Twofold Change Research Articles

Analysis of anti-osteoporosis function of chlorogenic acid by gene microarray profiling in ovariectomy rat model.

The aim of the present study was to clarify the effect of chlorogenic acid (CGA) on estrogen deficiency-induced osteoporosis based on micro-computed tomography (micro-CT) and potential mechanism of gene regulation via microarray profiling. Eighteen female Sprague–Dawley rats were divided randomly into sham-operated group, ovariectomy (OVX) plus saline vehicle group, and OVX plus CGA treatment group (CGA at 45 mg/kg/day). The loss of bone mass of the femoral metaphysis was evaluated by micro-CT to represent. Gene expression profiling was analyzed for bone marrow mesenchymal stem cells (BMSCs) of OVX and OVXT groups. Bioinformatics analysis was used to find the potential pathways regulated by CGA. OVX-induced osteoporosis could decrease femur bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecula number (Tb.N), and trabecular thickness (Tb.Th) and increased the trabecular separation (Tb.Sp) and structure model index (SMI) in the rats. Gene microarray profiling showed 121 differentially expressed genes in collected BMSCs between OVX and OVXT groups were identified with a threshold of a two-fold change and P<0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the potential mechanism of CGA and we observed that many mitogen-activated protein kinase (MAPK) pathway associtated genes were altered, suggesting this pathway may play an important role. CGA improved bone quality by modifying the BMD and trabecular microarchitecture. Differential expression genes were screened by gene microarray profile and the results suggested MAPK pathway might participate in the process of OVX-induced bone remodeling.

Profiles for long non-coding RNAs in ovarian granulosa cells from women with PCOS with or without hyperandrogenism.

What is the expression pattern of long non-coding RNAs (lncRNA) in ovarian granulosa cells of women with polycystic ovary syndrome (PCOS) with or without hyperandrogenism? Microarray screening of lncRNA was conducted in ovarian granulosa cells collected from women with PCOS with hyperandrogenism (PCOS-T) or without hyperandrogenism (PCOS-N) and control participants, with four samples in each group. This was followed by hierarchy clustering, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Several candidate lncRNA were randomly selected for quantitative polymerase chain reaction validation in another 54 patients. To predict the regulatory effect of lncRNA on hyperandrogenism, a co-expression network was plotted using differentially hexpressed lncRNA with statistical significance (≥ twofold; P < 0.05) in PCOS-T compared with PCOS-N. A total of 3000 and 1030 differentially expressed lncRNA (≥ twofold change) were detected in PCOS-T compared with control and PCOS-N, respectively. A total of 1361 differentially expressed lncRNA were detected in PCOS-N compared with controls. Corticotropin releasing hormone binding protein is consistently the up-regulated lncRNA with the highest fold-change in PCOS-T compared with either control or PCOS-N. Gene ontology and pathway analysis showed that dysregulated lncRNA in PCOS-T have a regulatory role in mitochondrial function by interacting with transcription factors such as YY1 and SIX5. The expression patterns of lncRNA in women with PCOS were ascertained by microarray. Many lncRNA were differentially expressed in PCOS-T compared with PCOS-N, suggesting that they may play a key role in steroid genesis and metabolism.

Physiological and transcriptomic responses of reproductive stage soybean to drought stress.

The dynamic alterations of the physiological and molecular processes in reproductive stage soybean indicated the dramatic impact caused by drought. Drought is a major abiotic stress that limits soybean (Glycine max) production. Most prior studies were focused on either model species or crops that are at their vegetative stages. It is known that the reproductive stage of soybean is more susceptible to drought. Therefore, an understanding on the responsive mechanisms during this stage will not only be important for basic plant physiology, but the knowledge can also be used for crop improvement via either genetic engineering or molecular breeding. In this study, physiological measurements and RNA-Seq analysis were used to dissect the metabolic alterations and molecular responses in the leaves of soybean grown at drought condition. Photosynthesis rate, stomata conductance, transpiration, and water potential were reduced. The activities of SOD and CAT were increased, while the activity of POD stayed unchanged. A total of 2771 annotated genes with at least twofold changes were found to be differentially expressed in the drought-stressed plants in which 1798 genes were upregulated and 973 were downregulated. Via KEGG analysis, these genes were assigned to multiple molecular pathways, including ABA biogenesis, compatible compound accumulation, secondary metabolite synthesis, fatty acid desaturation, plant transcription factors, etc. The large number of differentially expressed genes and the diverse pathways indicated that soybean employs complicated mechanisms to cope with drought. Some of the identified genes and pathways can be used as targets for genetic engineering or molecular breeding to improve drought resistance in soybean.

