Top PDF Long non-coding RNA expression profiling of mouse testis during postnatal development.

Long non-coding RNA expression profiling of mouse testis during postnatal development.

Long non-coding RNA expression profiling of mouse testis during postnatal development.

Mammalian testis development and spermatogenesis play critical roles in male fertility and continuation of a species. Previous research into the molecular mechanisms of testis development and spermatogenesis has largely focused on the role of protein-coding genes and small non-coding RNAs, such as microRNAs and piRNAs. Recently, it has become apparent that large numbers of long (.200 nt) non-coding RNAs (lncRNAs) are transcribed from mammalian genomes and that lncRNAs perform important regulatory functions in various developmental processes. However, the expression of lncRNAs and their biological functions in post-natal testis development remain unknown. In this study, we employed microarray technology to examine lncRNA expression profiles of neonatal (6-day-old) and adult (8-week-old) mouse testes. We found that 8,265 lncRNAs were expressed above background levels during post-natal testis development, of which 3,025 were differentially expressed. Candidate lncRNAs were identified for further characterization by an integrated examination of genomic context, gene ontology (GO) enrichment of their associated protein-coding genes, promoter analysis for epigenetic modification, and evolutionary conservation of elements. Many lncRNAs overlapped or were adjacent to key transcription factors and other genes involved in spermatogenesis, such as Ovol1, Ovol2, Lhx1, Sox3, Sox9, Plzf, c-Kit, Wt1, Sycp2, Prm1 and Prm2. Most differentially expressed lncRNAs exhibited epigenetic modification marks similar to protein- coding genes and tend to be expressed in a tissue-specific manner. In addition, the majority of differentially expressed lncRNAs harbored evolutionary conserved elements. Taken together, our findings represent the first systematic investigation of lncRNA expression in the mammalian testis and provide a solid foundation for further research into the molecular mechanisms of lncRNAs function in mammalian testis development and spermatogenesis.
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Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy

Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy

In this present study, the SW480 colon cancer cell line was used to study the LINC00261 cisplatin resistance in colon cancer cells. We found that LINC00261 was down-regulated in colon cancer cell lines and tissues, and it reduced according to the stage. Furthermore, we built the cisplatin resistance SW480 cell line, and found that LINC00261 expression was relatively low in the drug-resistant cell line compared to drug-sensitive cells. Moreover, our experiments demonstrated that LINC00261 overexpression in resistant cells could effectively reduce their drug resistance. In addition, we found that LINC00261 regulated colon cancer cell proliferation and migration, and promoted apoptosis. During the mechanism study, we speculated that LINC00261 might down-regulate nuclear b-catenin through restraining b-catenin from cytoplasm into nuclei or it could also promote b-catenin degradation. Studies reported that blocking the nuclear translocation of b-catenin could inhibit transcriptional activation of T cell factor (TCF), lymphoid enhancer factor (LEF), and expressions of other target genes (32,33). Nuclear accumulation of b-catenin can form TCF/LEF/ b-catenin complex, and in the nucleus, this complex further activates target genes such as Myc and CCND1, which are involved in oncogenic transformation. In this study, we speculated that LINC00261 down-regulated b-catenin in nuclei and promoted b-catenin degradation, inactivated Wnt/b-catenin pathway and downstream target genes, then inhibited TCF/LEF/b-catenin complex formation, and finally, repressed colon cancer and reduced the cisplatin resistance of tumor cells. Finally, by tumor formation in vivo experiment, we found that LINC00261 overexpres- sion effectively inhibited the formation and development of colon cancer. To conclude, our study revealed the role of LINC00261 in colon cancer cells drug resistance and might offer a new vision and direction for the treatment of colon cancer.
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Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice.

Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice.

