Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs

Da li holesterol vezan za hemoglobin utiče na anti-oksidativni enzimski sistem u humanim eritrocitima?

In a previous study, it was shown that the lipid fraction, which is occasionally observed in red blood cell hemolysates, represents cholesterol (Ch) associated with phospholipid firmly bound to haemoglobin (termed Hb-Ch). The current study was conducted to investigate whether Hb-Ch could affect the primary anti-oxidant enzyme defence system in human erythrocytes. Sixty healthy volunteers were used for the current study. Group 1 consisted of 28 subjects without or with a low level of Hb-Ch. Group 2 comprised 32 subjects with a considerably higher level of Hb-Ch. The activities of erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as the content of methaemoglobin (metHb) were measured in both groups. The results indicated that the amount of Hb-Ch neither influenced the activities of the erythrocyte anti-oxidant enzymes nor altered the level of metHb. However, a higher amount of Hb-Ch changed the correlations in the part of the anti-oxidant defence system relating to glutathione, suggesting increased peroxidative pressure from plasma lipids. Group 2 also had significantly increased concentrations of total plasma Ch and triglycerides. Together, these facts are strong indications that the anti-oxidant defence system in human erythrocytes finely retunes its composition according to plasma oxidative demands.U prethodnom radu pokazano je da lipidna frakcija koja se javlja u hemolizatu zdravih ljudi predstavlja holesterol (asosovan sa fosfolipidima) čvrsto vezan za hemoglobin (Hb-Ch). U ovom radu ispitivan je uticaj Hb-Ch na anti-oksidativni enzimski sistem u humanim eritrocitima. Određena je aktivnost superoksid-dizmutaze, katalaze, glutation-peroksidaze i glutation-reduktaze, kao i sadržaj met-hemoglobina (metHb) u eritrocitima 60 ljudi, podeljenih u dve grupe na osnovu količine Hb-Ch. Rezultati pokazuju da količina prisutnog Hb-Ch ne menja aktivnost merenih enzima, niti nivo metHb. Međutim, u grupi ispitanika sa povećanim sadržajem Hb-Ch zapažene su korelativne promene u delu anti-oksidativnog enzimskog sistema povezanog sa glutationom. U istoj grupi detektovane su i veće koncentracije ukupnog holesterola i triglicerida u plazmi, što zajedno ukazuje na povećani peroksidativni pritisak iz plazme. Ovi rezultati ukazuju da odbrambeni anti-oksidativni enzimski sistem u humanim eritrocitima prilagođava svoju organizaciju prema zahtevima iz svog okruženja.

The LSST AGN Data Challenge: Selection methods

Development of the Rubin Observatory Legacy Survey of Space and Time (LSST) includes a series of Data Challenges (DC) arranged by various LSST Scientific Collaborations (SC) that are taking place during the projects preoperational phase. The AGN Science Collaboration Data Challenge (AGNSCDC) is a partial prototype of the expected LSST AGN data, aimed at validating machine learning approaches for AGN selection and characterization in large surveys like LSST. The AGNSC-DC took part in 2021 focusing on accuracy, robustness, and scalability. The training and the blinded datasets were constructed to mimic the future LSST release catalogs using the data from the Sloan Digital Sky Survey Stripe 82 region and the XMM-Newton Large Scale Structure Survey region. Data features were divided into astrometry, photometry, color, morphology, redshift and class label with the addition of variability features and images. We present the results of four DC submitted solutions using both classical and machine learning methods. We systematically test the performance of supervised (support vector machine, random forest, extreme gradient boosting, artificial neural network, convolutional neural network) and unsupervised (deep embedding clustering) models when applied to the problem of classifying/clustering sources as stars, galaxies or AGNs. We obtained classification accuracy 97.5% for supervised and clustering accuracy 96.0% for unsupervised models and 95.0% with a classic approach for a blinded dataset. We find that variability features significantly improve the accuracy of the trained models and correlation analysis among different bands enables a fast and inexpensive first order selection of quasar candidatesComment: Accepted by ApJ. 21 pages, 14 figures, 5 table

