Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

Synthesis, kinetic studies, and QSAR of dinucleoside polyphosphate derivatives as human AK1 inhibitors.

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.

Life-history parameters of adult females of Merluccius capensis (Gadidae) off the south coast of South Africa.

This study explores the life-history parameters of female Merluccius capensis off South Africa (N = 1819) during 2014-2016, including gonadosomatic index (GSI), length-at-maturity, length-weight relationships, and condition indices (relative condition [k] and Fulton's condition factor [K]). We detected weak indications of two peaks of spawning within the year, the first in austral autumn from March to May, whereas the other in austral spring around August. GSI was slightly higher in spring and autumn, though still low at all maturity stages (≤7%), though the opposite was true for the actively spawning stage (≥7%) as well as access to less such data during winter- and summertime. The length (L) at 50% maturity was around 38 cm (L50), though differences occurred between the two applied staging methods, histology and visual (macroscopic) classification, when L approached infinity. The latter method presented underestimated length at maturity values at the 75 and 95 percentiles (48 and 60 cm) compared to the corresponding percentiles given by histology (50 and 65 cm). There were trivial across-method differences in L50. However, we found a clear reduction in L50 in view of published information in prior years when this estimate was 48 (1985), 42 (2008), 53 (2011), and 24.8 (2015) cm. Overall, L explained 90% of the variation in whole body weight (W). As the bootstrapped, grand mean growth coefficient was b = 2.98, indicating a slight allometric growth function, there were no significant variations between years, though an isometric growth existed for 2016 with b = 3.0, whereas for 2014 and 2015 this b was 2.98 and 2.93, respectively. In terms of demography, females <60 cm generally showed isometric growth (b = 3) as opposed to allometric growth (b = 2.95) at >60 cm. The relative condition index (k = 1) exhibited higher values than Fulton's K, which was 0.80. Overall, the maternal stock of M. capensis along the south coast seems to be in good condition and likely spawns throughout the year, but we found that the macroscopic data tend to give biased maturity ogives.

Imaging the human brain on oral contraceptives: A review of structural imaging methods and implications for future research goals.

Worldwide over 150 million women use oral contraceptives (OCs), which are the most prescribed form of contraception in both the United States and in European countries. Sex hormones, such as estradiol and progesterone, are important endogenous hormones known for shaping the brain across the life span. Synthetic hormones, which are present in OCs, interfere with the natural hormonal balance by reducing the endogenous hormone levels. Little is known how this affects the brain, especially during the most vulnerable times of brain maturation. Here, we review studies that investigate differences in brain gray and white matter in women using OCs in comparison to naturally cycling women. We focus on two neuroimaging methods used to quantify structural gray and white matter changes, namely structural MRI and diffusion MRI. Finally, we discuss the potential of these imaging techniques to advance knowledge about the effects of OCs on the brain and wellbeing in women.

Steganography in IoT: Information Hiding with Joystick and Touch Sensors.

This paper describes a multi-secret steganographic system for the Internet-of-Things. It uses two user-friendly sensors for data input: thumb joystick and touch sensor. These devices are not only easy to use, but also allow hidden data entry. The system conceals multiple messages into the same container, but with different algorithms. The embedding is realized with two methods of video steganography that work on mp4 files, namely, videostego and metastego. These methods were chosen because of their low complexity so that they may operate smoothly in environments with limited resources. It is possible to replace the suggested sensors with others that offer similar functionality.

Cognitive CAPTCHA Password Reminder.

In recent years, the number of personal accounts assigned to one business user has been constantly growing. There could be as many as 191 individual login credentials used by an average employee, according to a 2017 study. The most recurrent problems associated with this situation faced by users are the strength of passwords and ability to recall them. Researchers have proven that "users are aware of what constitutes a secure password but may forgo these security measures in terms of more convenient passwords, largely depending on account type". Reusing the same password across multiple platforms or creating one with dictionary words has also been proved to be a common practice amongst many. In this paper, a novel password-reminder scheme will be presented. The goal was that the user creates a CAPTCHA-like image with a hidden meaning, that only he or she can decode. The image must be in some way related to that individual's memory or her/his unique knowledge or experience. With this image, being presented each time during logging in, the user is asked to associate a password consisting of two or more words and a number. If the image is selected properly and strong association with a person's visual memory has been linked to it, the chances of recalling a lengthy password he/she created should not present a problem.