Cardiac pro-hypertrophic signaling together with cardiomyocyte damage leads to the acti- vation of resident fibroblasts and deposition of interstitial connective tissue. Indeed, both AngII and TAC induced myocardial collagen deposition and ablation of Malat-1 did not affect this process, ruling out an important role of this lncRNA in fibroblast activation. Additionally, the number of CD45 + cells in the pressure-overloaded heart was not affected by presence or absence of Malat-1, although a role for Malat-1 in pro-inflammatory cytokine production by HUVECS has recently been suggested [35]. On the cellular level, knockdown of Malat-1 results in alternative splicing of several genes [14], and in line with this, it was found that Malat-1 co- localizes with the splicing factors ASF/SF2 in nuclear speckles of mouse embryonic fibroblasts and cultured neurons [36]. Interestingly, a deficiency of ASF/SF2 has been shown to alter cardi- omyocyte function by affecting splicing of calcium/calmodulin-dependent kinase IIδ [37] and perturbation of mRNA splicing is a feature of heart failure [38]. Interestingly, we found alterna- tive splicing of Ndrg2, which shows skipping of exon 3 in hypertrophic mouse hearts [31], to be less common in Malat-1 KO mice both at baseline and after pressure overload. In contrast, alternative splicing of Eif4h was apparent after pressure overload as previously reported [32] but not affected by ablation of Malat-1. These data confirm that Malat-1 can influence splicing of individual mRNAs but do not indicate an important role of this effect in cardiac pressure overload. The named molecular and histological changes entail effects on cardiac morphology and function both in Malat-1 WT and KO mice. Our echocardiographic analysis is limited to systolic function, but in view of the comparable cardiomyocyte hypertrophy and myocardial fibrosis it appears unlikely that diastolic function is affected by absence of Malat-1. In conclu- sion, despite extensive phenotyping of cardiac function, morphology, histological appearance, and gene expression, no important differences could be found in the hearts of Malat-1 WT and KO mice after TAC- or AngII-induced cardiac pressure overload.
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Long Noncoding RNA Expression during Human B-Cell Development.

Long Noncoding RNA Expression during Human B-Cell Development.

Long noncoding RNAs (lncRNAs) have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre- B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n = 7), and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n = 6), respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 anti- sense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we ana- lysed their co-expression with well-characterized protein-coding genes, a method known as “guilt by association”. By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignan- cies that originate from distinct B-cell subpopulations.
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Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and in flammatory injury by up-regulation of PKR in keratinocytes

Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and in flammatory injury by up-regulation of PKR in keratinocytes

As an important epigenetic regulation mechanism of gene expression under environmental stress, lncRNAs have been found to play important roles in environmental response (19). Several techniques have been applied to study the molecular mechanisms underlying UVB-induced photo damage and skin carcinogenesis, and lncRNAs have been shown to be involved in this process (8). However, the specific functions of HOTAIR and the underlying mechanisms are still unknown in UVB-induced skin injury. The importance of lncRNAs has been impli- cated in many different contexts. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is neces- sary for PRC2 occupancy and histone H3 lysine-27 trimethylation of different genes in different chromosomes. PRC2 is a histone methyltransferase that implements epigenetic silencing during different processes, including cancer development (20).
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Long non-coding RNA contributes to direct conversion reprogramming

Long non-coding RNA contributes to direct conversion reprogramming

Over the last years, research led lncRNAs to go from transcriptional noise to important regulators of gene expression, being now known their association with many regulatory pathways by a wide set of mechanisms. Some of these RNAs play very important roles in cell differentiation, given their differential and temporal expression during tissue and organismal development. The discovery that terminally differentiated cells could revert to pluripotency or be redirected to multipotent progenitors of different lineages through forced expression of a specific onset of genes opened the way for direct conversion studies. The fact that various lncRNAs have been associated with differentiation, pluripotency and cancer indicates that these molecules might be useful tools in direct conversion. Manipulation of lncRNA expression during cell reprogramming could provide aid in overcoming current protocol limitations as the tumorigenic potential of iPSCs, low efficiencies and aging related epigenetic resistance. LncRNAs with influence in cell character transitions such as epithelial- mesenchymal transition (EMT) or mesenchymal-epithelial transition (MET) could contribute to direct conversion reprograming, as could lncRNAs that are specifically expressed in the reprogramming target cell line. We herein present two lncRNAs with the mentioned characteristics, Zeb2NAT and Pnky, respectively, as potential targets for improving fibroblast direct conversion reprogramming to multipotent hematopoietic and neural progenitors with a single pluripotency transcription factor (TF). Downregulation of Zeb2Nat during direct conversion seems to impair reprogramming efficiency, however if upregulating this lncRNA will have an improving effect is still under study. Getting unlimited patient-specific progenitor cells of different lineages from more accessible sources presents an enormous medical issue.
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Revealing potential long non-coding RNA biomarkers in lung adenocarcinoma using long non-coding RNA-mediated competitive endogenous RNA network

