling evidence suggest that n-3 PUFA reduces CVD risk and this is partly mediated by its potent triglyceride-lowering effects [n-3 PUFA has been shown to reduce triglyceride synthesis via inhibition of diacylglycerol acyltransferase, fatty acid synthase and acetyl CoA carboxylase enzymes [n-3 PUFA also enhance fatty acid β-oxidation via a peroxisome proliferator-activated receptor (PPAR)-α mediated pathway resulting in decreased substrate availability for triglyceride formation [n-3 PUFA suppress transcription of sterol regulatory element-binding protein-1c gene and hence, inhibit de novo lipogenesis [n-3 PUFA may also stimulate the post-endoplasmic reticulum presecretory proteolysis pathway, thereby increasing the degradation of newly synthesized apolipoprotein (apo) B [in vivo studies. We observed that a high-fish diet (1.23 g/day EPA and DHA) decreased non-fasting plasma triglycerides, with concomitant reductions in triglyceride-rich lipoprotein (TRL) apoB-100 concentration and production rate, compared with a low-fish diet in elderly men and women with moderate hyperlipidemia [in vitro and animal studies showing that n-3 PUFA suppressed TRL apoB-48 synthesis by either decreasing apoB-48 mRNA expression and/or increasing post-translational degradation of newly synthesized apoB-48 [n-3 PUFA on TRL apoB-48 metabolism [n-3 PUFA supplement compared with weight loss alone on TRL apoB-48 metabolism in a postprandial, non-steady state setting. The study was a 12-week, randomized intervention trial of a hypocaloric diet (energy deficit of ≥1900 kJ) and n-3 PUFA supplement  compared with a hypocaloric diet alone on TRL apoB-48 metabolism in obese men and women following ingestion of an oral fat load  [n-3 PUFA supplement decreased fasting and total postprandial TRL apoB-48 area under the curve (AUC) compared with weight loss alone. These changes were chiefly due to a significant reduction in basal TRL apoB-48 secretion rate, without a significant effect during the postprandial period [Fish oils are a rich source of the long-chain  effects ,3,4. n-3 (apo) B . These mipidemia   compared with medium-chain fatty acid  supplementation on fasting lipoprotein profile and metabolism in 12 overweight-obese men using a randomized, double-blind crossover study design [n-6 PUFA lowered fasting total, very low-density lipoprotein (VLDL) and LDL cholesterol, and total plasma, VLDL and LDL triglyceride concentrations compared with MCFA. Using a combination of the Particle Profiler computational model and compartmental modeling, the authors reported that the uptake to production ratio, but not lipolysis to production ratio, in VLDL was significantly higher with n-6 PUFA compared with MCFA supplementation [n-6 PUFA. The authors attributed this to an up-regulation of lipoprotein lipase (LPL) activity and hepatic uptake of VLDL, consequent on PPAR-activation by n-6 PUFA [n-6 PUFA diet on CVD outcomes also merits investigation.y design   intake on lipoprotein metabolism in humans. Early radioisotope studies demonstrated that decreasing SFA, together with increasing tabolism ,17 , compared with 20 g corn oil, on TRL apoB-100 and apoB-48 metabolism in obese, insulin resistant men [The length of the SFA may also have differential effects on lipoprotein metabolism. Saturated fats may be sub-classified into short-chained (<6 carbon atoms), medium-chained (8–10 carbon atoms) and long-chained fatty acids (>12 carbon atoms). In a randomized crossover study, Tremblay tant men   on lipoprotein metabolism in humans. Zheng  did not   on lipoprotein metabolism. Five weeks consumption of TFA derived partially hydrogenated fats was associated higher total and LDL cholesterol, and lower HDL cholesterol concentrations in post-menopausal women [To date, only one study has examined the impact of al women . These cal women . Long- tal women ,24. APOE is one of the more extensively investigated genes in CVD and APOE genotypes, E2, E3 and E4 are viewed as key genetic determinants of inter-individual variations in postprandial lipemia. In keeping with this concept, Liang et al. explored the interactions between APOE genotypes and plasma phospholipid EPA and DHA on plasma lipid and lipoprotein profiles in a cross-sectional analysis of 2340 Multi-Ethnic Study of Atherosclerosis (MESA) participants [APOE and EPA and DHA interactions with small-LDL concentration. Specifically, significant and positive associations were observed between EPA and DHA with small-LDL concentration in participants with E4, but not with E2 and E3. Therefore, the authors