5'-fluoro(di)phosphate-labeled oligonucleotides are versatile molecular probes for studying nucleic acid secondary structure and interactions by 19F NMR.

The high sensitivity of 19F nucleus to changes in the chemical environment has promoted the use of fluorine-labeled molecular probes to study structure and interactions of nucleic acids by 19F NMR. So far, most efforts have focused on incorporating the fluorine atom into nucleobase and ribose moieties using either monomer building blocks for solid-phase synthesis, or nucleoside triphosphates for enzymatic synthesis. Here, we report a simple and efficient synthesis of 5'-fluoromonophosphorylated and 5'-fluorodiphosphorylated oligodeoxyribonucleotides, which combines solid-phase and in-solution synthesis methods and requires only commercially available nucleoside phosphoramidites, followed by their evaluation as 19F NMR probes. We confirmed that the fluorine atom at the oligonucleotide 5' end did not alter the secondary structure of DNA fragments. Moreover, at the same time, it enabled real-time 19F NMR monitoring of various DNA-related biophysical processes, such as oligonucleotide hybridization (including mismatch identification), G-quadruplex folding/unfolding and its interactions with thrombin, as well as formation of an i-motif structure and its interaction with small-molecule ligands.

Steganography in IoT: Information Hiding with APDS-9960 Proximity and Gestures Sensor.

This article describes a steganographic system for IoT based on an APDS-9960 gesture sensor. The sensor is used in two modes: as a trigger or data input. In trigger mode, gestures control when to start and finish the embedding process; then, the data come from an external source or are pre-existing. In data input mode, the data to embed come directly from the sensor that may detect gestures or RGB color. The secrets are embedded in time-lapse photographs, which are later converted to videos. Selected hardware and steganographic methods allowed for smooth operation in the IoT environment. The system may cooperate with a digital camera and other sensors.

Improving the predictive potential of diffusion MRI in schizophrenia using normative models-Towards subject-level classification.

Diffusion MRI studies consistently report group differences in white matter between individuals diagnosed with schizophrenia and healthy controls. Nevertheless, the abnormalities found at the group-level are often not observed at the individual level. Among the different approaches aiming to study white matter abnormalities at the subject level, normative modeling analysis takes a step towards subject-level predictions by identifying affected brain locations in individual subjects based on extreme deviations from a normative range. Here, we leveraged a large harmonized diffusion MRI dataset from 512 healthy controls and 601 individuals diagnosed with schizophrenia, to study whether normative modeling can improve subject-level predictions from a binary classifier. To this aim, individual deviations from a normative model of standard (fractional anisotropy) and advanced (free-water) dMRI measures, were calculated by means of age and sex-adjusted z-scores relative to control data, in 18 white matter regions. Even though larger effect sizes are found when testing for group differences in z-scores than are found with raw values (p < .001), predictions based on summary z-score measures achieved low predictive power (AUC < 0.63). Instead, we find that combining information from the different white matter tracts, while using multiple imaging measures simultaneously, improves prediction performance (the best predictor achieved AUC = 0.726). Our findings suggest that extreme deviations from a normative model are not optimal features for prediction. However, including the complete distribution of deviations across multiple imaging measures improves prediction, and could aid in subject-level classification.

Reduced limbic microstructural integrity in functional neurological disorder.

BACKGROUND: Functional neurological disorder (FND) is a condition at the intersection of neurology and psychiatry. Individuals with FND exhibit corticolimbic abnormalities, yet little is known about the role of white matter tracts in the pathophysiology of FND. This study characterized between-group differences in microstructural integrity, and correlated fiber bundle integrity with symptom severity, physical disability, and illness duration. METHODS: A diffusion tensor imaging (DTI) study was performed in 32 patients with mixed FND compared to 36 healthy controls. Diffusion-weighted magnetic resonance images were collected along with patient-reported symptom severity, physical disability (Short Form Health Survey-36), and illness duration data. Weighted-degree and link-level graph theory and probabilistic tractography analyses characterized fractional anisotropy (FA) values across cortico-subcortical connections. Results were corrected for multiple comparisons. RESULTS: Compared to controls, FND patients showed reduced FA in the stria terminalis/fornix, medial forebrain bundle, extreme capsule, uncinate fasciculus, cingulum bundle, corpus callosum, and striatal-postcentral gyrus projections. Except for the stria terminalis/fornix, these differences remained significant adjusting for depression and anxiety. In within-group analyses, physical disability inversely correlated with stria terminalis/fornix and medial forebrain bundle FA values; illness duration negatively correlated with stria terminalis/fornix white matter integrity. A FND symptom severity composite score did not correlate with FA in patients. CONCLUSIONS: In this first DTI study of mixed FND, microstructural differences were observed in limbic and associative tracts implicated in salience, defensive behaviors, and emotion regulation. These findings advance our understanding of neurocircuit pathways in the pathophysiology of FND.