Revealing potential long non-coding RNA biomarkers in lung adenocarcinoma using long non-coding RNA-mediated competitive endogenous RNA network

phosphorylation. Of note, these modifications can mod- ulate the nuclear chromatin setting and is a well-known epigenetic mechanism for regulating gene expression pat- terns (19). Histone modifications influence various cellular processes including chromatin modification, DNA replica- tion and repair (20,21). Importantly, the modification of histones has been demonstrated to be a predictor for colorectal cancer (22), and gastric carcinomas (23). More- over, histone H4 modification has been indicated to play an important role in bronchial carcinogenesis and might be a candidate signature for therapeutic approaches in lung cancer (24). Accordingly, the GO term of histone modifica- tion might be closely associated with the LAD progression. Negative regulation of cell cycle was another signifi- cant GO term. To our knowledge, the hallmark of cancer is uncontrolled cell proliferation caused by dysregulation of cell-cycle (25). Disturbed surveillance of cell-cycle pro- gression, for example cellular evasion of cell-cycle check- points, which are driven by aberrant activation of cell-cycle regulators (cyclins and cyclin-dependent kinases), can cause the tumor development and progression (26). Activated cyclin-dependent kinase-6 and cyclin-dependent kinase-2 during the G1–S cell-cycle transition are key regulators in the modifications of retinoblastoma pathway (27), disruption of which shows strong cell-proliferative activity and has been shown to play important roles in the pathogenesis of lung cancer (28). More importantly, Cai et al. (29) have reported that miR-186 exerts a tumor- suppressive role in the development of LAD, partially through regulation of cell-cycle progression. Hence, it is plausible that the lncRNA biomarkers act as ceRNAs involving the negative regulation of cell cycle, which might exert important functions in the occurrence and progression of LAD.
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A long non-coding RNA regulating cellular reprogramming

A long non-coding RNA regulating cellular reprogramming

During the last decade scientists have been fighting to solve these problems. Some even published fabricated results declaring the discovery of a new way to reprogram cells simply using a low-acid bath (Obokata, Sasai, et al., 2014; Obokata, Wakayama, et al., 2014). As the years passed, more reprogramming cocktails have been described. In 2008, it was shown that reprogramming can be achieved without c-Myc and Klf4, by the ectopic expression of Oct3/4, Sox2, Nanog and Lin28 in human fibroblasts (Tomioka et al., 2010), although Nanog has been previously described as a dispensable factor (Takahashi & Yamanaka, 2006). It has also been described that cells characterized by the endogenous expression of at least one reprogramming factor can reprogram by the induction of the other factors, excluding the one endogenously expressed (Utikal et al., 2009, Giorgetti et al., 2010). At the same time Kim et al. reprogrammed both mouse (J. B. Kim, Sebastiano, et al., 2009) and human neural stem cells (J. B. Kim, Greber, et al., 2009), that naturally express Sox2, only by over-expressing Oct3/4. To induce reprogramming using ‘removable’ episomal vectors seems one of the safest processes, since the vector is not integrated in the genome and does not request any viral infection (J. Yu et al., 2009), but its efficiency is a concern. There is also the possibility of using a combination of small molecules to reprogram cells (Hou et al., 2013), but this was never done with human cells (Lin & Wu, 2015). iPS technology is still evolving for a safer and better use in human therapies.
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Long non-coding RNA profiling in laryngeal squamous cell carcinoma and its clinical significance: potential biomarkers for LSCC.

Long non-coding RNA profiling in laryngeal squamous cell carcinoma and its clinical significance: potential biomarkers for LSCC.

Pseudogenes are considered to arise from the mutation of transcripts or as a result of mistakes in transcription. Moreover, pseudogenes can be generated by the degeneration of protein- coding genes or integration of cDNA from reverse transcription, and can even be produced from other pseudogenes. Processed pseudogenes can be integrated by retrotransposition of mRNA [24–25]. The gene of AC026166.2-001 is a known processed pseudogene (ENSG00000233026). AC026166.2-001, a 344nt transcript, is an unspliced lncRNA expressed from the opposite strand within one intron of the gene SYN2. Many studies have demonstrated that pseudogenes are involved in the development of Figure 5. Survival curve of laryngeal carcinoma patients. (A) Survival curve of laryngeal carcinoma patients with varying expression levels of AC026166.2-001 (P = 0.029). Lower curve: LSCC patients with low expression (n = 63); upper curve: LSCC patients with high expression (n = 24). (B) Survival curves of laryngeal carcinoma patients (n = 87) with differing. RP11-169D4.1-001 expression levels (P = 0.025). Lower curve: LSCC patients with low expression (n = 47); upper curve: LSCC patients with high expression (n = 30). (C) Survival curves of laryngeal carcinoma patients according to T stage (P,0.001). (D) Survival curves of laryngeal carcinoma patients with and without cervical lymph node metastasis (P,0.001). (E) Survival curves of laryngeal carcinoma patients according to histological differentiation (P = 0.182).
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Identification and characterization of long non-coding RNAs related to mouse embryonic brain development from available transcriptomic data.