Effect of the Surgical Intervention of Laparoscopic Donor Nephrectomy on Proteomic Changes in Living Kidney Donors using either Propofol or Sevoflurane Anaesthesia

Introduction Surgical intervention i.e. ‘trauma’ induces perioperative stress impacting on systemic inflammatory and humoral responses that are associated with postoperative comorbidity. Different types of anaesthesia and anaesthetic agents are shown to have differential effects on the innate and adaptive immunity. In this study, the biological effect of the surgical intervention combined with propofol or sevoflurane anaesthesia in the healthy cohort of living kidney donors undergoing laparoscopic surgery was assessed by proteome profiling of blood plasma samples. Materials and Methods Plasma samples of healthy living donors (LD) participating in the VAPOR-1 trial were used. Sample points were: before surgery (T0), immediately after surgery (T1), and 24 hours after surgery (T2). LDs were anaesthetised with either propofol (n=19) or sevoflurane (n=17) and matched by age, gender, BMI, comorbidity and medication. Anaesthetic and hemodynamic management was strictly protocolised. Samples were subjected to proteome profiling by mass spectrometry followed by data analysis with MaxQuant software. Student’s T-Test and multiple testing correction were applied to detect significant changes in protein abundance between comparisons. Results Quantitative protein identification resulted in detection of 633 plasma proteins. A subset of 28 proteins showed statistically significant (P<0.05) expression level changes between time points. Proteins with over two-fold change comprised a smaller group of nine upregulated targets that are known to be involved in acute phase inflammatory response (CRP, SAA1, SAA2, LBP, SERPINA1, SERPINA3) and tissue regeneration (FGL1, LRG1, MAN1A1). These targets were common in all LDs, except MAN1A1 that was upregulated only in propofol anaesthesia. Discussion In this study, we have evaluated the effect of a major surgical intervention in a unique cohort of healthy subjects selected to donate a kidney, using systems biology by analysing individual sequential proteomic profiles. The predominant changes in profiles before and after surgery involved pathways of acute phase response and tissue remodelling. The highest detected changes in protein levels were found to be independent of anaesthesia type, while proteome profiles also displayed a number of moderate level anaesthesia-specific changes. More proteins with statistically significant level changes were detected one day after surgery (T2) when compared to immediately after surgery (T1), in both anaesthesia groups. Conclusion This study is a first attempt to gain better insight in molecular effects of surgical intervention in a selected healthy ‘control’ group using systems biology. Quantitative proteome analysis identified upregulated proteins in response to laparoscopic intervention independent of anaesthetic type, which known to be involved in acute phase inflammatory response, immunogenicity and tissue remodelling.

Superovulation alters the expression of endometrial genes critical to tissue remodeling and placentation.

