Lophelia pertusa coral cover and morphology and for the cover of the associated sponges Mycale lingua and Geodia sp. Our results showed that the SE side was a better habitat for L. pertusa (including live and dead colonies). Low cover of Geodia sp. was found on both sides of Tisler Reef. In contrast, Mycale lingua had higher percentage cover, especially on the NW side of the reef. Bush-shaped colonies of L. pertusa with elongated branches were the most abundant coral morphology on Tisler Reef. The highest abundance and density of this morphology were found on the SE side of the reef, while a higher proportion of cauliflower-shaped corals with short branches were found on the NW side. The proportion of very small L. pertusa colonies was also significantly higher on the SE side of the reef. The patterns in coral spatial distribution and morphology were related to local hydrodynamics—there were more frequent periods of downwelling currents on the SE side—and to the availability of suitable settling substrates. These factors make the SE region of Tisler Reef more suitable for coral growth. Understanding the impact of local hydrodynamics on the spatial extent and morphology of coral, and their relation to associated organisms such as sponges, is key to understanding the past and future development of the reef.This study demonstrates how cold-water coral morphology and habitat distribution are shaped by local hydrodynamics, using high-definition video from Tisler Reef, an inshore reef in Norway. A total of 334 video frames collected on the north-west (NW) and south-east (SE) side of the reef were investigated for  Cold-water coral (CWC) ecosystems are protected in national and international waters. They are considered vulnerable marine ecosystems   and CTD casts on both the SE and NW sides of Tisler Reef  in relation to local hydrodynamics. We also assessed the densities of different coral growth morphologies on each side of the reef.Wagner et al.  found thCWCs at Tisler Reef were first discovered in 2002  during ALKOR cruise 232 in 2003. Data were processed using Fledermaus software resulting in a grid with pixel size of 8.22 × 8.22 m.R/V Lophelia from Tjärnö Marine Biological Laboratory  was used to deploy the ROV Sperre SubFighter 7500 DC to record high-definition video transects at Tisler Reef on 22 and 23 May 2014. Two video transects were collected on the SE end (dive 1 and 2) with a length of 239 m and 203 m, respectively. On the NW end, a single transect was collected (dive 3) (Table The 3) Table  conducteA Sony FCBH11 HD camera with two Sperre 200-W HMI lights was used to collect the video footage from an altitude of ~ 1 m. Video signals were transmitted over an optical fibre and recorded on compact flash cards using a nanoFlash recorder (Convergent Design). The ROV moved at an average speed of 0.7 knots. Two laser beams, spaced by 5 cm, were used as a reference to calculate the transect width as well as to measure the colony size. Navigation data from a Kongsberg Simrad USBL system type HPR 410P, a Simrad dGPS instrument and a Furuno satellite compass were integrated in the software package Olex to provide ROV navigation and transect position data.A video frame was extracted every 5 s from the video footage using the software VLC7. This extraction frequency is based on an average speed of 0.7 knots, meaning that a frame is analysed every ~ 1.8 m along the transect. The average surface area covered per image for each dive was calculated using the calibrating tool in Coral Point Count (see below) (Kohler and Gill L. pertusa) and sponges  , the coral colonies (live and dead ua) Fig.  was calcua) Fig. . This CPL. pertusa. The first is a compact “cauliflower” shape, which results from live coral branches growing symmetrically in multiple directions  transformation was applied to the percentage cover data of the substrate classes L. pertusa (live and dead) and the sponges (Geodia sp. and M. lingua). This transformation decreases the relative importance of high percentage coral cover values  and L. pertusa (live and dead), for which the Pearson product-moment correlation coefficient (r) was calculated in R. Since different morphology classes  were attributed to every coral colony, density was used instead of percentage cover. The densities of L. pertusa morphology classes were calculated by dividing the number of colonies by the average surface area per sub-transect.A 0.5 logL. pertusa, M. lingua, Geodia sp. and the different substrates) and L. pertusa morphology classes differed between the different locations.Statistical analyses used the PRIMER 6 software package   Fig. . The aveL. pertusa was weakly correlated  with Geodia sp. A stronger positive correlation  was found between dead L. pertusa and Geodia sp. Cover of both live and dead L. pertusa was strongly positively correlated with cover of M. lingua .Live cover of −2) than on the SE side   and small (n = 463) colonies had on average between 1 and 50 branches which were not large enough to visually assign a morphology class. Therefore, only the medium (n = 527) and large (n = 174) colonies were described as a function of shape and branch length. There were significant differences in coral morphology between the NW and SE side of the reef   on their skeleton prevents attachment of sessile epibiotic species . These high temperatures recorded at shallow depths between 90 and 120 m have resulted in mass mortality events , whereas a negative correlation occured at Sotbakken and Traena reefs (Purser et al. Geodia sp, M. lingua can use living L. pertusa as a substrate; they can grow within a colony (Lavaleye et al. M. lingua is much higher than that of Geodia sp. Interestingly, a higher percentage cover of M. lingua was found on the NW side of Tisler Reef. This finding seems surprising, as a higher percentage of L. pertusa, on which M. lingua can grow, was recorded on the SE side (Figs. M. lingua a competitive advantage (Rützler and Muzik A positive correlation between the cover of live and dead de Figs. b, 5c, d.L. pertusa in a CWC reef or on a CWC carbonate mound. De Clippele et al. (This study highlights differences in the spatial distribution of live versus dead coral framework and morphology within a reef, which are mostly related to variations in the substrate and local hydrodynamics. Buhl-Mortensen et al.  and Wheee et al.  showed te et al.  observede et al.  highlighe et al.  indicatee et al. . This st


Small-cell lung cancer (SCLC) is a malignant cancer with the ability to metastasize quickly. The relationship between tumor size and the distant metastasis patterns of Extensive-Stage Small Cell Lung Cancer (ES-SCLC) has not been reported.The aim of this study was to determine the different distant metastasis patterns as they related to tumor size in ES-SCLC.We used Surveillance, Epidemiology, and End Results (SEER) population-based data collected from 2010 through 2013 to identify 11058 ES-SCLC patients with definite evidence of distant metastases. Multivariate logistic regression analysis was used to demonstrate the association between tumor size and distant metastasis patterns including bone, liver, brain, and lung metastases. Age, race, sex, and N stage were also selected in the logistic regression model.P = 0.003) and patients with tumors ≥7 cm have a higher risk of lung metastasis . In addition, patients with tumors ≥7 cm had a lower risk of brain metastasis and liver metastasis than patients with tumors ≤3 cm . Interestingly, there was no correlation between a larger tumor and a higher risk of metastasis. However, the tumor metastasis pattern did have some correlation with age, gender, race and N-status.Subtle differences in metastasis patterns were found among patients based on different tumor sizes. Patients with tumors 3–7 cm have a higher risk of bone metastasis compared with those that have tumors ≤3 cm  in ES-SCLC is clinically valuable. Small-cell lung cancer (SCLC) is an extremely aggressive malignancy with approximately 31,000 cases diagnosed annually in the United States. It comprises 14% of total lung cancer diagnoses . SCLC isIn this study, the Surveillance, Epidemiology, and End Results (SEER) database was used to analyze the relationship between the size of the tumor and the sites of distant metastasis.This study was a retrospective study with data obtained from the SEER registry of the US National Cancer Institute . The SEEThis study was in compliance with the Helsinki Declaration and approved by the Ethics Committee of the Shandong Cancer Hospital. This study was based on the SEER database and did not involve personal privacy information so no informed consent was required.P < 0.05 was considered statistically significant.The variables of age, race, gender, tumor size, N-stage, and metastatic site were included in a multivariate logistic regression analysis to examine the association between clinically relevant factors and specific metastatic patterns. The binomial logistic regression models were used to assess the comparative risk of metastasis. SPSS 22.0  was used for data analysis. All statistical tests were bilateral and From 2010 to 2013, 11,058 ES-SCLC patients were reported in the SEER database; their clinical characteristics are presented in All the possible combinations of metastasis patterns are summarized in The metastatic sites were identified as bone metastasis, brain metastasis, liver metastasis, and lung metastasis. According to the most recent Eighth Edition of the Tumor, Node, and Metastasis (TNM) Classification of Lung Cancer, tumors ≤1 cm, 1–2 cm, 2–3 cm, 3–4 cm, 4–5 cm, 5–7 cm, and >7 cm are staged as T1a, T1b, T1c, T2a, T2b, T3, and T4, respectively. In the T stage, ≤3 cm is classified as a T1 tumor, 3–7 cm tumors are classified as T2-T3, and tumors ≥7 cm are classified as T4 tumors . PatientP = 0.851), and race and gender were not an independent risk factor