Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially in vivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.

Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic dsDNA helix could be advantageous for cell replication such as bacterial binary fission and cell mitosis without the need for topoisomerase or helicase to consume additional energy.

We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function - sensing when the correct amount of DNA has been packaged. The three-dimensional structure of the gp6 SizA mutant, which packages a smaller chromosome, reveals significant differences in that region.

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.

Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well-established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling, we demonstrate that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 nonredundant genes that significantly discriminate the tumor stroma from nontumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix, and transforming growth factor beta (TGFβ) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increased expression of collagen 4A1/COL4A1, laminin gamma 2/LAMC2, osteopontin/SPP1, KIAA0101, and TGFβ2 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of osteopontin, TGFβ2, and laminin in the stroma of ICC was significantly correlated with overall patient survival. More important, multivariate analysis demonstrated that the stromal expression of osteopontin was an independent prognostic marker for overall and disease-free survival.

CONCLUSIONS

The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidate biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression.

Many DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids. Concatemerization arises from either replication or homologous recombination. Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system. The nuclease component is related to the exonuclease of phage lambda and is common to viruses with linear double-stranded DNA genomes. To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized 34.1 gene from Bacillus subtilis phage SPP1, expressed it in Escherichia coli, purified the protein, and determined its enzymatic properties. Like lambda exonuclease, Chu (the product of 34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg(2+)-dependent reaction, and shows a preference for 5'-phosphorylated DNA ends. A model for viral recombination, based on the phage lambda Red recombination system, is proposed.

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression.

Hepatocyte-specific Pten deficient (Pten KO) mice possess almost the same hepatic lesions histologically as human NASH and are thought to represent some limited NASH patients. We analyzed a comprehensive gene expression of hepatocytes derived from 10- to 35-week-old Pten KO mice using the DNA microarray technology to find out the candidate genes related to development and aggravation of human NASH. Spp1, Vnn1, Itga6, Abcd2, Auh, Acox1, Pdk4, Cpt1a, Lcn2, Igfbp2, Gstm6, Socs3, Tgm2, and Aldh9a1 were regarded as the candidate genes related to inflammation. The candidate genes of fibrosis were Spp1, Ctgf, and Cyp2c39 and moreover Cidec and Spp1 were regarded as the candidate genes of carcinogenesis. To confirm that these genes contribute to the etiology of some human NASH, further investigations using human liver samples are needed.

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and are involving in diverse cellular processes. However, their roles in reprogramming of gene expression profiles during lineage commitment and maturation of mesenchymal stem cells (MSCs) remain poorly understood. In the current study, we characterize the expression of a lncRNA, HoxA-AS3, during the differentiation of MSCs. We showed that HoxA-AS3 is increased upon adipogenic induction of MSCs, while HoxA-AS3 remains unaltered during osteogenic induction. Silencing of HoxA-AS3 in MSCs resulted in decreased adipogenesis and expression of adipogenic markers, PPARG, CEBPA, FABP4 and ADIPOQ. Conversely, knockdown of HoxA-AS3 expression in MSCs exhibited an enhanced osteogenesis and osteogenic markers expression, including RUNX2, SP7, COL1A1, IBSP, BGLAP and SPP1. Mechanistically, HoxA-AS3 interacts with Enhancer Of Zeste 2 (EZH2) and is required for H3 lysine-27 trimethylation (H3K27me3) of key osteogenic transcription factor Runx2. Our data reveal that HoxA-AS3 acts as an epigenetic switch that determines the lineage specification of MSC.

Tailed icosahedral bacteriophages and other viruses package their double-stranded DNA inside a preformed procapsid. In a large number of phages packaging is initiated by recognition and cleavage by a viral packaging ATPase (terminase) of the specific pac sequence (pac cleavage), which generates the first DNA end to be encapsidated. A sequence-independent cleavage (headful cleavage) terminates packaging, generating a new starting point for another round of packaging. The molecular mechanisms underlying headful packaging and its processivity remain poorly understood. A defined in vitro DNA packaging system for the headful double-stranded DNA bacteriophage SPP1 is reported. The in vitro system consists of DNA packaging reactions with highly purified terminase and SPP1 procapsids, coupled to a DNase protection assay. The high yield obtained enabled us to quantify directly the efficiency of DNA entry into the procapsids. We show that in vitro DNA packaging requires the presence of both terminase subunits. The SPP1 in vitro system is able to efficiently package mature SPP1 DNA as well as linear plasmid DNAs. In contrast, no DNA packaging could be detected with circular DNA, signifying that in vitro packaging requires free DNA extremities. Finally, we demonstrate that SPP1 in vitro DNA packaging is independent of the pac signal. These findings suggest that the formation of free DNA ends that are generated by pac cleavage in vivo is the rate-limiting step in processive headful DNA packaging.

Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

BACKGROUND

We have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features.

RESULTS

Here we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue.

CONCLUSIONS

In summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.

An understanding of the genetic control of porcine female reproductive performance would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing three to five more viable piglets per litter than the European Large White breed. This difference in prolificacy is attributed to the Meishan's superior prenatal survival levels. The present study utilized a three-generation cross in which the founder grandparental animals were purebred Meishan and Large White pigs in a scan for quantitative trait loci (QTL) on porcine chromosome 8 (SSC8) associated with reproductive performance. Reproductive traits, including number of corpora lutea (ovulation rate), teat number, litter size, and prenatal survival, were recorded for as many as 220 F2 females. Putative QTL for the related traits of litter size and prenatal survival were identified at the distal end of the long arm of SSC8. A physiological candidate gene, SPP1, was found to lie within the 95% confidence interval of these QTL. A suggestive QTL for teat number was revealed on the short arm of SSC8. The present study demonstrates, to our knowledge, the first independent confirmation of QTL for fecundity on SSC8, and these QTL regions provide a crucial starting point in the search for the causal genetic variants.

BACKGROUND

Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days.

RESULTS

Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection.

CONCLUSIONS

The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion.

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.

A role for matrix proteins has previously been proposed in the pathogenesis of arterial calcification in the setting of atherosclerosis, and a link has been suggested between osteoporosis and arterial calcification. Our aim has been to investigate whether matrix Gla protein (MGP) T-138C, osteopontin (SPP1) T-443C, and Asp94Asp single nucleotide polymorphisms are associated with the development of arterial calcification and bone density. The individual effects of the MGP and SPP1 polymorphisms with coronary calcification are weak and not statistically significant. Bone mineral density differences at both the hip and spine do not vary statistically by genotype for any of the polymorphisms studied. Given the significant role of both MGP and SPP1 in arteriosclerosis, further research in higher risk, older populations are needed to determine fully the way in which MGP and SPP1 polymorphisms are associated with disease.

Interferon tau (IFNT), the pregnancy recognition signal in ruminants, abrogates the uterine luteolytic mechanism to ensure maintenance of function for the corpora lutea to produce progesterone (P4). IFNT also suppresses expression of classical IFN-stimulated genes by uterine lumenal epithelium (LE) and superficial glandular (sGE) epithelium but, acting in concert with progesterone, affects expression of a multitude of genes critical to growth and development of the conceptus. The LE and sGE secrete proteins and transport nutrients into the uterine lumen necessary for conceptus development, pregnancy recognition signaling, and implantation. Secretions include arginine and secreted phosphoprotein 1 (SPP1). Arginine can be metabolized to nitric oxide and to polyamines or act directly to activate the mechanistic target of rapamycin cell signaling pathway to stimulate proliferation, migration, and mRNA translation in trophectoderm cells. SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, adhesion, and migration of conceptus trophectoderm cells during implantation. Thus, arginine and SPP1 mediate growth, migration, cytoskeletal remodeling, and adhesion of trophectoderm essential for pregnancy recognition signaling and implantation. This minireview focuses on components of histotroph that affect conceptus development in the ewe.

Secreted phosphoprotein-1 (SPP1) promotes cancer cell survival and regulates tumor-associated angiogenesis and inflammation, both central to the pathogenesis of malignant pleural effusion (MPE). Here, we examined the impact of tumor- and host-derived SPP1 in MPE formation and explored the mechanisms by which the cytokine exerts its effects. We used a syngeneic murine model of lung adenocarcinoma-induced MPE. To dissect the effects of tumor- versus host-derived SPP1, we intrapleurally injected wild-type and SPP1-knockout C57/BL/6 mice with either wild-type or SPP1-deficient syngeneic lung cancer cells. We demonstrated that both tumor- and host-derived SPP1 promoted pleural fluid accumulation and tumor dissemination in a synergistic manner (P<0.001). SPP1 of host origin elicited macrophage recruitment into the cancer-affected pleural cavity and boosted tumor angiogenesis, whereas tumor-derived SPP1 curtailed cancer cell apoptosis in vivo. Moreover, the cytokine directly promoted vascular hyper-permeability independently of vascular endothelial growth factor. In addition, SPP1 of tumor and host origin differentially affected the expression of proinflammatory and angiogenic mediators in the tumor microenvironment. These results suggest that SPP1 of tumor and host origin impact distinct aspects of MPE pathobiology to synergistically promote pleural fluid formation and pleural tumor progression. SPP1 may present an attractive target of therapeutic interventions for patients with MPE.