Epidemiologic data suggest that in vitro fertilization (IVF) is associated with an increased risk of disorders of placentation including preeclampsia and fetal growth restriction. Specifically, studies have demonstrated that singleton pregnancies conceived following a fresh embryo transfer are at an increased risk of delivering an infant with low birth weight compared to those conceived following a frozen embryo transfer. The mechanism responsible for this association remains unclear. Procedures utilized in IVF have also been linked with epigenetic changes and gene expression changes in both fetal and maternal tissues. Data suggest that modifications in the maternal endometrium can lead to disordered trophoblast invasion and placentation. This study examines the effect of ovarian stimulation on endometrial gene expression and DNA methylation during the window of implantation to examine potential pathways playing a role in the adverse outcomes associated with IVF. Endometrial biopsies were obtained from oocyte donors and age-matched naturally cycling women 11days following oocyte retrieval in donors or 12days following luteinizing hormone (LH) surge in naturally cycling women. Global gene expression was analyzed via Affymetrix Human Gene 1.1 ST array and confirmed with RT-qPCR. DNA methylation was assessed with the Infinium DNA methylation 450K BeadChip. Analysis of endometrial gene expression from 23 women (11 oocyte donors and 12 controls) demonstrated 165 genes with a greater than twofold change in expression between donors and controls. While there were 785 genes with significant differential methylation in the endometrium of donors when compared with control subjects, none of the genes with altered expression showed significant changes in DNA methylation. Analysis of the differentially expressed genes showed enrichment for genes involved in endometrial remodeling including PLAT, HSPE2, MMP2, and TIMP1. Validation studies using RT-qPCR found a 73% reduction in expression of heparanase 2 (HSPE2) an enzyme associated with both angiogenesis and cell invasion, a greater than twofold increase in tissue-type plasminogen activator (PLAT), a serine protease participating in matrix degradation, and a 70% increase in MMP2, a gelatinase involved in collagen and fibronectin breakdown. Superovulation alters expression of genes critical to endometrial remodeling during early implantation. Such changes could lead to altered trophoblast migration and impaired endovascular invasion. These findings offer a potential mechanism for the adverse perinatal outcomes observed following embryo transfer during fresh IVF cycles.

Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD

To enhance our understanding of molecular mechanisms and mine novel biomarkers of non-alcoholic fatty liver disease (NAFLD), RNA sequencing was performed to gain hepatic expression profiles of mRNAs and miRNAs in NAFLD and normal rats. Using DESeq with thresholds of a two-fold change and a false discovery rate (FDR) less than 0.05, 336 mRNAs and 21 miRNAs were identified as differentially expressed. Among those, 17 miRNAs (e.g., miR-144-3p, miR-99a-3p, miR-200b-3p, miR-200b-5p, miR-200c-3p, etc.) might serve as novel biomarkers of NAFLD. MiRNA target genes (13565) were predicted by the miRWalk database. Using DAVID 6.8, the intersection (195 genes) of differentially expressed mRNAs and miRNA-predicted target genes were enriched in 47 gene ontology (GO) terms and 28 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Using Cytoscape, pathway interaction and protein-protein interaction (PPI) networks were constructed, and hub genes (e.g., Abcg8, Cyp1a1, Cyp51, Hmgcr, etc.) associated with NAFLD were obtained. Moreover, 673 miRNA-mRNA negative regulatory pairs were obtained, and networks were constructed. Finally, several representative miRNAs and mRNAs were validated by real-time qPCR. In conclusion, potential molecular mechanisms of NAFLD could be inferred from integrated analysis of mRNA and miRNA profiles, which may indicate novel biomarkers of NAFLD.

Next generation sequencing for miRNA profile of spleen CD4+ T cells in the murine model of acute asthma.

To explore the miRNAs profile of CD4+ T lymphocytes in asthma via next generation sequencing. In the murine model of acute asthma, spleen CD4+ T lymphocytes were sorted, in which small RNAs were extracted and sequenced. Novel miRNAs were measured with real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). A total of 127 miRNAs were found to exhibit at least twofold change. In the 262 predicted novel miRNAs, 14 novel miRNAs were measured in qRT-PCR in the sorted CD4+ T cells or in the differentiated Th1/Th2 cells and novel miR-11 (xxx-m0228-3p) was significantly decreased in the sorted CD4+ T cells from the murine model of asthma and in the Th2 cells. Aberrant miRNAs profile in the CD4+ T lymphocytes from acute asthma was documented.

Ocnus is essential for male germ cell development in Drosophila melanogaster.