for lung metastasis . Compared with patients with tumors ≤3 cm, there is a higher risk of bone metastasis in patients with tumors 3–7 cm . Patients with tumors ≥7 cm have a higher risk of lung metastasis  but a lower risk of brain and liver metastases than patients with tumors ≤3 cm  P < 0.001; OR 0.747, 95% CI [0.672–0.830], P < 0.001). The relationships between the metastasis pattern and age, and gender, race, and N-status were simultaneously observed. Patients ≥65 years old had a lower risk of bone metastasis and brain metastasis  and a higher risk of lung metastasis (P < 0.001). Compared with whites, blacks, and others ethnic patients had a lower risk of bone metastasis and liver metastasis  and black races had a higher risk of brain metastasis (P = 0.004). We also found that the higher the N stage, the higher the risk of bone and liver metastasis  and only patients with N3 staging had a higher risk of lung metastasis (P < 0.001). However, with a higher N stage, the risk of brain metastasis was reduced (P < 0.001)  .Small-cell lung cancer (SCLC) is characterized by a rapid doubling time and early, widespread metastasis. The incidence of distant metastasis at the time of the initial diagnosis of SCLC was more than 60% and the most common metastatic sites were the liver, bone, brain, lung, and adrenal glands . The typp  <  0.001), the concentration of neuron-specific enolase , and histopathological types  were the independent risk factors for bone metastasis in patients with lung cancer (p = 0.003) and not with the larger tumor size of ≥7 cm . Previous research has examined three steps necessary for lung cancer cells to metastasize to bone: (i) escape from the primary tumor; (ii) entering the circulation; and (iii) colonizing the bone (P < 0.001) had a significantly lower probability than those with tumors ≤3 cm to develop brain metastasis. Similarly, a higher N stage correlated with a lower risk of brain metastasis . SCLC is a tumor that develops brain metastasis very early, typically because small cell lung cancer originates from pulmonary neuroendocrine cells and other potential candidate cells, such as alveolar type 2 cells  . The prog cancer . Anotherg cancer . The risthe bone . At the the bone . We hypothe bone . At the the bone , which ithe bone . A studythe bone . Our res 2 cells . There i 2 cells . It is p 2 cells . Other s 2 cells . Due to P < 0.001). Additional research is necessary to identify why patients with tumors ≥7 cm are not prone to develop distance metastasis, especially to the liver. For lung metastasis, we found that age, tumor size, and N stage were independent risk factors and that it was more likely for lung metastasis to occur when the tumor was ≥7 cm  and the N stage was higher . It is possible that the larger tumor size is associated with a higher probability of lymph node metastasis and local disease extension   and a higher risk of lung metastasis (P < 0.001). Ethnic patients had lower risk of bone metastasis and liver metastasis when compared with whites, blacks, and others  and black races had a higher risk of brain metastases (P = 0.004). The correlation between these clinical factors and distant metastatic sites may involve differences in population characteristics and further exploration is warranted. We also found that higher N stages  correlated with a higher risk of bone and liver metastasis. This is consistent with current reports that the volume and number of metastatic lymph nodes are closely related to the site of metastasis  and liver metastasis was the most common site of hematogenous metastasis in ES-SCLC . Some stnvasion) . Finallytastasis .There are some limitations to this study. First, it is not possible to assess the impact of chemotherapy, radiation therapy, or smoking status on metastasis because this information is missing from the SEER database. Secondly, the aim of this study was to determine the different distant metastasis patterns based on tumor size in ES-SCLC and we only analyzed the relationship between tumor size and the distant metastatic patterns. When extracting data from the SEER database, we did not extract the corresponding information about survival rates. Thirdly, this study is a non-randomized study and although our sample size is large, there are inherent defects in any retrospective study. Fourth, the sites of metastasis that were analyzed were limited to the bone, lungs, liver, and brain. Although the common sites of SCLC metastasis are bone, liver, lungs, and brain , metastaThe pattern of distant metastasis of ES-SCLC is related to the tumor size and the tumor size is indicative of the metastatic site. Larger tumor sizes did not correlate with a higher risk of distant metastasis, but the size is related to the pattern of distant metastasis. The study of different distant metastasis patterns based on tumor size and other clinical features  in ES-SCLC is clinically valuable.10.7717/peerj.8163/supp-1Supplemental Information 1Click here for additional data file.


New Zealand dairy farmers place cows in uncovered off-paddock facilities for a part  of the day to reduce their impact on pasture during winter. Lying time is a key indicator of whether these hybrid pasture systems are meeting welfare requirements. While it is easy to measure lying time in a research setting using accelerometers, it is not yet common to measure it on farm, and more convenient indicators are needed. We investigated the lying behaviour of dairy cows as bedding quality deteriorated over a five-week period to determine what measures may be useful as farmer-friendly indicators of cow welfare. The daily lying time of the two groups of cows on bedding according to normal farm practice (NFP) declined over the five weeks and was significantly less than that of cows on fresh bedding during the last 10 days of the trial. The fresh woodchip bedding contained less than 65% moisture throughout the trial. By week 3 and week 4, the moisture content of the lying surface exceeded 75% for NFP Group 2 and Group1, respectively. A bedding moisture threshold of 75% assessed by a ‘gumboot score’ and cows lying rather than grazing when on pasture may be useful farmer-friendly indicators of cow welfare because of their relationship with reduced lying time.2/cow, with 6 h/d on pasture for 5 weeks. Lying times were recorded continuously for 60 cows per group using accelerometers. Bedding moisture content was measured weekly. Data for each NFP group were analysed and compared with those of their respective FB group using repeated measures. The lying time declined over five weeks from 11.6 h/day during the first week to 5.6 h/day during the fifth week . The moisture content of the bedding increased over the five weeks and was significantly higher for both NFP groups  than those of the respective FB groups. The percentage of total time lying when in the paddock increased for the NFP groups, reaching 15% in the last week of the trial. During five weeks on a stand-off pad, bedding quality deteriorated, and cows lay down less, to such an extent that welfare was compromised.Bedding quality and cow lying time were measured during five weeks in a normal farm practice (NFP) off-paddock system with no bedding refreshment. Two groups of 100 non-lactating dairy cows were compared to groups of 8 cows with fresh bedding (FB). The cows were on a woodchip pad for 18 h/d at a space allowance of 5.4 m New Zealand has a pasture-based dairy system; however, the temporary use of off-paddock facilities is becoming more commonplace, especially to reduce environmental impacts. This is particularly done to protect the soil and consequently pasture production from treading damage such as pugging . Having Lying behavior is important for adult cattle and has been used as a measure of cow comfort in numerous studies. After a period in which cows were prevented from both lying down and feeding, cows delayed feeding to lie down . In addiThe key environmental variable which determines lying time and hence welfare and appropriate facility usage is bedding quality. Access to dry bedding has been shown to be important for dairy cows . In uncoIn order to define good practice and to understand the welfare risks associated with the use of stand-off pads, we investigated the lying behaviour of dairy cows as bedding quality deteriorated over a five-week period. The aim of this study was to develop measures that farmers could use to determine if cows are receiving adequate rest and whether their welfare requirements are being met. To do this, we measured a suite of animal and bedding quality factors to determine their relationship to cow lying time.Prior to the start of this study, ethical approval was obtained from the Ruakura Animal Ethics Committee (protocol no. 13566), as required by the New Zealand Animal Welfare Act 1999.2/cow, again within the normal on-farm stocking rates for such a stand-off pad in New Zealand [In this study, 216 healthy non-lactating dairy cows were divided into two groups of 100 cows, paired with two groups of 8 cows, balanced for breed, liveweight, age and expected calving date . Cows we Zealand . For each normal farm practice group (NFP) of 100 cows, a paired group of 8 cows each was kept at the same stocking density on 6 × 7.2 m pens with woodchips, which were refreshed three times a week, and represented the fresh bedding (FB) groups. Throughout the study, FB 1 measures were collected at the same time as NFP 1 measures and, likewise, for FB 2 and NFP 2 measures. Because of practical constraints and resource limitations, the incorporation of a proper control was not feasible. Instead, the smaller FB groups served as comparative groups.2) and wind speed (m/s), were recorded throughout the trial at 10 min intervals using two portable weather stations  located in an unsheltered area within 10 m of the stand-off pads.Weather conditions, including