RUNX2 is an essential transcription factor for osteoblast differentiation, because osteoblast differentiation is completely blocked in Runx2-deficient mice. However, it remains to be clarified whether RUNX2 is sufficient for osteoblast differentiation during embryogenesis. To address this issue, Runx2 transgenic mice were generated under the control of the Prrx1 promoter, which directs the transgene expression to mesenchymal cells before the onset of bone development. The transgene expression was detected in the cranium, limb buds, and the region from the mandible to anterior chest wall. The skull became small and the limbs were shortened depending on the levels of the transgene expression. Early onset of Runx2 expression in the cranial mesenchyme induced mineralization on E13.0, when no mineralization was observed in wild-type mice, and resulted in craniosynostosis as shown by the closure of sutures and fontanelles on E18.5. Col1a1 and Spp1 expressions were detected in the mineralized regions on E12.5-13.5. The limb bones were hypoplastic and fused, and ectopic bones were formed in the hands and feet. Col2a1 expression was inhibited but Col1a1 expression was induced in the limb buds on E12.5. In the anterior chest wall, ectopic bones were formed through the process of intramembranous ossification, interrupting the formation of cartilaginous anlagen of sternal manubrium. These findings indicate that RUNX2 is sufficient to direct mesenchymal cells to osteoblasts and lead to intramembranous bone formation during embryogenesis; Runx2 inhibits chondrocyte differentiation at an early stage; and that Runx2 expression at appropriate level, times and spaces during embryogenesis is essential for skeletal development.

Madin-Darby Bovine Kidney cells cultured with 150 μm of Wy-14 643 (WY, PPARα agonist) or twelve long-chain fatty acids (LCFA; 16 : 0, 18 : 0, cis-9-18 : 1, trans-10-18 : 1, trans-11-18 : 1, 18 : 2n-6, 18 : 3n-3, cis-9, trans-11-18 : 2, trans-10, cis-12-18 : 2, 20 : 0, 20 : 5n-3 and 22 : 6n-3) were used to uncover PPAR-α target genes and determine the effects of LCFA on expression of thirty genes with key functions in lipid metabolism and inflammation. Among fifteen known PPAR-α targets in non-ruminants, ten had greater expression with WY, suggesting that they are bovine PPAR-α targets. The expression of SPP1 and LPIN3 was increased by WY, with no evidence of a similar effect in the published literature, suggesting that both represent bovine-specific PPAR-α targets. We observed the strongest effect on the expression of PPAR-α targets with 16 : 0, 18 : 0 and 20 : 5n-3.When considering the overall effect on expression of the thirty selected genes 20 : 5n-3, 16 : 0 and 18 : 0 had the greatest effect followed by 20 : 0 and c9t11-18 : 2. Gene network analysis indicated an overall increase in lipid metabolism by WY and all LCFA with a central role of PPAR-α but also additional putative transcription factors. A greater increase in the expression of inflammatory genes was observed with 16 : 0 and 18 : 0. Among LCFA, 20 : 5n-3, 16 : 0 and 18 : 0 were the most potent PPAR-α agonists. They also affected the expression of non-PPAR-α targets, eliciting an overall increase in the expression of genes related to lipid metabolism, signalling and inflammatory response. Data appear to highlight a teleological evolutionary adaptation of PPAR in ruminants to cope with the greater availability of saturated rather than unsaturated LCFA.