Identification and characterization of long non-coding RNAs related to mouse embryonic brain development from available transcriptomic data.

As shown in Figure 1, the transcripts analyzed in this study were assembled using a widely used yet modified protocol [41]. Briefly, we used TopHat [42] to align reads of embryonic brain RNA-seq datasets. Then, we used Cufflinks [43] to assemble transcripts into known gene models or novel gene models by cufflinks guided by known gene annotations. The assembled transcripts were then merged by the Cuffmerge utility provided by the Cufflinks package, resulting in the assembly of 678,324 nonredundant transcript isoforms from 321,413 loci in embryonic brain, which provided the unique basis for further filtering putative transcripts and characterizing expression. These transcripts were concurrent- ly annotated by Cuffcompare program in Cufflinks suite, of which known transcripts were used as reference to screen for novel lncRNAs. Small RNAs were filtered out using a minimum length threshold of 200 nt, further decreasing the number of transcripts to 421,379. In order to obtain a reliable dataset of putative lncRNAs, single exon genes were filtered out, unless supporting evidence from at least two developmental time points was available. Same procedure was also used in another study [44]. We also removed transcripts with Reads Per Kilobase per Million mapped reads (RPKM) ,0.3 (refer to Materials and Methods). Applying the threshold, the number of transcripts in embryonic brain decreased to 72,544. Next, we removed transcripts that were likely to be assembly artifacts or PCR run-on fragments (refer to Materials and Methods). Among the different classes, only those annotated by ‘‘u’’, ‘‘i’’, ‘‘j’’ and ‘‘x’’ were retained, which represent novel intergenic, intronic, alternative spliced and cis-antisense transcripts, respectively. But here, most analysis focused on intergenic, intronic and cis-antisense lncRNAs.
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Long non-coding RNA expression profiles in hereditary haemorrhagic telangiectasia.

Long non-coding RNA expression profiles in hereditary haemorrhagic telangiectasia.

can be used to relate the lncRNA transcriptome to biology using well-known mRNA function and gene ontology (GO) terms. Using GREAT, three interesting and statistically significant GO terms were the result: blood vessel morphogenesis, blood vessel development, and vasculogenesis. All three were located inside the top 6 resulting GO terms. Blood vessel morphogenesis is defined as: ‘The process in which the anatomical structures of blood vessels are generated and organized. The blood vessel is the vasculature carrying blood’. Blood vessel development is defined as: ‘The process whose specific outcome is the progression of a blood vessel over time, from its formation to the mature structure’; and vasculogenesis: ‘The differentiation of endothelial cells from progenitor cells during blood vessel development, and the de novo formation of blood vessels and tubes’. These GO term results are interesting as HHT manifestations are thought to result from imbalanced angiogenesis. The results may point to a causal effect of lncRNAs regulating mRNAs central to vasculogenesis, angio- genesis, and perhaps even HHT telangiectasia formation.
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Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells.

Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells.