air temperature (°C), relative humidity (%), rainfall (mm), solar radiation  set to record Once a week, gait score for the same 60 cows was recorded on a 5-point scale (based on Thomsen ) when thBedding measures from 10 stratified 25 × 40 cm quadrat samples across the gate, middle, edge and trough areas of each pad were taken weekly. Similarly, five samples were taken from the FB 1 and two pens at the same time. Samples of approximately 100 g were taken, weighed and dried at 70 °C for two days and then reweighed to calculate the percentage of dry matter (reported as moisture content).0—Clean, dry appearance woodchips—not distorted by standing on and nothing sticking to boots1—Mixture mud/woodchips, some mud sticking to the bottom of boots, spreading slightly on standing2—Surface soft/muddy, spreads on standing, and boots are left muddy 3—Surface may be runny or deep mud, i.e., it covers feet and stays on boots or it is difficult to lift the feet up out of “mud”A visual score on a scale of 0–3, ranging from 100% wood chips to greater than 75% mud/manure was taken along with a “Gumboot score” on a scale of 0–3 to describe the degree of muddiness of the lying surface:Animal measures and bedding quality data for each NFP group were analysed separately with those of their appropriate FB group . Unless otherwise stated, all analyses were conducted using GenStat18 version 19 .The data from the data loggers were downloaded using the HOBOware Pro software  and converted to daily summaries of lying behaviour using the SAS software  code desThe bedding quality data were analysed using repeated measures, formulated as a mixed model. The correlation between bedding measurements made on an individual quadrat over time was modelled using a power model of order 1. The fixed effects of Group , Area , Time and their interactions were tested, with the model selected by sequentially dropping statistically non-significant terms (tested at the 5% significance level). Fisher’s protected least significant difference at the 5% level was used to compare the NFP and FB group means. The weekly data for an average dirtiness score, i.e., total dirtiness/5, were analysed using repeated measures, formulated as a mixed model with effects for Group, Time and Group by Time interaction. The correlation between the measurements taken for an individual cow over time was modelled using a power model of order 1. Fisher’s protected least significant difference at the 5% level was used to compare the NFP and FB group means over time (Time). The average daily temperature over the trial was 9.4 °C . There was some rain on 21 of the 35 days of the trial; however, only on 8 days it was over 5 mm . These r1,25 = 351.56; p < 0.001; 32,223 = 8.27; p < 0.001) and NFP 2  groups were statistically significantly lower than those of the FB groups for the last 10 days  and after a fortnight on the pad , respectively. The daily lying time for the NFP groups declined over the five weeks from 11.6 h/d during the first week to 5.6 h/d during the fifth week  and after 2 weeks on the pad  for the NFP Group 2 .The percentage of cows that achieved 8 h lying decreased over time and was significantly different from the corresponding percentage in the FB groups on the same days considering total daily lying time, i.e., for the last 10 days for the NFP Group 1 (The decline in daily lying time was mainly driven by a decline in lying time on the pad. For NFP Group 2 (and to a lesser extent NFP Group 1), the time spent lying in the paddock increased after approximately two weeks, however, this did not compensate for the reduction of time spent lying on the pad. The percentage of time spent lying on the pad declined from over 65% in week 1 to less than 30% in week 5 . At the 3,27 = 6.85; p = 0.001: Trough 63.5%; Middle 65.1%; Gate 67.1%; Edge 68.4%; LSD (5%) ≈ 2.4). The moisture content of the bedding increased over the five weeks and was significantly higher for both NFP groups  than for the FB groups from week 1  and the gumboot scores , were significantly increased  and week 2 (NFP Group 1), the mean visual  produced a surface that cows were reluctant to lie on within three weeks and resulted in cows being severely deprived of lying. Lying times of less than 6 h per day have been shown to cause stress-induced perturbations of the hypothalamic–pituitary–adrenal axis  or exposFor comparison, the small group of cows on a pad with refreshed woodchips achieved lying times of 10.5 h/d throughout the five weeks of the trial. This lying time is similar to those other studies have measured with shorter exposures to woodchip pads  and for There was a significant effect of rain and time of rainfall on the average daily lying time. The lying time decreased 13 min per day per mm of rainfall. There was also a cumulative effect of the rainfall on bedding moisture content, particularly from rainfall events in the preceding 48 h, which had a resulting effect on cow lying times. Many studies have shown that cows prefer a dry surface to lie on ,13, and As in other winter studies ,32, cowsAs daily lying time on the pad decreased, cows lay down more when in the paddock, a consistent response even with shorter periods of stand-off than those used in this study ,32, SchüIn conclusion, during five weeks in a typical New Zealand stand-off system, bedding surfaces became unsuitable for lying, so that cows failed to meet the recommended daily lying times. We identified several indicators that could assist farmers to manage the quality of the bedding in such a system. A threshold of 75% bedding moisture correlated with the ability of the cows to achieve an adequate lying time, and this could be readily assessed by a combination of the visual and gumboot scores. Cow lying behaviour assessed in the paddock was an additional indicator of cow welfare due to its relationship with reduced lying times on the stand-off pad.


Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications. Sputum is a valuable source of cells, proteins, lipids, nucleic acids and oxidative products, and it is an ideal sample for studying lung physiology –4. AssesDespite the advantages, relatively few eukaryotic gene expression studies from sputum have been published to date, partly due to the challenges involved in the isolation of sputum RNA. Sputum samples are often difficult to obtain, need to go through a lengthy processing protocol, and therefore generally yield RNA of low quantity and quality with respect to RNA isolated from other biological samples. The ubiquitous presence of bacteria in sputum samples aggravates the problems even further.Previously, we have developed a combined sputum processing/RNA extraction/reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocol to maximize the quantity and the quality of sputum RNA in order to enhance detection of transcripts by qPCR . The eliIn this study we aimed to improve the efficiency of eliminating contaminating DNA without affecting RNA quality. Tested modifications to common RNA isolation procedures included prolonged DNase treatment and bead vortexing of sputum cell fractions during cell lysis, a recently developed technique for disrupting tough-to-lyse bacteria in biological samples.6 inflammatory cells per cryotube. From each sputum sample as many aliquots were prepared as possible. Aliquots were centrifuged and finally the pellet was re-suspended in 150 μL RNAlater solution  and stored at -80°C. The research protocol was approved by the Hungarian Scientific and Research Ethics Committee of the Medical Research Council , and all subjects gave written informed consent to participation in the study.Induced sputum samples were collected from 10 clinically stable patients with chronic obstructive pulmonary disease (COPD)  during routine ambulatory visits. Sputum induction and processing with dithiothreitol (DTT) was performed as previously described . Briefly6 sputum cells using either (i) Trizol Reagent , (ii) NucleoSpin TriPep kit , (iii) Direct-zol RNA MiniPrep kit  or (iv) RNeasy Plus Mini kit  as previously described [g for 1 min samples were loaded onto the corresponding RNA isolation column.RNA isolation was performed from 10escribed  with theIntegrity of the isolated sputum RNA was estimated using the cycle threshold (Ct) values of different base pair (bp)-length amplicons of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in qPCR assays as previously described .DNase digestion (1 hour and 24 hours) was performed after RNA isolation using the Turbo DNA-free kit .]