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

Finding robust biomarkers for Parkinson disease (PD) is currently hampered by inherent technical limitations associated with imaging or antibody-based protein assays. To circumvent the challenges, we adapted a staged pipeline, starting from our previous proteomic profiling followed by high-throughput targeted mass spectrometry (MS), to identify peptides in human cerebrospinal fluid (CSF) for PD diagnosis and disease severity correlation. In this multicenter study consisting of training and validation sets, a total of 178 subjects were randomly selected from a retrospective cohort, matching age and sex between PD patients, healthy controls, and neurological controls with Alzheimer disease (AD). From ∼14,000 unique peptides displaying differences between PD and healthy control in proteomic investigations, 126 peptides were selected based on relevance and observability in CSF using bioinformatic analysis and MS screening, and then quantified by highly accurate and sensitive selected reaction monitoring (SRM) in the CSF of 30 PD patients versus 30 healthy controls (training set), followed by diagnostic (receiver operating characteristics) and disease severity correlation analyses. The most promising candidates were further tested in an independent cohort of 40 PD patients, 38 AD patients, and 40 healthy controls (validation set). A panel of five peptides (derived from SPP1, LRP1, CSF1R, EPHA4, and TIMP1) was identified to provide an area under curve (AUC) of 0.873 (sensitivity = 76.7%, specificity = 80.0%) for PD versus healthy controls in the training set. The performance was essentially confirmed in the validation set (AUC = 0.853, sensitivity = 82.5%, specificity = 82.5%). Additionally, this panel could also differentiate the PD and AD groups (AUC = 0.990, sensitivity = 95.0%, specificity = 97.4%). Furthermore, a combination of two peptides belonging to proteins TIMP1 and APLP1 significantly correlated with disease severity as determined by the Unified Parkinson's Disease Rating Scale motor scores in both the training (r = 0.381, p = 0.038)j and the validation (r = 0.339, p = 0.032) sets. The novel panel of CSF peptides, if validated in independent cohorts, could be used to assist in clinical diagnosis of PD and has the potential to help monitoring or predicting disease progression.

Amyloid plaques in Alzheimer's disease (AD) mice are surrounded by activated microglia. The functional role of microglia activation in AD is not well understood; both detrimental and beneficial effects on AD progression have been reported. Here we show that the population of activated microglia in the cortex of the APPswe/PS1dE9 mouse AD model is divided into a CD11c-positive and a CD11c-negative subpopulation. Cd11c transcript levels and number of CD11c-positive microglia increase sharply when plaques start to occur and both parameters continue to rise in parallel with the age-related increasing plaque load. CD11c cells are localized near plaques at all stages of the disease development and constitute 23% of all activated microglia. No differences between these two populations were found in terms of proliferation, immunostaining intensity of Iba1, MHC class II, CD45, or immunoproteasome subunit LMP7/β5i. Comparison of the transcriptome of isolated CD11c-positive and CD11c-negative microglia from the cortex of aged APPswe/PS1dE9 with WT microglia showed that gene expression changes had a similar general pattern. However, a differential expression was found for genes involved in immune signaling (Il6, S100a8/Mrp8, S100a9/Mrp14, Spp1, Igf1), lysosome activation, and carbohydrate- and cholesterol/lipid-metabolism (Apoe). In addition, the increased expression of Gpnmb/DC-HIL, Tm7sf4/DC-STAMP, and Gp49a/Lilrb4, suggests a suppressive/tolerizing influence of CD11c cells. We show that amyloid plaques in the APP/PS1 model are associated with two distinct populations of activated microglia: CD11c-positive and CD11c-negative cells. Our findings imply that CD11c-positive microglia can potentially counteract amyloid deposition via increased Aβ-uptake and degradation, and by containing the inflammatory response.

The objective of this study was to identify genes that are related to pathogenesis of carcinoma in situ (CIS) to invasive cervical cancer with the use of oligonucleotide microarray and reverse transcription-polymerase chain reaction (RT-PCR). Each two cases of normal cervix, CIS, and invasive cervical cancer were investigated with DNA microarray technology. Differential gene expression profiles among them were analyzed. Expression levels of selected genes from the microarray results were confirmed by RT-PCR. The expressions of 15,286 genes were compared and 458 genes were upregulated or downregulated by twofold or more compared with each other group. Among 458 genes, 22 genes were upregulated and 40 genes were downregulated by twofold or more in invasive cervical cancer group compared with CIS group. RT-PCR analysis confirmed upregulation of 18 genes and downregulation of 5 genes in invasive cervical cancer group. RBP1, TFRC, SPP1, SAA1, ARHGAP8, and NDRG1, which were upregulated, and GATA3, PLAGL1, APOD, DUSP1, and CYR61, which were downregulated, were considered as candidate genes associated with invasion of cervical cancer.