Assessing the impact of a construction virtual reality game on design review skills of construction student

Being able to perform an effective design review is a fundamental skill that every design or construction student should possess. Performing a design review requires critical thinking, analysis, and communication skills. To support the acquisition of these skills, this study investigated the impact of a virtual reality game, the design review simulator (DRS), in a classroom environment. To assess the impact of the game, the authors performed a crossover experiment with 120 students. The students were split into groups and were asked to perform design reviews in two treatment conditions, 2D drawings or the DRS. A knowledge test was administered at the end of different treatments to collect quantitative data on the students’ performance. Results from this research indicate that the DRS supported students in identifying a higher number of design mistakes. Furthermore, students that played with the DRS after performing 2D drawings reviews scored significantly higher in the knowledge test. The research results contribute to the growing knowledge base on the implementation of VR in the classroom and in supporting students in meeting learning objectives related to design review skills. In particular, the research illustrated that the benefits of VR found in the construction industry in terms of improved communication, user involvement, and feedback collection could be translated into the classroom environment

Silicon Alleviates Iron Deficiency in Barley by Enhancing Expression of Strategy II Genes and Metal Redistribution

The beneficial effects of silicon (Si) have been shown on plants using reduction-based strategy for iron (Fe) acquisition. Here we investigated the influence of Si on Fe deficiency stress alleviation in barley (Hordeum vulgare), a crop plant which uses the chelation-based strategy for Fe acquisition. Analyses of chlorophyll content, ROS accumulation, antioxidative status, concentrations of Fe and other micronutrients, along with the expression of Strategy II genes were studied in response to Si supply. Si successfully ameliorated Fe deficiency in barley, diminishing chlorophyll and biomass loss, and improving the activity of antioxidative enzymes, resulting in lowered reactive oxidative species accumulation in the youngest leaves. Alleviation of Fe deficiency stress correlated well with the Si-induced increase of Fe content in the youngest leaves, while it was decreased in root. Moreover, Si nutrition lowered accumulation of other micronutrients in the youngest leaves of Fe deprived plants, by retaining them in the root. On the transcriptional level, Si led to an expedient increase in the expression of genes involved in Strategy II Fe acquisition in roots at the early stage of Fe deficiency stress, while decreasing their expression in a prolonged stress response. Expression of Strategy II genes was remarkably upregulated in the leaves of Si supplied plants. This study broadens the perspective of mechanisms of Si action, providing evidence for ameliorative effects of Si on Strategy II plants, including its influence on accumulation and distribution of microelements, as well as on the expression of the Strategy II genes

Cerebral autoregulation: every normal subject is the average, the rest is noise?

Cerebral autoregulation (CA) refers to the ability of the brain to maintain blood flow approximately constant, when blood pressure changes. The assessment of CA presents many challenges and often leads to considerable dispersion among CA measures even within groups of healthy adults. This results in great difficulty in setting a clear border between normal and impaired CA. The current work considers if the diversity of results seen in normal subjects is just "noise", or might provide useful information that could be exploited in developing more robust clinical assessment methods. In this paper we ask how between- and within-subject variability in CA compare, and how this changes between rest and a protocol with augmented variability in blood pressure. The latter was motivated by the previous observation that increased variability (unsurprisingly) leads to more robust estimates of the relationship between blood pressure and flow. In the current work, random inflations of thigh-cuffs (TC) were used to provoke this increased challenge to the CA system

LAMMER kinase contributes to genome stability in Ustilago maydis

PMCID: PMC4526389Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1Q488*) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1Q488* rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1Q488* mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1Q488* mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.This work was supported in part by National Institutes of Health grants GM042482 and GM079859 to WKH. MM, DBN and MK were supported in part by grant 173005 from the Ministry of Education, Science and Technological Development, Republic of Serbia. J P-M was supported in part by grant BIO2014-55398-R from the Spanish government.Peer Reviewe

Does cholesterol bound to haemoglobin affect the anti-oxidant enzyme defence system in human erythrocytes?