. To check for contaminating human genomic DNA, GAPDH primers were used, which give a 121 bp long PCR product for cDNA and a 210 bp product for genomic DNA [12]. PCR was performed with Phire DNA Polymerase kit  as described previously . In pilot experiments alternative DNA polymerases, such as the 5PRIME HotMaster Taq DNA Polymerase kit , the Phusion DNA (Thermo Fisher Scientific) and the Pfu DNA Polymerase kits (Thermo Fisher Scientific) were also tested.The presence of contaminating bacterial DNA was checked by PCR using universal bacterial primers producing a 466 bp long amplicon ,14. To c14eviously . In piloAmplicons were identified by electrophoresis  on 2% agarose gel  cast in TBE buffer . For visualization 7 μL GR Safe nucleic acid stain  was used.0.5 μg of total RNA in 20 μL reaction volume was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Life Technologies) as previously described . Random Real-time PCR (qPCR) reactions were carried out with 2 μL cDNA sample in a total reaction mixture of 20 μL containing either 10 μL 2× iTaq Universal SYBR Green Supermix  and 250 nmol each of GAPDH forward and reverse primers giving rise to 75, 121, 225 and 406 bp long amplicons 9) or 10 μL 2× iTaq Universal Probes Supermix (Bio-Rad) and 1 μL GNB2L1 Taqman probe (Life Technologies Hs00914568_g1) as reference gene. Amplification in qPCR assays was performed as previously described [ or 10 μLt-test. Calculations were performed by GraphPad Prism 4.0 . A p value <0.05 was considered significant.Data are presented as mean±standard error of mean (SEM). Data distribution was analyzed by the Kolmogorov-Smirnov test. Ct values were compared using one-way analysis of variance with Newman-Keuls test for multiple comparisons. Comparisons between two groups were performed by the Student’s An integral part of the study protocol was to check for the presence of contaminating bacterial DNA in the isolated sputum RNA. PCR using universal bacterial primers was the method of choice and for this to be informative we had to make sure that no exogenous bacterial DNA was introduced during cDNA synthesis. Thus, a suitable DNA polymerase that was free from contaminating bacterial DNA had to be selected first. In pilot PCR experiments using a complete reaction mix but no RNA template it was found that the 5PRIME and the Phusion polymerases are contaminated with bacterial DNA, while the Phire and the Pfu kits presented no such problems . The PhiAs demonstrated previously, strictly following the manufacturer’s recommendations, none of the RNA isolating methods tested was capable to yield DNA-free RNA from sputum as the recommended 1-hour long DNase treatment proved insufficient to eliminate contaminating bacterial DNA from the samples . ProlongNext, we tested whether vortexing in the presence of various beads during the cell lysis step of RNA isolation protocols could facilitate the removal of bacterial DNA. We found that vortexing sputum cells with bashing beads and subsequent loading of the lysate onto the gDNA eliminator spin column of the RNeasy Plus Mini kit was necessary and sufficient to completely eliminate DNA from the samples . NotablyBead vortexing was less effective in combination with other RNA isolation methods  allows the efficient and complete elimination of bacterial DNA from samples using the RNeasy Plus Mini kit protocol. Bead bashing obviates the need for repeated or prolonged DNase digestion, and, as a consequence, RNA of better quality can be obtained, as evidenced by the improved threshold values for both short and long amplicons in qPCR assays.Although most RNA isolation protocols incorporate steps for eliminating DNA, those measures, if at all, only work for DNA already in solution. Certain bacteria are resistant to lysis buffers, therefore DNA within intact bacterial cells can escape measures implemented against DNA contamination during RNA isolation. Cells that withstand the cell lysis step could be a potent source for contaminating DNA during downstream applications that involve boiling samples, such as PCR ,16. The Efficient disruption of bacterial cells before RNA isolation may be a plausible explanation why the use of the gDNA eliminator column alone was insufficient, while in combination with bashing beads treatment it was sufficient for the removal of bacterial DNA from sputum samples using the RNeasy Plus Mini kit. This, however, does not explain why RNA isolated using either the Tripep or the Direct-Zol kits still contained significant amounts of DNA, despite pretreatment of samples with beads. These methods employ solid phase columns for DNA removal as well, but in contrast to the gDNA eliminator column in the RNeasy Plus Mini kit, those columns are incapable of completely eliminating DNA, that is why their respective protocols include an additional DNase step to work efficiently. Thus, it appears that the gDNA eliminator column uses some additional proprietary feature to eliminate the entire DNA in solution. Another plausible explanation for the behavior of these kits was mentioned by Korfhage et al., who demonstrated that highly fragmented nucleic acids often yield high levels of residual gDNA, because the smaller sized fragmented gDNA co-purifies with RNA . Taken tTesting DNA content left in the isolates we found that certain commercially available DNA polymerases are contaminated with DNA, confirming earlier observations ,19. It iIt is well known that using low-quality RNA may strongly compromise results of qPCR . The RNAAlthough bacterial cell walls can be disrupted by various treatments including enzymes, detergents and even sonication , bead bePreviously we demonstrated that by adapting the conditions of isolation to the special requirements of sputum, the quantity RNA can be greatly increased. In our present work we could show, that instead of repeated DNAse digestion, vortexing sputum cells in the presence of bashing beads improves the quality of sputum RNA, making it compatible with gene expression studies. Our results highlight the importance of selecting a suitable DNA polymerase for cDNA synthesis. We speculate that our findings can be applied to other types of respiratory samples, for example, bronchoalveolar lavage. Just like sputum, lavage is also a rich source of cells and macromolecules, but it may also contain bacteria whose removal poses a similar challenge during RNA isolation.Obtaining appropriate amounts of high-quality RNA is a critical step in all gene expression studies, including the ones that utilize sputum. Although in our previous study  we alreaS1 FigThe presence of contaminating bacterial DNA in the first two reactions is evidenced by the respective PCR product in lane 2 and 3. M: molecular weight marker.(TIF)Click here for additional data file.S2 FigError bars indicate SEM.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 TableThe amount of remaining bacterial DNA was visualized by gel electrophoresis; the semi-quantitative results are indicated (DNase treatment was omitted in each case).(PDF)Click here for additional data file.


In eukaryotes, most genes code for multiple transcript isoforms that are generated through the complex and tightly regulated process of RNA splicing. Despite arising from identical precursor transcripts, alternatively spliced RNAs can have dramatically different functions. Transcriptome complexity is elevated further by the production of circular RNAs (circRNAs), another class of mature RNA that results from the splicing of a downstream splice donor to an upstream splice acceptor. While there has been a rapid expansion of circRNA catalogs in the last few years through the utilization of next generation sequencing approaches, our understanding of the mechanisms and regulation of circular RNA biogenesis, the impact that circRNA generation has on parental transcript processing, and the functions carried out by circular RNAs remains limited.Here, we present a visualization and analysis tool, SpliceV, that rapidly plots all relevant forward- and back-splice data, with exon and single nucleotide level coverage information from RNA-seq experiments in a publication quality format. SpliceV also integrates analysis features that assist investigations into splicing regulation and transcript functions through the display of predicted RNA binding protein sites and the configuration of repetitive elements along the primary transcript.https://github.com/flemingtonlab/SpliceV and can be installed from PyPI using pip.SpliceV is an easy-to-use splicing visualization tool, compatible with both Python 2.7 and 3+, and distributed under the GNU Public License. The source code is freely available for download at The online version of this article (10.1186/s12859-019-2865-7) contains supplementary material, which is available to authorized users. The majority of mammalian genes code for multiple transcript isoforms that contribute substantially to the vast complexity of both the mammalian transcriptome and proteome E. T. , 38). Ea. Ea38]).While each exon-intron boundary inherently contains a splice site, contiguous exons are not always spliced together. Retained introns (Y. ), skippeThe same splicing reaction that generates mature mRNAs can also fuse a downstream splice donor to an upstream splice acceptor (much like tying the end of a string to the beginning), in effect circularizing the transcript . These cExploring the relationship between linear and circular RNA isoforms of a common parental gene can be facilitated by utilizing Next Generation Sequencing (NGS) technology. NGS based approaches have provided the framework to study the abundance of individual transcript isoforms at a large scale, allowing investigators to compare circular and linear isoform abundance. However, the majority of bioinformatic pipelines require prior knowledge of transcript structure. While useful for broad scale interpretations, these approaches fail to resolve the abundances of both linear and circular isoforms of each gene, the function of which can dramatically differ from one another. Between linear transcripts alone, alternatively spliced isoforms can code for proteins that are truncated , lack sphttps://github.com/flemingtonlab/SpliceV and can be installed from PyPI using the Python package manager, pip. SpliceV is written with a GNU 3.0 public license, provided with anonymous download and installation. Full usage information can be found in Additional file Our software package is written in Python 3 but is backwards compatible with Python 2.7, relying only upon the third-party libraries, matplotlib , and pysRay et al [ALU repeat elements impact RNA secondary structure, we have also incorporated the option to add a track of ALU elements to the plot.SpliceV generates plots of coverage, splice junctions, and back-splice junctions with customizable parameters, depicting expression of both the linear and circular isoforms of a given gene. Standard formats  are accepted as input files. BAM files are sequentially accessed by our software . In practice, this means that SpliceV first determines the chromosomal coordinates that mark the beginning and end of the input gene. Next, it extracts reads that fall within that range from each BAM file (one BAM file at a time). As