The ocnus (ocn) gene encodes a protein abundant in the testes, implying its role in testis development. When Drosophila melanogaster is infected with the endosymbiont wMel Wolbachia, which affects the spermatogenesis of its hosts, ocn is downregulated in the third-instar larval testes, suggesting a role of ocn in spermatogenesis. In this study, we knocked down ocn in the testes and found that the hatch rates of embryos derived from ocn-knockdown males were significantly decreased, and 84.38% of the testes were much smaller in comparison to controls. Analysis of the smaller testes showed no germ cells but they had an extended hub. Using RNA-sequencing (RNA-Seq), we identified 69 genes with at least a twofold change (q-value < 5%) in their expression after ocn knockdown; of these, eight testes-specific and three reproduction-related genes were verified to be significantly downregulated using quantitative reverse transcription-PCR. Three genes (orientation disruptor, p24-2 and CG13541) were also significantly downregulated in the presence of Wolbachia. Furthermore, 98 genes were not expressed when ocn was knocked down in testes. These results suggest that ocn plays a crucial role in male germ cell development in Drosophila, possibly by regulating the expression of multiple spermatogenesis-related genes. Our data provide important information to help understand the molecular regulatory mechanisms underlying spermatogenesis.

Making the hidden seen: A narrative analysis of the experiences of Assumption of Care at birth

BackgroundBeing involved in any child protection system is complex and multifaceted and none more so than in non-voluntary Assumption of Care which occurs in New South Wales when, for child protection concerns, the Department of Family and Community Services removes a newborn baby from her/his mother. ObjectiveThis research studied childbearing women’s and professionals’ experiences of Assumption of Care at birth to increase understanding of individual participants’ stories, how they made sense of meanings and how these experiences framed their lives. MethodA narrative inquiry framework guided interviews with four groups: childbearing women, midwives, social workers and Family and Community Services case managers. Holistic form was used for reading, interpreting and analysing the narratives. FindingsThis research found unwanted emotional (isolation, shame, guilt, loss, disenfranchised grief) and physical consequences (depression, substance abuse complications) for women experiencing an Assumption of Care at the time of birth. There were also conflicting ethical and moral positions for the professionals involved. The use/abuse of power, concealment of facts and disenfranchised grief were identified as intertwined plots that caused or increased tensions. DiscussionBoth the women and the professionals felt pressure from trying to achieve competing and overlapping roles. The unwanted effects of Assumption of Care are exacerbated by the current child protection and maternity care systems. ConclusionTo address the tensions raised in this study, we suggest a two-fold change to maternity care for women at risk of an Assumption of Care: a therapeutic justice model of maternity care and continuity of midwifery care with a dedicated midwife. Introducing these changes could increase women’s and children’s safety and wellbeing.

MicroRNA-mediated interacting circuits predict hypoxia and inhibited osteogenesis of stem cells, and dysregulated angiogenesis are involved in osteonecrosis of the femoral head.

MicroRNAs (miRNAs) are associated with various pathologic conditions and can serve as diagnostic or therapeutic biomarkers. This study tried to identify the differentially expressed miRNAs to predict the possible pathomechanisms involved in osteonecrosis of the femoral head (ONFH). We compared the peripheral blood miRNAs in 46 patients with ONFH and 85 healthy controls by microarray and droplet digital polymerase chain reaction (ddPCR). Putative interacted networks between the differentially responded miRNAs were analyzed by web-based bioinformatics prediction tools. Microarray identified 51 differentially expressed miRNAs with at least twofold change (upregulation in 34 and downregulation in 17), and the results were validated by ddPCR using six selected miRNAs. Bioinformatics genetic network analysis focusing on the six miRNAs found the upregulated miR-18a and miR-19a are associated with angiogenesis after induction of ischemia; the upregulated miR-138-1 can inhibit osteogenic differentiation of mesenchymal stem cells; the most targeted genes, p53 and SERBP1, are associated with hypoxia and hypofibrinolysis. This study combined the miRNA analysis with the bioinformatics and predicts that hypoxia, inhibited osteogenesis of stem cells, and dysregulated angiogenesis might be orchestrated through the miRNA interacting circuits in the pathogenesis of ONFH.