proposed that the interaction with LDL chiefly reflects a synergistic effect of the APOE E4 genotype and n-3 PUFA on LDL metabolism [APOE and phospholipid EPA interactions with HDL cholesterol and the concentrations of large and total HDL particles [APOE E2 genotype and EPA on HDL metabolism [n-3 PUFA supplementation, but whether this is secondary to plasma triglyceride-lowering is not known [APOE genotype and lipid responses [APOE genotype and/or other genotypes on lipoprotein metabolism with dietary interventions.icipants . The auttabolism . There itabolism . On balaarticles . Specifitabolism . The undot known . In addiot known , no studesponses . Furthern-3 PUFA. This knowledge provides insights into the importance of regulating lipoprotein metabolism as a means to improve the plasma lipid profile and lower CVD risk. Further studies are required to better understand the cardiometabolic effects of other dietary fatty acids. As part of this, consideration should be given to study design, dose and duration of intervention, and choice of food or nutrients substituted and those displaced, in order to optimally test a proposed hypothesis. Importantly, a holistic multi-nutrient approach, in particular healthy dietary patterns such as the Mediterranean diet, in place of individual nutrient substitution should also be applied and studied. This approach integrates the complexity of diet and potential interactions between nutrients and bioactive components and may further inform guidelines for CVD health and risk reduction. The major advances in understanding the mechanisms of action of dietary fatty acids on lipoprotein metabolism have focused on


Investigators from Rothschild Foundation Hospital in Paris report language outcome in children after (left) dominant-hemisphere hemispherotomy for Rasmussen Syndrome. Investigators from Rothschild Foundation Hospital in Paris report language outcome in children after (left) dominant-hemisphere hemispherotomy for Rasmussen Syndrome. Six children underwent surgery at ages 4.1 to 8.4 years. Preceding duration of epilepsy was short, mean of 1.2 years. Four experienced pre-operative aphasia with decreased verbal comprehension indices (VCI) (4/6) and performance reasoning index (5/6). Mean duration of follow-up was 5.6 years. Longitudinal follow-up of cognition and speech is reported. Post-operatively VCI normalized over 3-8.3 years. The authors describe a specific linguistic profile: normal verbal comprehension, and weakness of grammatical judgment, word repetition, statement production, semantic verbal fluency and metaphonological abilities. Working memory scores were also weak, though not meeting statistical significance.The authors suggest this study emphasizes the ability of the right hemisphere to functionally reorganize language over a long period of time following surgery, support decision making toward hemispherotomy in left-sided Rasmussen syndrome and can aid post-operative follow-up and rehabilitation strategies. COMMENTARY. Rasmussen syndrome is a rare chronic progressive neurological disease of presumed immune basis characterized by unilateral inflammation and atrophy of a cerebral hemisphere, drug-resistant focal epilepsy, progressive hemiplegia and cognitive decline . To dateCareful pre-operative neuropsychological testing and in the cooperative child language functional MRI can lateralise language, making Wada testing, long deemed the gold-standard, no longer essential. Interval imaging may show transfer/re-localisation of language dominance to the contra-lateral hemisphere over the disease course, giving the epilepsy surgery team more confidence in the prospects of post-hemispherotomy language recovery and more confidence to proceed. Earlier onset seizures may increase the chances of this transfer but it is not known if earlier surgery could better drive this at a younger age with more retained neural plasticity, or whether it is better to allow function to become established naturally in the unaffected hemisphere, accepting that cognitive decline proceeds inexorably .The present study is encouraging; all children regained speech and long-term follow-up demonstrated on-going language recovery. Rehabilitation is essential after surgery and the patterns of language recovery delineated here can also inform therapy programs and goal-setting. Further, the parallel to the cognitive gains 5-years out from temporal lobe surgery, remind us the importance of long-term follow-up studies in informing our decision-making in the acute disease setting .