BAM files are indexed , this process never requires loading of the entire file into memory, and we have no reason to believe that a personal laptop computer would have difficulty running SpliceV on many BAM files at once. Because junction calling sensitivity can be improved using specialized software, canonical and back-splice junction information can be extracted directly from BAM files or input separately as BED-formatted files containing the coordinates and quantities of each junction. The user is provided the flexibility of normalizing expression of each exon across all samples or for exon normalization to be confined within each sample . As introns are generally much larger than exons, an option to reduce intron size by a user-defined amount is also provided. In an effort to guide interpretation of gene specific splicing patterns, predicted or empirically determined RNA binding protein binding sites can be added to the plots Fig.  by supplay et al . BecauseSPPL2A  and user supplied ALU element sites  . Whole EHere we present a new tool, SpliceV, that facilitates investigations into transcript biogenesis, isoform function and the generation of publication quality figures for the RNA biologist. SpliceV is fast (taking full advantage of the random access nature of BAM files), customizable , and can filter data and make cross-sample comparisons. It is modular in structure, allowing for the inclusion of new features in future package releases. SpliceV should provide value to the toolkit of investigators studying RNA biology and function and should speed the time frame from data acquisition, data analysis to publication of results.Project name: SpliceVhttps://github.com/flemingtonlab/SpliceVProject home page: Operating system: Platform independentProgramming language: PythonOther requirements: Python 2.7 or Python 3.0+License: GNU Public LicenseAny restrictions to use by non-academics: License neededAdditional file 1:Command line parameters for SpliceV. (PDF 251 kb)Additional file 2:An example pipeline to generate the required files for SpliceV analysis. (PDF 148 kb)


Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA–RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA–RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.To maintain genome integrity, segmented double-stranded RNA viruses of the  Reoviridae family of double-stranded (ds) RNA viruses encompass important human, animal and plant pathogens  with an AKTA pure system  connected to a DAWN HELEOS (Wyatt).σNS–RNP complex was prepared by mixing σNS (175 μM monomer) with 20mer unstructured RNA 70 μM,  to ensurI(q) versus momentum transfer q , were collected using SEC-SAXS on beamline B21 at Diamond Light Source  over a range of q of 0.004 < q < 0.442 Å−1. A total of 50 μl of each sample (∼175 μM) was loaded onto a 2.4 ml Superdex 200 Increase 3.2 column mounted on Agilent HPLC High Pressure Liquid Chromatography (HPLC) system, and the eluent was flowed through the SAXS beam at 0.04 ml/min. The SEC buffer used as the background was collected after one SEC column volume. SAXS data were collected at 1 s intervals using a Pilatus 2M detector  at a distance of 3.9 m and an X-ray wavelength of 1 Å. Guinier plot fit and real space inversions were performed using Primus (gR) were estimated using AUTORG  samples were prepared in a similar manner, whereby σNS was incubated with saturating amounts of 20mer or 40mer RNA , as descg Primus . Radii og AUTORG  in slow g AUTORG  and DAMF−1. Data were processed with MassLynx v4.1, Driftscope 2.5 (Waters) and Massign  with platinum/gold-plated borosilicate capillaries prepared in-house. Typical instrument parameters were: capillary voltage, 1.2–1.6 kV; cone voltage, 40 V; trap collision voltage, 6 V; transfer collision voltage, 10 V; trap DC bias, 20 V; backing pressure, 4.5 mbar; IMS gas pressure, 0.5 mbar; traveling wave height, 7 V; and traveling wave velocity, 250 ms Massign  using ar Massign ) were sen IMPACT . Estimat2. Raw Anisotropy (r) values were calculated as follows:Fluorescence anisotropy measurements with AlexaFluor488 dye-labeled RNAs  were perWhere Kobs on buffer ionic strength can be expressed as:+M] is the monovalent counterion concentration (in this case Na+), m’ is the number of ion pairs formed, ψ is defined as the thermodynamic extent of counterion binding and Kobs,1M is the non-electrostatic contribution to the dissociation constant, defined as the Kobs at 1 M NaCl, when the polyelectrolyte effect is minimal  against log[M+] relates to the number of counterions released upon binding as follows:The dependence of  minimal ,32. The PE, can therefore be determined as:obs is the total free energy of binding, and ΔGnPE is the non-electrostatic (non-polyelectrolyte) contribution. As the parameter ψ varies between different polynucleotides, we estimated the amount of salt bridges using empirically determined values, poly(U) = 0.68 and poly(A) = 0.78 as the upper and lower limits of salt bridges involved in complex formation (The electrostatic (poly-electrolyte) contribution to free energy of binding, ΔGormation ,32. As Normation , a range−1, containing 3′-donor (ATTO532) and 5′-acceptor (ATTO647N) dye fluorophores as a FRET pair (EFRET(app)) were calculated as follows:A dual-labeled RNA stem-loop was designed with an MFE of folding of −8.9 kcal molRET pair . Dual-laIATTO532 and IATTO647N are donor and acceptor emission intensities, respectively. For probing RNA:RNA interactions between S5f and S11f fragments (2) and snap-cooled to 4°C for 20 min, ensuring complete stem-loop folding prior to co-incubation. A total of 10 nM of S5f and S11f were incubated at 37°C in annealing buffer  and immediately analyzed by electrophoresis on a native 15% acrylamide-TBE gel, run at 80 V at 4°C, and stained with 0.01% (w/v) SYBR gold. Gels were imaged using a fluorescence scanner as described above, using 488 nm excitation.Where ragments , 100 nM Equimolar amounts of 18-nt Cy3- and Cy5-labeled non-complementary RNAs (10 nM each)  were inc−1.Circular dichroism (CD) experiments were performed on a Chirascan plus spectrometer (Applied Photophysics). Samples were prepared by dialyzing protein solutions against 10 mM phosphate buffer pH 7.4, 50 mM sodium fluoride. Spectra were recorded over a wavelength range of 190–260 nm, with a bandwidth of 1 nm, step size of 1 nm and a path length of 1 mm. An average of three scans were used for the final spectra. Thermal stability was analyzed by monitoring the CD signal at 222 nm during heating from 20°C to 70°C with a heating rate of 1°C minDual-labeled stem-loop  was heatE, were calculated from background-corrected photon counts in the donor channel and acceptor channel after donor excitation . Single-pair FRET measurements were performed on a custom-built confocal microscope with multiparameter fluorescence detection and pulsed interleaved excitation (PIE)  as descrPAM software package , as described in detail elsewhere (−1) using the emission spectra of a neon glow lamp taken before and after each Raman measurement. The Raman contribution from corresponding buffer was subtracted, and the spectra were corrected for non-Raman background.Raman spectra of RNA, σNS and RNP and their corresponding buffers were acquired on a modular multi-channel Raman spectrograph Jobin Yvon–Spex 270M in 90° scattering geometry using 532 nm line of a continuous-wave solid-state Nd:YVOlsewhere . Raman min vitro  of ∼8 nm than the apoprotein (Rh ∼ 5 nm)  Figure . This diRg) values for σNS apoprotein and RNP complex were 5.5 ± 0.03 nm and 7.6 ± 0.05 nm, respectively  value from 19.9 to 41.6 nm . A typical ESI-MS spectrum shows σNS hexamers along with additional oligomeric species, in agreement with previous analysis of σNS  Figure . In cont2) are in good agreement with the CCS value estimated for the apoprotein SAXS envelope (102.2 ± 0.5 nm2)  closely matches the size of the SAXS envelope generated for the RNP complex (119.4 ± 1.0 nm2). Thus, simultaneous binding of two RNAs results in the formation of stable, elongated octameric σNS–RNP complexes.We then used ion-mobility mass-spectrometry to estimate rotationally averaged collision cross-sections (CCSs) for each observed species . We comp) Figure . This isEFRET(app)) of 0.73 ± 0.03, suggesting that the stem-loop is folded. The RNA hexameric shift had EFRET(app) of 0.48 ± 0.11, and this value decreased further for the octameric shift (EFRET(app) = 0.28 ± 0.05), indicating that octameric σNS has a greater capacity for destabilizing RNA structure than hexameric σNS FRET to analyze the FRET states of discrete populations of protein-free and oligomer-bound RNAs using the dual-labeled RNA construct described above Figure . For steSimilarly, we analyzed RNA stem-loop destabilization by σNS Figure . While tEnsemble FRET experiments conducted at a wider range of RNA and protein concentrations further demonstrate that NSP2 is more efficient at destabilizing RNA than σNS . CollectG = −3.8 kcal mol−1), and a stable hairpin structure    Figure . All thr) Figure .2+ ions, which stabilize RNA structures , there is a 10- to 30-fold decrease in affinity of both NSP2 and σNS for the metastable and stable RNAs   . Despiteroteins) , with earoteins)  reveals Reoviridae family may exploit distinct mechanisms of regulating RNA chaperone activities underpinning genome segment assortment. We propose that the stability of RNA structure together with distinct unwinding mechanisms underpins the observed selectivity of RNA–RNA interactions. This may serve to regulate selection of genomic RNA segments to achieve assembly of a complete set of cognate genomic RNAs with high fidelity.Our results suggest that different members of the https://www.sasbdb.org/), ID numbers: SASDDT5 (σNS–RNP), SASDDU5 (σNS apoprotein).SAXS models reported in this paper can be accessed at SASBDB (Supplementary DataClick here for additional data file.