In a previous study, it was shown that the lipid fraction, which is occasionally observed in red blood cell hemolysates, represents cholesterol (Ch) associated with phospholipid firmly bound to haemoglobin (termed Hb-Ch). The current study was conducted to investigate whether Hb-Ch could affect the primary anti-oxidant enzyme defence system in human erythrocytes. Sixty healthy volunteers were used for the current study. Group 1 consisted of 28 subjects without or with a low level of Hb-Ch. Group 2 comprised 32 subjects with a considerably higher level of Hb-Ch. The activities of erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as the content of methaemoglobin (metHb) were measured in both groups. The results indicated that the amount ofHb-Ch neither influenced the activities of the erythrocyte anti-oxidant enzymes nor altered the level of metHb. However, a higher amount ofHb-Ch changed the correlations in the part of the anti-oxidant defence system relating to glutathione, suggesting increased peroxidative pressure from plasma lipids. Group 2 also had significantly increased concentrations of total plasma Ch and triglycerides. Together, these facts are strong indications that the anti-oxidant defence system in human erythrocytes finely retunes its composition according to plasma oxidative demands

Estimating confidence intervals for cerebral autoregulation: a parametric bootstrap approach

Cerebral autoregulation (CA) refers to the ability of the brain vasculature to control blood flow in the face of changing blood pressure. One of the methods commonly used to assess cerebral autoregulation, especially in participants at rest, is the analysis of phase derived from transfer function analysis (TFA), relating arterial blood pressure (ABP) to cerebral blood flow (CBF). This and other indexes of CA can provide consistent results when comparing groups of subjects (e.g. patients and healthy controls or normocapnia and hypercapnia) but can be quite variable within and between individuals. The objective of this paper is to present a novel parametric bootstrap method, used to estimate the sampling distribution and hence confidence intervals (CIs) of the mean phase estimate in the low-frequency band, in order to optimise estimation of measures of CA function and allow more robust inferences on the status of CA from individual recordings. A set of simulations was used to verify the proposed method under controlled conditions. In 20 healthy adult volunteers (age 25.53.5 years), ABP and CBF velocity (CBFV) were measured at rest, using a Finometer device and Transcranial Doppler (applied to the middle cerebral artery), respectively. For each volunteer, five individual recordings were taken on different days, each approximately 18 min long. Phase was estimated using TFA. Analysis of recorded data showed widely changing CIs over the duration of recordings, which could be reduced when noisy data and frequencies with low coherence were excluded from the analysis (Wilcoxon signed rank test p = 0.0065). The TFA window-lengths of 50s gave smaller CIs than lengths of 100s (p &lt; 0.001) or 20s (p &lt; 0.001), challenging the usual recommendation of 100s. The method adds a much needed flexible statistical tool for CA analysis in individual recordings.</p

Increased blood pressure variability upon standing up improves reproducibility of cerebral autoregulation indices

Background: Dynamic cerebral autoregulation, that is the transient response of cerebral blood flow to changes in arterial blood pressure, is currently assessed using a variety of different time series methods and data collection protocols. In the continuing absence of a gold standard for the study of cerebral autoregulation it is unclear to what extent does the assessment depend on the choice of a computational method and protocol. Methods: We use continuous measurements of blood pressure and cerebral blood flow velocity in the middle cerebral artery from the cohorts of 18 normotensive subjects performing sit-to-stand manoeuvre. We estimate cerebral autoregulation using a wide variety of black-box approaches (ARI, Mx, Sx, Dx, FIR and ARX) and compare them in the context of reproducibility and variability. Results: For all autoregulation indices, considered here, the ICC was greater during the standing protocol, however, it was significantly greater (Fisher’s Z-test) for Mx (p &lt; 0.03), Sx (p &lt; 0.003) and Dx (p &lt; 0.03). Conclusions: In the specific case of the sit-to-stand manoeuvre, measurements taken immediately after standing up greatly improve the reproducibility of the autoregulation coefficients. This is generally coupled with an increase of the within-group spread of the estimates.</p