Proliferative vitreoretinopathy (PVR), a severe blinding disease characterized by the formation of epiretinal membranes through a defective wound repair process, occurs as a complication of rhegmatogenous retinal detachment [1,2]. PVR is the primary reason for failure of initially suc- cessful retinal re-attachment surgery due to the recurrent preretinal or epiretinal membrane traction, which further leads to retinal redetachment and dramatic visual loss [3]. Several cell types are involved in the pathogenesis of PVR, including retinal pigment epithelial (RPE) cells, fibroblasts (primarily derived from RPE cells), glial cells, and inflammatory cells [4]. In all these cell types, RPE cells is thought to play the principal role in the pathogenesis of PVR as it is recognized as the largest cellular component of the epiretinal membranes in PVR patients [5]. In the settings of PVR development, RPE cells which exposed to the vitreous (which is rich of cytokines and growth factors) are detached from Bruch’s membrane and migrate into the vitreous through the retina tear [6]. In this process, RPE cells undergo a process known as epi- thelial-mesenchymal transition (EMT), an orchestrated series of events in which fully differen- tiated epithelial cells undergo transition and acquire a mesenchymal phenotype. Afterwards, RPE cells gradually participate in the formation of fibrotic membrane on the retina. These membrane contracts under the stimulation of growth factors/cytokines in the vitreous, and fur- ther leads to traction retinal detachment [7]. Therefore, fully understanding of the mechanisms of EMT in RPE cells is required for identifying potential therapeutic targets in treating PVR. Currently, an increasing number of studies were subjected to explore the mechanism and the treatment of EMT in RPE cells. ARPE-19 cells, a human RPE cell line, is frequently used to establish the EMT model because it simuates the EMT process of RPE cells solidly and sonsi- tantly, though it lacks some of RPE features [7–15]. Currently, various growth factors/cyto- kines, intracellular signaling pathways, transcription factors, and microRNAs are indicated to play significant roles in EMT of RPE cells [7–12,16]. However, it is not clear whether long non- coding RNAs (LncRNAs) contribute to EMT of RPE cells.
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Clustering Rfam 10.1 : clans, families, and classes

Clustering Rfam 10.1 : clans, families, and classes

It is natural to ask whether the hierarchical clustering trees can be used to identify significant structure-based clusters, and whether the families with strong similarities share biological functions. The Rfam clans have been constructed manually and consist of groups of families sharing common ancestors too divergent to be aligned, or also presenting good alignments but distinct functions so that they could not be included in the same Rfam family [3]. Some of these clans clearly share a common evolutionary ancestry, considering that similarities in their biological functions were experimentally verified. Hierarchical clustering, on the other hand, suggests how Rfam families that are not contained in clans could be related to each other and how clans may be organized at even higher levels of aggregation. It thus provides a more inclusive annotation.
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Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

The LRV-Lae L494 sequence was obtained by combination of total small RNA sequencing and specific-primer sequencing from cDNA. A library of small RNAs ( ,42 nt) was generated from total RNA (purified with Trizol, described above) using the method described by Atayde and co-workers [29]. Recent genome sequence data of the Lae L147 line reveals an absence of genes required for RNAi, consistent with the evolutionary position of L. aethiopica within the Leishmania clade shown previously to lack RNAi [27], and thus the small RNAs represent primarily degradation products (unpublished data), as seen previously in similar studies in Leishmania tarentolae which also lacks RNAi [27,30]. The library was sequenced using Illumina HiSeq2000 technology, yielding 35.9 million reads. The 59 and 39 adapters were trimmed from the data and then mapped to the sequence of the Lae L494 PCR products described above and/or the L. major LRV2. From this analysis, three large contigs were obtained, and the remaining regions of the LRV2 were obtained by PCR amplification across the gaps and sequencing. This strategy allowed us to get the complete 5193 bp LRV-Lae L494 genome sequence (GenBank accession number: KF757256).
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Enhancing reprogramming and transdifferentiation through long non-coding RNAs

Enhancing reprogramming and transdifferentiation through long non-coding RNAs

XI stained with DAPI in blue. Scale bar was set for 50μm. Images acquired through the Carl Zeiss Axiovert 200M fluorescence microscope. Images were treated posteriorly by using Fiji software. ..... 35 Figure 3.16 - Illustration of LNAs GapmeRs transfection protocol with cellular reprogramming protocol performed afterwards in 3yr and WI38 human fibroblasts. LNAs GapmeRs transfection protocol was performed in day 1 and 2. Cellular reprogramming protocol started in day 2 with 293T transfection and ended in day 6 with second infection of human fibroblasts. ....................................... 36 Figure 3.17 - Representation of CRISPR/CAS9 technique used to delete NORAD gene. (A) Norad plasmids in red designed to cut in a specific location for NORAD deletion. (B) Two par of primers in yellow designed to detect if NORAD was successfully deleted through electrophoresis. (C) If NORAD was successfully deleted a band of 507bp in electrophoresis gel would be detected. (D) If NORAD deletion was not accomplished two bands of 434bp and 5703bp would appear in electrophoresis gel. 38 Figure 6.1 - RT-qPCR results of hKLF4 expression levels of WI38 and 3yr human fibroblasts presented as ΔΔC t normalized using non OSKM lentiviral transduced cells as control and hGAPDH
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Iron deprivation in synechocystis: inference of pathways, non-coding RNAs, and regulatory elements from comprehensive expression profiling