Non-Coding RNA Scientific Board wishes you an exciting and fruitful read.We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently. We hope to keep you up-to-date with non-coding RNA research works that are outside your study area. The Cell, Eimer et al. showed that an intronic triplet expansion can trigger the biogenesis of short interfering RNAs (siRNAs), leading to the transcriptional downregulation of the afflicted gene [Although it is known that trinucleotide repeat expansions cause a number of neuromuscular disorders , the exact mechanisms through which gene functions are affected remain uncertain for most of these diseases. In a recent issue of ted gene .Arabidopsis ISOPROPYL MALATE ISOMERASE LARGE SUBUNIT 1 (IIL1) gene leads to its downregulation and a growth defect at elevated temperatures. Here, they demonstrated that this downregulation is associated with a transcriptionally repressed chromatin state characterized by an abundance of histone H3 lysine 27 trimethylation (H3K27me3) marks. Apparently, the triplet expansion leads to the production of double-stranded RNAs, which are converted to siRNAs by DICER-LIKE 3 (DCL3). The siRNAs, through the RNA-dependent DNA methylation (RdDM) pathway and the polycomb repressive complex (PRC), direct the repressive epigenetic modification at the IIL1 locus.Previously, Sureshkumar Balasubramanian’s group showed that an intronic GAA/TTC repeat expansion at the These findings suggest that siRNA-mediated silencing could be a possible mechanism for diseases such as FRDA. Future studies will shed light on whether the underlying mechanisms of certain repeat expansion disorders are conserved across systems and/or kingdoms.Cell [RNA molecules lead fascinating lives, and from the beginning until the end, they encounter many interacting partners. We have entered the era of RNA interactomics, and new technologies are necessary to profile these interactions toward illuminating their physiological relevance. The XRNAX (protein-crosslinked RNA extraction) method, recently published in Cell , expandsSimilar to plethora techniques for mapping RNA–protein interactions, XRNAX utilizes UV irradiation to crosslink protein and RNA, in vivo, with TRIzol extraction performed to harvest the complexes. However, unlike other approaches, the authors collect the interphase between the aqueous and non-aqueous phases, which are usually carefully omitted in the standard TRIzol extraction protocol, and enrich the desired RNA–protein complexes. The high-throughput mass spectrometric and next-generation sequencing analyses of these complexes delineated both the interacting proteome and transcriptome, respectively, with such resolution as to identify a novel RNA binding motif. Uniquely, XRNAX achieved the first identification of the human non-coding RNA binding proteome, with over 700 proteins identified, including BRCA1 and confirmed interactions for TP53, which was previously a controversial interacting partner for non-coding RNA.XRNAX is the newest member of a family of RNA interactomics techniques, offering great potential to uncover the secrets of the complex lives of non-coding RNAs.Telomerase is a ribonucleoprotein complex that extends the chromosomal termini known as the “telomere” by adding DNA repeats and thus neutralizing the continuous shortening of DNA due to incomplete DNA replication. The telomeric repeat addition processivity ascends from the species-specific usage of telomerase RNA (TR) template domain, while the catalytic activity is mediated by telomerase reverse transcriptase (TERT) activity. However, in the absence of a three-dimensional model of telomerase, the exact mechanism of telomerase-regulated telomere lengthening remained elusive.Recent cryo-Electron Microscopy (EM) models of both human  and TetrThese models have improved our understanding about the mechanistic properties of the telomerase holoenzyme and should provide paths to explore structure-function relationship of this important ribonucleoprotein complex for developing new therapeutics.MALAT1 gene in MMTV-PyMT (mouse mammary tumor virus-polyomavirus middle T antigen) mice leads to a higher existence of circulating tumor cells (CTC) and causes an upregulation of tumor-promoting transcription factors such as TEAD1 (TEA domain family member 1). They demonstrated that MALAT1 inhibits the interaction between TEAD1 and its cofactor YAP (yes-associated protein 1) by blocking the transactivation domain of TEAD1. In further consequence, MALAT is able to downregulate metastasis, promoting proteins such as integrin β4 (ITGB4) and vascular endothelial growth factor (VEGFA) [The dysregulated expression of long non-coding RNAs (lncRNAs) influences the development of many different cancer types. In a recently published study, Kim et al. have shown that an overexpression of lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) promotes breast cancer metastasis. Knockout of the  (VEGFA) .The results of Kim et al. are in strong contrast to other related publications due to their remarkable model systems and experiments. The function of MALAT1 as a tumor-suppressing factor refutes the previous hypotheses and shows the tasks of lncRNAs from a different angle. The new insights into the regulatory mechanisms of lncRNAs may allow new therapeutic options for breast cancer patients in the future.Long non-coding RNA (ncRNA) transcripts have several of the same features as protein-coding mRNA, yet long ncRNAs are often less efficiently spliced than mRNAs. This observation has fueled debates on whether splicing controls the function and localization of long ncRNAs. Krchnáková et al. have addressed this important question using ncRNA-a2 , an actiBy a more specific analysis of splice-site sequences, the authors showed a positive correlation between the strength of the 5′ splice-site and the polypyrimidine tract and long ncRNA splicing efficiency. The authors provide evidence that long ncRNAs are more dependent on basic splice-site sequences than mRNAs due to the decreased productive binding of SR proteins to long ncRNAs.In addition, the authors reported that removing the intron from ncRNA-a2 does not affect its enhancer-like function, suggesting that splicing is not essential for function for at least a group of long ncRNAs.Communication is a key feature in cardiovascular biology, and cells in multicellular organisms communicate with each other via a number of mechanisms, including direct cell–cell contact, cell–matrix interaction, long-range signals, electrical signals, and extracellular chemical molecules. In this sense, extracellular vesicles and exosomes represent an emerging field of investigation. Cheng et al. demonstrated that myocardial microRNAs (myomiRs) carried in circulating exosomes (cardiosomes) allow a systemic response to cardiac injury . The autAnnals of Surgery, Meijer et al. provided very exciting evidence that plasma levels of miR-181a-5p might actually offer a promising biomarker potential that can predict the response to FOLFIRINOX in patients with pancreatic cancer [Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, and the majority of patients present with advanced disease, when the disease is mostly unresectable. In PDAC patients, the first-line treatment consists of a combination regimen of 5-fluorouracil, oxaliplatin, irinotecan, and leucovorin (FOLFIRINOX), or gemcitabine (GEM) plus nanoparticle albumin-bound paclitaxel (nab-paclitaxel). However, unfortunately, only a very small subset of patients respond to such treatments, and an appropriate selection of patients who might benefit from such treatment modalities requires the availability of biomarkers that can guide decision-making, which is currently lacking. In a recent issue of In this study, the authors used a microarray-based profiling approach to discover deregulated miRNAs, in both pre-chemotherapy and post-chemotherapy plasma specimens, in PDAC patients. Based upon their progression-free survival (PFS) following FOLFIRINOX treatment, the investigators identified a panel of nine candidate plasma miRNAs that could predict the response to this first-line therapy. Thereafter, the expression of these nine miRNAs was validated in an independent patient cohort, which led to the identification of the most significant miRNA biomarker: miR-181a-5p. The plasma levels of miR-181a-5p were significantly downregulated in patients with non-progressive disease following FOLFIRINOX therapy. Furthermore, in multivariate analysis, a combination model comprising of miR-181a-5p expression and CA19-9 levels significantly correlated with improved PFS and overall survival. To add another layer of specificity, the authors were also able to confirm the correlation between the expression of this miRNA between tissue and plasma specimens, highlighting its potential as a non-invasive biomarker.These results are truly exciting, as these provide a glimpse into the potential of miRNAs, especially as non-invasive, liquid biopsy biomarkers, for making a significant clinical impact for appropriate patient selection, which