Response Predictors to Calcineurin Inhibitors in Patients with Primary Membranous Nephropathy

Background: Currently, there is an urgent need to find ways of identifying primary membranous nephropathy (PMN) patients who are likely to benefit from calcineurin inhibitors (CNI) or who are resistant to them. In this study, we employed nano-HPLC-MS/MS analysis to identify serum biomarkers that predict the clinical response to CNI therapy in PMN patients. Methods: The endpoint was complete remission (CR) after CNI treatment. PMN patients were grouped into no-remission (NR) or CR groups to screen predictive candidates using the nano-HPLC-MS/MS analysis. Results: Compared with NR patients, 3 upregulated proteins and 5 downregulated proteins were found to present a twofold change in CR patients’ serum. Serum amyloid A1 protein (SAA1) was further validated by ELISA; it was decreased in patients in the NR group compared with patients in the CR group, but SAA1 in patients in these groups was lower than in healthy controls and minimal change disease patients. The area under the receiver operating characteristic (ROC) curve of SAA1 was used to distinguish PMN NR patients from those in remission and was 0.901, with a sensitivity of 78.3% and specificity of 86.8%, similar to that of the phospholipase A2 receptor (PLA2R) antibody. Combining SAA1 with the PLA2R antibody, the area under the ROC curve was 0.956, which was higher than that of SAA1 or the PLA2R antibody alone. Conclusions: Serum SAA1 may be a candidate PMN biomarker that can be used to discriminate CNI NR cases from remission patients. The combination of SAA1 and the PLA2R antibody increases the accuracy of diagnosis.

Plasma proteomic analysis reveals altered protein abundances in cardiovascular disease

BackgroundCardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis.MethodsLC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change.ResultsPeptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested.ConclusionsThis proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD.

Mutations in Neisseria gonorrhoeae grown in sub-lethal concentrations of monocaprin do not confer resistance.

Neisseria gonorrhoeae, due to its short lipooligosaccharide structure, is generally more sensitive to the antimicrobial effects of some fatty acids than most other Gram negative bacteria. This supports recent development of a fatty acid-based potential treatment for gonococcal infections, particularly ophthalmia neonatorum. The N. gonorrhoeae genome contains genes for fatty acid resistance. In this study, the potential for genomic mutations that could lead to resistance to this potential new treatment were investigated. N. gonorrhoeae strain NCCP11945 was repeatedly passaged on growth media containing a sub-lethal concentration of fatty acid myristic acid and monoglyceride monocaprin. Cultures were re-sequenced and assessed for changes in minimum inhibitory concentration. Of note, monocaprin grown cultures developed a mutation in transcription factor gene dksA, which suppresses molecular chaperone DnaK and may be involved in the stress response. The minimum inhibitory concentration after exposure to monocaprin showed a modest two-fold change. The results of this study suggest that N. gonorrhoeae cannot readily evolve resistance that will impact treatment of ophthalmia neonatorum with monocaprin.

High frequency of multiclass HCV resistance-associated mutations in patients failing direct-acting antivirals: real-life data.

Direct-acting antiviral (DAA) therapy has dramatically increased sustained virological response rates in HCV-infected patients. However, resistance-associated substitutions (RAS) interfering with NS3- and NS5A-targeted therapy, still emerge. This real-life study analysed the type and frequency of RAS in rare cases of patients failing DAA regimens in 12 clinical centres in Israel. Blood samples and clinical data from 49 patients who failed various DAAs were collected. RAS identified in the NS3 and NS5A regions by population (Sanger) and next-generation sequencing (NGS) were compared by treatment regimen and HCV subtypes. The majority (71.4%, 35/49) of patients were infected with the genotype (GT)1b strain, while 12.2% (6/49) carried GT1a and 14.3% GT3a/b (7), GT4a (1) and GT1b/GT3a. RAS were identified in 85.7% (42/49) of failures, of which 90.5% (38/42) were clinically relevant RAS (known to be associated with a specific GT and DAA in patients failing therapy or those with more than twofold change in in vitro replicon assays). The most abundant RAS were 168A/E/Q/G/N/V (32.6%, 16/49) identified in NS3, and 93H/N (61.2%, 30/49), 31I/M/V (34.7%, 17/49) and 30R/H/K (12.2%, 6/49), identified in NS5A. Significantly more clinically relevant RAS were identified in NS5A (82.2%, 37/45) than in NS3 (35.7%, 10/28; P<0.01). While RAS were identified in all GT1a, GT3b and GT4a failures (100%, 10/10), only 71.8% (28/39) of GT1b or GT3a failures had RAS (P=0.09). In four cases, NGS identified additional clinically relevant RAS and in one patient, NGS deciphered coexistence of GT3a and GT1b infections. Our findings, together with additional real-life data, will contribute to the optimization of retreatment in DAA failure, when cost-related and suboptimal regimens must be employed.