Iron deprivation in synechocystis: inference of pathways, non-coding RNAs, and regulatory elements from comprehensive expression profiling

A coordinated inverse regulation between genes involved in the metabolism of carbon and nitrogen has been reported before (Muro- Pastor et al. 2001; Osanai et al. 2006). Since both processes, nitrogen reduction and carbon fixation, depend on iron-containing enzymes, a coordinated regulation might reflect the prioritization of one path- way over the other depending on the environmental conditions. The inference of such coordinated regulation is supported by several other observations. For instance, a mutant lacking the metal-regulated gene A (mrgA), encoding a protein that exerts a role in the mobilization of iron from bacterioferritins (Shcolnick et al. 2007), showed a similar downregulation of genes involved in carbon fixation and upregulation of nitrite/nitrate transporters compared with wild-type strain even under normal conditions. The levels of expression of these genes is probably due to the impaired mobilization of iron from the cell res- ervoirs and thus explains why the mrgA knockout presents transcript levels resembling those of iron-stressed cells (Shcolnick et al. 2009). A further link between carbon fixation and iron limitation is likely pro- vided by the sll0217-sll0218-sll0219 operon, which is involved in ac- climation to low-carbon, high-light stress and whose expression under such stress conditions reduced photosystem II damage (Hihara et al. 2001; Zhang et al. 2009). In iron-limiting conditions, photosystem II protective mechanisms can be expected to be especially important due to the observed reduction in the number of photosystem I complexes. Surprisingly, however, it was downregulated in our study. Thus, the repression of these genes might be caused either because of their iron content or because of their association to carbon fixation. This latter affirmation is supported by the detected upregulation of ndhR, a neg- ative regulator of genes involved the CO 2 uptake system of Synecho-
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SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes.

SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes.

Immunofluorescence Staining of Testis Tissue Sections Testes from neonatal (postnatal day (PND) 0 and PND 3), juvenile (PND 7 and PND 14) and adult (.6 weeks of age) mice were dissected and fixed in 4% PFA for 2 hours (neonatal testes) or overnight (juvenile and adult testes) at 4uC. Fixed tissues were processed for paraffin embedding and 5 m m serial sections were collected. Sections were deparaffinized in xylene (2615 min), rehydrated in a graded ethanol series (26100% for 10 min, 1695% for 5 min, 1680% for 5 min, 1670% for 5 min, 1650% for 5 min, 1625% for 5 min) and rinsed in 16DPBS. Slides were then incubated in sodium citrate antigen retrieval buffer (10 mM Sodium Citrate, 0.05% Tween-20, pH 6) for 30 min at 97.5 uC and allowed to cool to room temperature. After rinsing twice in 16 DPBS containing 0.1% Tween-20 (DBPS-T), unspecific binding sites in tissue sections were saturated by incubation with blocking buffer (16 DPBS containing 3% bovine serum albumin, 0.1% Triton X-100 and 5% normal serum from the host species of the secondary antibody) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and added to tissue sections for 90 min at room temperature. Isotype matched normal IgG at a comparable concentration was used instead of primary antibody in negative controls (see Figures S1 and S2). After washing the slides in DPBS-T 36 for 5 min, samples were incubated with the appropriate secondary antibody (goat IgG or donkey IgG) conjugated to AlexaFlour-488 or AlexaFluor-568 dye (all Molecular Probes, Invitrogen) for 45 min at room temperature. Sections were washed 365 min with DPBS-T and 165 min with DPBS before mounting with Vectashield Mount- ing Medium with DAPI (H-1200, Vector Laboratories, Burlin- game, CA).
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Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology

Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology

A number of genes encoding proteins of interest were found to be up- or downregulated under stress conditions in the present study. One such transcript encodes ornithine decarbox- ylase (ODC), a key enzyme of polyamine biosynthesis that is elevated in rapidly proliferating cells. Overexpression of ODC has been associated with carcinogenesis in mammals (4, 39). ODC is also a marker of liver neoplasia in winter flounder (P. americanus) (32), and its upregulation in stressed gilthead sea bream may cause a predisposition to tumors, an issue of some importance for aquaculture. Interestingly, two of the main proteins in iron metabolism, ferritin heavy and light subunit and the transferrin receptor, were downregulated by cortisol. Oxidative stress and iron metabolism are interrelated in mul- tiple ways. Ferritin is an intracellular molecule that stores iron in a soluble nontoxic form and was found to be induced in response to hypoxia in a gene expression profiling study in G. mirabilis (18) and repressed in response to quinone-induced oxidative stress in a study of murine B6 fibroblasts (17). The transferrin receptor is an iron transport protein that was like- wise found to be repressed in murine fibroblasts in response to oxidative stress (17). Because it is known that intracellular iron can be a rate-limiting factor for cell growth and proliferation (35), a downregulation of intracellular iron levels may be associated with the suppression of growth observed under stress conditions. Furthermore, elevated levels of transferrin receptor were found in Atlantic salmon macrophages activated by infection with Piscirickettsia salmonis (49), suggesting that its downregulation could contribute to immunosuppression. In humans, transferrin is used as a clinical marker as its concen- tration level correlates with the total mass of immature ery- throid cells (47). Potentially, it could also be employed as a diagnostic marker in aquaculture. For this purpose, it will be important to determine whether the decrease of the transferrin receptor seen during stress primarily reflects a decrease in the number of hematopoietic cells or a lowering of intracellular iron levels.
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Analysis of topological organization of chromatin during spermatogenesis in mouse testis

Analysis of topological organization of chromatin during spermatogenesis in mouse testis

One of the striking observations is the absence of a detectable halo in the round spermatids at all the EtBr con- centrations following 2.0 M NaCl and its appearance fol- lowing 2.4 M NaCl extractions. The lack of halo indicates a chromatin organization in round spermatids that is remark- ably different from other testicular cell types, even elongat- ing spermatids. It was difficult to unequivocally distinguish between the elongating/elongated spermatids and testicular sperm in the EtBr-stained nucleoids. Therefore, these cell types were scored together. The occurrence of typical biphasic change in the nucleoid and the dimensions of halo after 2.0 M NaCl extraction imply that topologically nega- tive supercoiling of DNA persists, even at higher degrees of spermatid differentiation. A curious aspect of this finding is that, though elongating spermatid (and testicular sperm) is a later and expectedly more condensed state of chromatin than round spermatid, it is the latter which is more resistant to the salt extraction. It seems unlikely that the apparent lack of loop domains is due to the large number of attach- ment points on the nuclear matrix. The spermatogenic cal- endar of the mouse shows that, while transition proteins (TPs) are transcribed in the round spermatids, the prota- mines (P1, P2) appear only in the elongating spermatids (Hecht, 1990). It is possible that, in the process of transi- tion, the removal of histones by the protamines may relieve the constraints in the DNA, as it progresses from round spermatid to the elongating type. The retention of (-)ve supercoiled state and nucleosome structure in the elongat- ing spermatid and testicular sperm suggests that the resid- ual presence of histones is enough to generate the nucleosomal organization.
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Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

Agrobacterium genetically modifies plants in nature to cause crown gall disease [1]. Under lab- oratory conditions, Agrobacterium also can transform practically any eukaryotic species, from fungal to human cells [2]. To initiate infection, Agrobacterium exports single-stranded mole- cules of the transferred DNA (T-DNA) into its target cells, as well as several types of virulence (Vir) protein effectors that actively participate in the transformation process [3, 4]. One such exported bacterial effector is VirF [5], which is an F-box protein [6]. Studies suggest that VirF recognizes and induces degradation by the ubiquitin/proteasome system (UPS) of the plant protein VIP1 and its associated bacterial effector VirE2 [7, 8], which likely package the T-DNA into a transfer (T) complex for nuclear import and chromatin targeting [9–11]. Thus, one func- tion of VirF may be to uncoat these associated proteins from the T-DNA molecule via the SCF VirF pathway prior to integration of the T-DNA into the host cell genome [7, 8]. Interest- ingly, because VirF is a host range determinant, it is not essential for infection of some plant species [12, 13], which presumably encode their own F-box proteins [14] that can fulfill this function, such as VBF in Arabidopsis [14].
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