is an important step forward as we usher into the era of precision medicine for patients suffering from a lethal malignancy such as pancreatic cancer.A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene, encoding a likely guanine nucleotide exchange factor, was uncovered in 2011 as an associative allele for hereditary amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). C9orf72 repeats lie within a first intron and can be retained in transcripts. Repeat-associated RNAs from both sense and antisense strands are observed in patient-derived cells, with some experimental evidence for their translation. Thus, peptides from either strand are possible, with poly-PR dipeptides produced from antisense RNAs.Science [A 15th February paper published in Science  evaluateThe developmental context of the experiments may help capture snapshots of progressive neuroinflammation, leading to cell death—a feat that is not possible using post-mortem tissue. Future experiments will help determine whether this largely transgene-based approach is germane to the molecular pathology associated with human disease.Cancer Cell [Long non-coding RNAs (lncRNA) play an important role in tumorigenesis. Epigenetic alterations have been established as one of the hallmarks of tumorigenesis, but their involvement on lncRNA gene regulation was still unclear. In a recent issue of cer Cell , Wang etEPIC1) has been identified as the most epigenetically activated lncRNA in several cancers. Breast cancer patients with EPIC1 hypomethylation and increased EPIC1 expression have the worst survival. Further analysis indicated that EPIC1 epigenetic activation associated with luminal B and Her2 subtypes of breast cancer. EPIC1 has been shown to promote cell proliferation, anchorage independent growth, cell cycle progression in breast cancer, and in vivo xenograft tumor growth, suggesting the oncogenic function of EPIC1, and thus could be a good target for breast cancer treatment. RNA sequencing and other molecular analysis revealed that oncogenic function of EPIC1 is through the association with the MYC protein. The authors demonstrated that EPIC1 interacts in the nucleus with the 148–220 aa region of MYC through its 129–283 nt region and specifically regulates MYC’s occupancy on a subset of MYC targets, enhancing its heterodimerization with MAX and its activity as transcription factor.Epigenetically induced lncRNA  were found to accumulate in the saturated status, which were then identified as “stable introns” [Previously, Morgan et al. performed RNA-seq analysis on two introns” . ECM33 iIn the same issue, a companion paper from Sherif Abou Elela’s group also demonstrated the surprising function of introns on cell response to starvation in yeast . However


The RNA exosome is an evolutionarily conserved 3′–5′ exoribonucleolytic protein complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA exosome, few studies have been performed on the localization and expression of exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level.Lithobates catesbeianus and analysis of the differential subcellular localization of RNA exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four exosome subunits in different organs. In addition to being part of the RNA exosome complex, its subunits might participate independently of the complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species.We report the identification of exosome subunits in The online version of this article (10.1186/s13104-019-4077-7) contains supplementary material, which is available to authorized users. The eukaryotic RNA exosome is an essential multisubunit ribonuclease complex that participates in degradation or processing of all classes of RNA in the nucleus and cytoplasm. Nuclear RNA exosome is involved in 3′–5′ processing of rRNAs, snRNAs and snoRNAs, degradation of hypomodified tRNAs, and cryptic unstable transcripts (CUTs), whereas cytoplasmic RNA exosome is responsible for the degradation of both normal and aberrant mRNA species, in addition to being involved in degradation of interfering RNAs and ncRNAs , 2.The RNA exosome was first identified in yeast and subsequently found in other eukaryotes as well as in archaea –6. The eSaccharomyces cerevisiae exosome subunit yRRP6 has been shown to have its expression levels decreased from vegetative growth to meiosis and spore formation [ormation . Based oLithobates catesbeianus is characterized by morphofunctional testicular changes that occur in the seasonal reproductive cycle, divided in two phases: quiescent (winter) and active (summer), making the bullfrog testis an interesting model to study spermatogenesis [As in other amphibians, the spermatogenesis of the bullfrog ogenesis –12.L. catesbeianus for the study of the RNA exosome in testis during spermatogenesis and in non-reproductive tissues. We analyzed the localization of RNA exosome subunits in testis, focusing on interstitial, and the primordial germ cells (PGCs). Our data show that, except for RRP6, the localization of exosome subunits is similar in the testicular cells during reproductive and quiescent periods in PGCs. In addition, contrary to testis, a significant variation in the levels of exosome subunits was detected when analyzing non-reproductive tissues in the same periods, suggesting that individual RNA exosome subunits might play different roles during cell differentiation.In this work, we used Lithobates catesbeianus) were obtained from RANAMAT frog farm in Matão , in March (summer) and July (winter). The animals were divided into summer  and winter  groups, respectively.Ten adult male bullfrogs , and 5 ng/mL of the protease inhibitors Pepstatin, Leupeptin, Aprotinin, Antipain and Chymostatin). Crude extracts were clarified by centrifugation at 13,000 rpm for 20 min. Protein concentrations were determined with bicinchoninic acid (Sigma-Aldrich).Bullfrog frozen organs were homogenized directly in lysis buffer . Western blot was performed by incubation of the membranes with the primary antibodies against human proteins and anti-rabbit secondary antibody. Anti-human actin was used as internal control. The immunoblots were developed using the Luminata Forte Western HRP Substrate (Millipore) and quantification was performed using ImageJ software.L. catesbeianus, Xenopus and human.CLUSTAL Omega  was usedL. catesbeianus testis, we first compared the amino acid sequences of the bullfrog RNA exosome subunits, obtained from a Bullfrog Annotation Resource for the Transcriptome (BART) derived from de novo assembled bullfrog RNA-seq data [Xenopus and human , Xl (Xenopus laevis—NP_001089320.1) and Hs (Homo sapiens—NP_057126.2). (*), identity; (:), strong similarity; (.), weak similarity. The amino acids residues involved in the RNA interactions, in the subunits interactions and those that are possible targets of posttranslational modifications are highlighted. CLUSTAL Omega was used for the sequence alignment [Figure S2. Multiple sequence alignment of LcRRP6. The full sequence of LcRRP6 and its putative orthologues from Xenopus and human were aligned. Numbers correspond to amino acid position in each protein. Proteins access numbers: Xt , Xl (Xenopus laevis—NP_001084822.1) and Hs (Homo sapiens—NP_001001998.1). (*), identity; (:), strong similarity; (.), weak similarity. The amino acids residues present in the active site and those that are probable targets of posttranslational modifications were highlighted. CLUSTAL Omega was used for the sequence alignment [Figure S3. Multiple sequence alignment of LcRRP42. The full sequence of LcRRP42 and its putative orthologues from Xenopus and human were aligned. Numbers correspond to amino acid position in each protein. Proteins access numbers: Xt , Xl (Xenopus laevis—NP_001086766.1) and Hs (Homo sapiens—BC012831). (*), identity; (:), strong similarity; (.), weak similarity. The amino acids residues involved in the interactions with the hexamer and RRP4 were highlighted. CLUSTAL Omega was used for the sequence alignment [Figure S4. Multiple sequence alignment of LcRRP43. The full sequence of LcRRP43 and its putative orthologs from Xenopus and human were aligned. Numbers correspond to amino acid position in each protein. Proteins access numbers: Xt , Xl (Xenopus laevis—NP_001079391.1) and Hs (Homo sapiens—NP_852480.1). (*), identity; (:), strong similarity; (.), weak similarity. The amino acids residues involved in the interactions with the hexamer and that are probably targets of posttranslational modifications were highlighted. CLUSTAL Omega was used for the sequence alignment [Figure S5. Photomicrographs of seminiferous lobules of L. catesbeianus from summer group  and winter group  stained by H&E. The seminiferous lobules (SL) from winter (C) are larger than those from summer (A). However, the interstitial tissue from summer (A) is more developed and contains numerous Leydig cells (asterisks) than that from winter (C). Both groups  show primordial germ cells  located in the lobular periphery. In high magnification, PGCs with large polymorphous nucleus (n) and evident nucleoli (thin arrow) are surrounded by Sertoli cells (S). Bars: A, C—45 µm; B, D—25 µm; insets—6 µm. Table S1. Antibodies against human homologs used to identify bullfrog exosome subunits. Table S2. Sequence conservation of Lithobatescates beianus RNA exosome subunits relative to Homo sapiens, Xenopus laevis, or Xenopus tropicalis.lignment . Figure lignment . Figure lignment . Figure lignment . Figure