Proteomics detection of S100A6 in tumor tissue interstitial fluid and evaluation of its potential as a biomarker of cholangiocarcinoma.

Tumor interstitial fluid contains tumor-specific proteins that may be useful biomarkers for cancers. In this study, we identified proteins present in cholangiocarcinoma interstitial fluid. Proteins derived from three samples of tumor interstitial fluid and paired samples of adjacent normal interstitial fluid from cholangiocarcinoma patients were subjected to two-dimensional liquid chromatography with tandem mass spectrometry. Candidate proteins were selected based on a greater than twofold change in expression levels between tumor interstitial fluid and normal interstitial fluid. Upregulation of six proteins in tumor interstitial fluid, including S100 calcium binding protein A6 (S100A6), S100 calcium binding protein A9, aldo-keto reductase family 1 member C4, neuropilin-1, 14-3-3 zeta/delta, and triosephosphate isomerase was assessed by western blot and immunohistochemistry. Their potential as markers was evaluated in human cholangiocarcinoma tissue arrays, and in serum using enzyme-linked immunosorbent assay. Expression of S100A6 was higher in tumor interstitial fluid than in normal interstitial fluid and showed the highest positive rate (98.96%) in cholangiocarcinoma tissues. Serum levels of S100A6 did not differ between cholangitis and cholangiocarcinoma patients, but were significantly higher than in healthy individuals ( p < 0.0001). In cholangiocarcinoma cases, S100A6 level was associated with vascular invasion ( p = 0.007) and could distinguish cholangiocarcinoma patients from healthy individuals as effectively as the carbohydrate antigen 19-9. In addition, potential for drug treatment targeting S100A6 and other candidate proteins was also demonstrated using STITCH analysis. In conclusion, proteomics analysis of tumor interstitial fluid could be a new approach for biomarker discovery, and S100A6 is a potential risk marker for screening of cholangiocarcinoma.

Ethylene causes transcriptomic changes in Synechocystis during phototaxis.

Ethylene is well known as a plant hormone, but its role in bacteria is poorly studied. We recently showed that Synechocystis sp. Strain PCC 6803 has a functional receptor for ethylene, ethylene response 1 (Etr1), that is involved in various processes such as phototaxis in response to directional light and biofilm formation. Here, we use RNA sequencing to examine the changes in gene transcripts caused by ethylene under phototaxis conditions. Over 500 gene transcripts across many functional categories, of approximately 3700 protein‐encoding genes, were altered by application of ethylene. In general, ethylene caused both up‐ and downregulation of genes within a functional category. However, the transcript levels of amino acid metabolism genes were mainly upregulated and cell envelope genes were mostly downregulated by ethylene. The changes in cell envelope genes correlate with our prior observation that ethylene affects cell surface properties to alter cell motility. Ethylene caused a twofold or more change in 62 transcripts with the largest category of upregulated genes annotated as transporters and the largest category of downregulated genes annotated as glycosyltransferases which sometimes are involved in changing the composition of sugars on the cell surface. Consistent with changes in cell envelope, glycosyltransferase, and transporter gene transcripts, application of ethylene altered the levels of specific sugar moieties on the surface of cells. Light signaling from Etr1 involves two proteins (Slr1213 and Slr1214) and a small, noncoding RNA, carbon stress‐induced RNA1 (csiR1). Application of ethylene caused a rapid, but transient, decrease in the transcript levels of etr1, slr1213, and slr1214 and a rapid and prolonged decrease in csiR1 transcript. Deletion of Slr1214 caused a large increase in csiR1 transcript levels and ethylene lowered csiR1 transcript. These data combined with prior reports indicate that ethylene functions as a signal to affect a variety of processes altering the physiology of Synechocystis cells.