Single nucleotide polymorphisms (SNPs) located in microRNA (miRNA) genes (miR-SNPs) have attracted increasing attention in recent years due to their involvement in the development of various types of cancer. Therefore, a systematic review on this topic was needed. From 55 scientific publications we collected 20 SNPs, which are located within 18 miRNA encoding genes and have been associated with 16 types of cancer. Among 20 miRNA gene polymorphisms 13 are located within the premature miRNA region, five within mature, and two within mature seed miRNA region. We graphically visualized a network of miRNA-cancer associations which revealed miRNA genes and cancer types with the highest number of connections. Our study showed that, despite a large number of variations currently known to be located within miRNA genes in humans, most of them have not yet been tested for association with cancer. MicroRNA SNPs collected in this study represent only 0.43% of known miRNA gene variations (20/4687). Results of the present study will be useful to researchers investigating the clinical use of miRNAs, such as the roles of miRNAs as diagnostic markers and therapeutic targets. MicroRNAs (miRNAs) are a class of non-protein coding RNAs that post-transcriptionally regulate expression of the target mRNAs . During Single nucleotide polymorphisms (SNPs) located in miRNA encoding gene (miR-SNPs) have recently come into focus regarding their possible role in the development of cancer. It is known that miR-SNPs can influence the transcription of the target gene, alter the processing of pri-miRNA or pre-miRNA and affect interaction between miRNA and mRNA . MoreoveSeveral meta-analyses related with a specific type of cancer or a couple of related types of cancer  have alLiterature mining was performed to systematically review published miR-SNPs associated with cancer. Seventy-five collected associations between miRNA and types of cancer were then visualized as a network in order to identify hubs with a higher number of connections. Workflow of the study and main results are shown in hsa-mir-146a and hsa-mir-196a-2, which were described in 13 and 14 individual studies, respectively.A publication search was performed in order to create a list of cancer-associated SNPs, which are located within miRNA encoding genes. Among 148 papers collected, 93 were not included in the analysis due to exclusion criteria. The literature analysis of 55 publications, including 16 meta-analyses that involved 70 papers, revealed 75 associations between miR-SNPs and cancers types. The review revealed 20 SNPs within 18 miRNA encoding genes which have been associated with 16 different types of cancer. Details regarding 75 associations are presented in the A total of 75 associations between miRNA and cancers were visualized as a network as shown in hsa-mir-196a-2) [hsa-mir-608) [hsa-mir-423) [hsa-mir-27a) [hsa-mir-149) [hsa-mir-202) [hsa-mir-196a-2 (8), hsa-mir-149 (7), and hsa-mir-146a (6). Identified hubs could be important for further studies investigating the role of miRNA in cancer.Among 75 SNP-cancer associations, 50 present increased and 15 decreased risk of a particular cancer. The remaining ten associations include six miR-SNPs associated with a risk role in overall survival in both gastric cancer and non-small cell lung cancer in Asian patients rs11614913; -196a-2) , increas-196a-2)  and decrmir-423) , better mir-27a) , improve; -196a-2We also gathered information on ethnic origin and the size of tested population (number of cases and controls) . More thin silico screening of the human genome revealed a large number of polymorphisms located within miRNA encoding genes (n = 4687). Collected miRNA SNPs in this study (n = 20) represent only 0.43% of all short variations currently known to be located in human miRNome. Most miRNA variations have not yet been tested for associations with phenotypic variability and disease development. MicroRNA polymorphisms could now be tested for association with cancer, particularly those located within both miRNA genes and cancer-associated protein-coding host genes. Among 18 miRNA genes, included in this review, ten genes are located within introns of protein-coding genes. Six miRNA genes overlap non-coding transcriptional units: four miRNA genes overlap regions encoding for ncRNAs , one overlaps another miRNA gene located on the reverse strand, and one overlaps a non-coding host gene located on the reverse strand . Instead, we suggest that scientists expand the research to a large number of untested miR-SNPs. The majority of published polymorphisms within miRNAs are located either in pre-mature or mature regions, whereas only two are located within the mature seed region. The impact of SNPs, particularly those located within the mature seed region, should also be explored. Collected miRNA SNPs could be now integrated into various bioinformatics tools; for instance, our previously developed bioinformatics tool miRNA SNiPer .A list of miRNA SNPs was obtained using miRNA SNiPer tool, version 5 ,25, whicIn conclusion, the present literature analysis provides an overview of miRNA polymorphisms associated with cancer and offers suggestions for further research in this area. Studies of miRNA polymorphisms play an important role in the field of cancer research and will be even more significant in the future. The present list of cancer associated SNPs located in miRNA genes will be useful to researchers investigating clinical use of miRNAs such as the roles of miRNAs as diagnostic markers and therapeutic targets.


Cilia are microtubule based organelles enveloped by a specialised extension of plasma membrane. Serving as cellular antennae, cilia are essential for perceiving extracellular cues and mediating downstream signal transduction crucial for normal cellular physiology and development. These functions are mediated in large part by the specialised ciliary membrane, which differs in composition from the plasma membrane, housing many of the receptors, channels and effectors that mediate cilium-based signalling. However, the molecular mechanisms underlying the establishment, maintenance and regulation of the ciliary membrane remain poorly understood.Caenorhabditis elegans ciliated sensory neuronal model, employing assays for cilium integrity (dye filling assay) and functionality .In this project we are seeking to identify and functionally characterize genes with ciliogenic roles in ciliary membrane transport and organisation. Specifically, we are taking a candidate gene approach using the rab-5-related endocytic gene mutants with defects in cilium integrity and function. One of these, rabs-5, encodes a RAB-5 effector, which we found is expressed mainly in ciliated cells. rabs-5 mutants possess short cilia and an expanded periciliary membrane compartment, indicative of defects in ciliary membrane homeostasis. Transgenic expression of a rabs-5(WT) construct rescues the cilium integrity and foraging defects of rabs-5 mutants, confirming the association of rabs-5 with cilia-related functions.Using predicted loss-of-function alleles, we screened 55 mutants of evolutionarily conserved membrane organising and trafficking genes and identified 4 C. elegans, a number of RAB-5-dependent downstream effectors have been identified as potential regulators of ciliogenesis and ciliary membrane homeostasis.By taking a reverse genetics candidate gene approach in