G-quadruplexes (G4s) are noncanonical DNA/RNA structures formed by guanine-rich sequences. Recently, G4s have been found not only in aptamers but also in the genomic DNA and transcribed RNA. In this study, we identified new RNA oligonucleotides working as aptamers by focusing on G4-forming RNAs located within the pre-mRNA. We showed that the G4 in the 5′ UTR and first intron of VEGFA bound to the protein encoded in VEGFA gene, VEGF165, with high affinity. Moreover, G4-forming RNAs located within the PDGFA and the PDGFB introns bound to PDGF-AA and PDGF-BB, respectively, indicating that G4 in the pre-mRNA could be an aptamer. It had been reported that the putative G4-forming RNA sequences are located in some parts of most genes, thus our strategy for aptamer identification could be applicable to other proteins. It has been reported that some G4-forming RNAs in 5′ UTRs are involved in translation control; however, G4-forming excised intronic RNA function has not been revealed previously. Therefore, these findings could not only contribute to the identification of RNA aptamers but also provide new insights into the biological functioning of G4-forming RNAs located within intronic RNA sequences. RNAs fold into specific complex three-dimensional structures to provide catalytic and molecular recognition abilities. These functional RNAs have been identified in both natural and artificial RNAs . Artificin silico selection of aptamers have been reported [in vitro selection. Aptamers can also be identified from genomic information without in vitro selection. Riboswitches, which sense metabolites to control gene expression, translation, splicing, and RNA stability, are known to be natural aptamers [SELEX enables the identification of aptamers against small molecules, peptides, proteins, and whole cells, including non-immunogenic and toxic targets . Modifiereported . Althougaptamers ,12. Riboaptamers .We have previously reported that G4-forming DNAs that are located within the gene promoter region of a target protein may act as DNA aptamers against the target proteins . The DNAin vitro.We considered the G4 structure as a good scaffold for target protein recognition because G4 has twice the negative charge density of double helices  to interVEGFA by QGRS Mapper [VEGFA first intron contains eight putative G4-forming RNA regions . Of these putative G4 RNAs, two G4 RNAs (VEGFA intronic G4_1 and G4_2) were randomly selected and chemically synthesized. Then we investigated the binding abilities of synthesized RNA that had putative G4 structures -mediated translation initiation . Therefoin vitro, Electromobility shift binding assay (EMSA) was performed. The fluorescein-labeled VEGFA intronic G4_1 was used for binding analysis against VEGF165 protein. We examined VEGFA intronic G4_1 and its mutant mixed with a serial dilution of VEGF165 by non-denaturing-PAGE. As a result for fluorescence detection, we observed the band shift in the mixtures of VEGFA intronic G4_1 and 1000–2000 nM VEGF165 , to improve the function of aptamers based on genetic algorithms. Using ISM, we can improve the binding ability of G4-forming RNA by optimization of G-run and loop sequences because of the loop sequence in the G4 aptamer are involved in the binding to the target protein [The G4 RNAs identified here bound not only to VEGF165, but also PDGF-AA, and PDGF-BB. However, we have developed a sequence mutation method, which we termed as  protein . In addi protein ,33,34,35VEGF165, PDGF-AA, and PDGF-BB were purchased from R & D systems . Human alpha-Thrombin was purchased from Haematologic Technologies Inc. . All RNAs were purchased from Greiner Japan  or Hokkaido System Science .VEGFA, PDGFA, and PDGFB were obtained from the UCSC genome browser [Transcribed RNA sequences of  browser  and puta browser . The synFirst, the chemically synthesized RNA oligonucleotides were labeled with fluorescein dye using the 5′ EndTag™ nucleic acid labeling system . 0.8 nmol RNAs were used for fluorescein labeling and the labeling procedures were performed according to a prescribed protocol. After that, the ethanol precipitated RNAs were dissolved in 10 μL TE buffer.Next EMSA was performed for evaluation of binding ability of RNAs. Fluorescein labeled RNAs were diluted into 2 μM in Tris-HCl potassium chloride buffer  and folded at 65 °C for five min and then allowed to cool to room temperature for 30 min. Heat treated RNAs (f.c. 1 μM) and several concentrations of VEGF165  were mixed in Tris-HCl potassium chloride buffer. For inhibition of RNA degradation, 1 U of RNase OUT  was mixed into the samples then the mixtures were incubated at room temperature for 30 min. After incubation, ten microliters of the mixtures were electrophoresed on 15% polyacrylamide gel in TBE buffer, followed by fluorescein scanning the gel using Typhoon8600 .2HPO4, 2 mM KH2PO4, pH 7.4) at 65 °C for five min, and then allowed to cool to room temperature for 30 min. PBS served as both the running and aptamer dilution buffers. SPR signals were used for the construction of the dissociation constant , and Kd was estimated by curve fitting using BIAevaluation software .The binding affinities of the G4-forming RNAs for VEGF165, PDGF-AA, and PDGF-BB were analyzed at 25 °C on a Biacore T200 instrument . VEGF165 , PDGF-AA , and PDGF-BB  were immobilized on a CM5 sensor chip  by the amine coupling procedure, and the RNAs were injected over the surface. Prior to use, all RNAs were denatured in PBS buffer  spectra were recorded on a Jasco-820 spectropolarimeter  using a quartz cell of 10 mm optical path length  and an instrument scanning speed of 500 nm/min. All RNA samples were diluted to 2 μM in PBS buffer. These RNA samples were then folded by heat treatment as described above. The CD spectra are representations of 10 averaged scans taken at 25 °C.VEGFA bind to VEGF165 with high affinity. Moreover, G4-forming RNAs located within PDGFA and PDGFB introns bound to PDGF-AA and PDGF-BB, respectively. Mutation analysis of G4-forming RNAs-VEGF165 interaction indicated that the G4 structure was important for binding to VEGF165. These results indicated that G4s in the pre-mRNA would be aptamers against the target protein that is encoded in the pre-mRNA. The potential G4-forming RNA sequences are located in RNA regions of many of the kinds of genes and the physiological function of the G4 RNAs has not been revealed. Taken together, our present research provides not only the methodology of G4-forming RNA aptamer identification, but also new insights on the physiological function of the G4-forming RNAs.In this study, we identified new RNA oligonucleotides that formed G4 located within the 5′ UTR and first intron of


Dear Editor,Pseudomonas aeruginosa (PA14) provides sequence-specific elimination of invading DNA2. Recently, a paper published in Cell Research reported that this system targets mRNA through a non-canonical mechanism3. Here, we implement the proposed design rules for generating CRISPR-RNA (crRNA)-guides that target RNA and test whether the proposed RNA-targeting activity provides immunity against RNA phage infection. Our experiments reveal that the type I-F CRISPR system provides protection from DNA, but not RNA phages.The type I-F CRISPR–Cas system from 5, Li et al.3 suggest that single-stranded RNA recognition proceeds via recognition of a “PAM-like” motif (5′-GGN-3′) on the opposite side of the complex and that this is followed by detection of a 12-nt “core” sequence , none of the MS2 targeting CRISPRs were capable of knocking down RNA phage infection. This biological result is supported by recent structural, biochemical, and biological assays which all show that Cas2/3 nuclease recruitment requires a conformational change in the Csy complex that is dependent on displacement of a non-complementary strand during crRNA-guided base pairing to the target DNA4. We acknowledge that CRISPR mechanisms are remarkably diverse and we remain open to alternative mechanisms for target recognition and Cas2/3 recruitment, however, Høyland-Kroghsbo et al.8 recently repeated the work by Li et al. but did not find evidence for RNA targeting in PA14. Collectively, these results suggest that type I-F CRISPR systems are capable of crRNA-guided detection and destruction of dsDNA, but not RNA.Importantly, the work presented here was not designed to replicate the previously published work by Li et al., but rather our intention was to determine if the non-canonical crRNA-guided recognition rules described by these authors could be generally applied for programable defense against RNA phages. Unlike the type III CRISPR systems described by Silas et al.Supplemental Material