Altered expression of microRNA during fracture healing in diabetic rats.

ObjectivesDiabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM.MethodsClosed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint.ResultsMicroarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls.ConclusionsOur findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM.Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139–147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1.

Flow speed has little impact on propulsive characteristics of oscillating foils

Experiments are reported on the performance of a pitching and heaving two-dimensional foil in a water channel in either continuous or intermittent motion. We find that the thrust and power are independent of the mean freestream velocity for two-fold changes in the mean velocity (four-fold in the dynamic pressure), and for oscillations in the velocity up to 38\% of the mean, where the oscillations are intended to mimic those of freely swimming motions where the thrust varies during the flapping cycle. We demonstrate that the correct velocity scale is not the flow velocity but the mean velocity of the trailing edge. We also find little or no impact of streamwise velocity change on the wake characteristics such as vortex organization, vortex strength, and time-averaged velocity profile development---the wake is both qualitatively and quantitatively unchanged. Our results suggest that constant velocity studies can be used to make robust conclusions about swimming performance without a need to explore the free-swimming condition.

Expression and clinical role of long non-coding RNA in high-grade serous carcinoma

ObjectiveTo profile long non-coding RNA (lncRNA) expression at the various anatomic sites of high-grades serous carcinoma (HGSC) and in effusion-derived exosomes. MethodsLncRNA profiling was performed on 60 HGSC specimens, including 10 ovarian tumors, 10 solid metastases and 10 malignant effusions, as well as exosomes from 30 effusion supernatants. Anatomic site-related expression of ESRG, Link-A, GAS5, MEG3, GATS, PVT1 H19, Linc-RoR, HOTAIR and MALAT1 was validated by quantitative PCR and assessed for clinical relevance in a series of 77 HGSC effusions, 40 ovarian carcinomas, 21 solid metastases and 42 supernatant exosomes. ResultsSignificantly different (p<0.05) expression of 241, 406 and 3634 lncRNAs was found in comparative analysis of the ovarian tumors to solid metastases, effusions and exosomes, respectively. Cut-off at two-fold change in lncRNA expression identified 54 lncRNAs present at the 3 anatomic sites and in exosomes. Validation analysis showed significantly different expression of 5 of 10 lncRNAs in the 4 specimen groups (ESRG, Link-A, MEG3, GATS and PVT1, all p<0.001). Higher ESRG levels in HGSC effusions were associated with longer overall survival in the entire effusion cohort (p=0.023) and in patients with pre-chemotherapy effusions tapped at diagnosis (p=0.048). Higher Link-A levels were associated with better overall (p=0.015) and progression-free (p=0.023) survival for patients with post-chemotherapy effusions. Link-A was an independent prognostic marker in Cox multivariate analysis in the latter group (p=0.045). ConclusionsWe present the first evidence of differential LncRNA expression as function of anatomic site in HGSC. LncRNA levels in HGSC effusions are candidate prognostic markers.

Identification and expression analysis of microRNAs in medaka gonads

Gonad development is a highly regulated, coordinated biological process and increasing evidences have indicated that microRNA (miRNA) may be involved in this dynamic program. Medaka (Oryzias latipes) is a good model for reproductive research as it has distinct sex determining genes, however, research in gonadal miRNAs is lacked. In this study, two small RNA libraries from the ovaries and testes were constructed and sequenced. A total of 285 conserved and 388 novel miRNAs were obtained, among which 142 mature miRNAs were significantly (> two-fold change) up or down regulated in the testis compared to the ovary. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis showed that miR-430c, miR-26a and miR-202-5p were expressed in a gonad-specific or sex-biased pattern. Fluorescence in situ hybridization (FISH) indicated that miR-202-5p was present throughout spermatogenesis and was only detected at the early stages of oogenesis, this sex biased expression pattern suggested that miR-202-5p might be a crucial candidate in male differentiation and development. Our study provides the repertoire, a comprehensive annotation of miRNAs from gonads and a reference for functional studies of miRNAs in medaka.