Cognitive Based Authentication Protocol for Distributed Data and Web Technologies.

The objective of the verification process, besides guaranteeing security, is also to be effective and robust. This means that the login should take as little time as possible, and each time allow for a successful authentication of the authorised account. In recent years, however, online users have been experiencing more and more issues with recalling their own passwords on the spot. According to research done in 2017 by LastPass on its employees, the number of personal accounts assigned to one business user currently exceeds 191 profiles and keeps growing. Remembering these many passwords, especially to applications which are not used every week, seems to be impossible without storing them either on paper, in a password manager, or saved in a file somewhere on a PC. In this article a new verification model using a Google Street View image as well as the user's personal experience and knowledge will be presented. The purpose of this scheme is to assure secure verification by creating longer passwords as well as delivering a 'password reminder' already embedded into the login scheme.

Federated Compressed Learning Edge Computing Framework with Ensuring Data Privacy for PM2.5 Prediction in Smart City Sensing Applications.

The sparse data in PM2.5 air quality monitoring systems is frequently happened on large-scale smart city sensing applications, which is collected via massive sensors. Moreover, it could be affected by inefficient node deployment, insufficient communication, and fragmented records, which is the main challenge of the high-resolution prediction system. In addition, data privacy in the existing centralized air quality prediction system cannot be ensured because the data which are mined from end sensory nodes constantly exposed to the network. Therefore, this paper proposes a novel edge computing framework, named Federated Compressed Learning (FCL), which provides efficient data generation while ensuring data privacy for PM2.5 predictions in the application of smart city sensing. The proposed scheme inherits the basic ideas of the compression technique, regional joint learning, and considers a secure data exchange. Thus, it could reduce the data quantity while preserving data privacy. This study would like to develop a green energy-based wireless sensing network system by using FCL edge computing framework. It is also one of key technologies of software and hardware co-design for reconfigurable and customized sensing devices application. Consequently, the prototypes are developed in order to validate the performances of the proposed framework. The results show that the data consumption is reduced by more than 95% with an error rate below 5%. Finally, the prediction results based on the FCL will generate slightly lower accuracy compared with centralized training. However, the data could be heavily compacted and securely transmitted in WSNs.

Eigenfaces-Based Steganography.

In this paper we propose a novel transform domain steganography technique-hiding a message in components of linear combination of high order eigenfaces vectors. By high order we mean eigenvectors responsible for dimensions with low amount of overall image variance, which are usually related to high-frequency parameters of image (details). The study found that when the method was trained on large enough data sets, image quality was nearly unaffected by modification of some linear combination coefficients used as PCA-based features. The proposed method is only limited to facial images, but in the era of overwhelming influence of social media, hundreds of thousands of selfies uploaded every day to social networks do not arouse any suspicion as a potential steganography communication channel. From our best knowledge there is no description of any popular steganography method that utilizes eigenfaces image domain. Due to this fact we have performed extensive evaluation of our method using at least 200 000 facial images for training and robustness evaluation of proposed approach. The obtained results are very promising. What is more, our numerical comparison with other state-of-the-art algorithms proved that eigenfaces-based steganography is among most robust methods against compression attack. The proposed research can be reproduced because we use publicly accessible data set and our implementation can be downloaded.

Synthesis of Trifluoromethylated Purine Ribonucleotides and Their Evaluation as 19F NMR Probes.

Protected guanosine and adenosine ribonucleosides and guanine nucleotides are readily functionalized with CF3 substituents within the nucleobase. Protected guanosine is trifluoromethylated at the C8 position under radical-generating conditions in up to 95% yield and guanosine 5'-oligophosphates in up to 35% yield. In the case of adenosine, the selectivity of trifluoromethylation depends heavily on the functional group protection strategy and leads to a set of CF3-modified nucleosides with different substitution patterns (C8, C2, or both) in up to 37% yield. Further transformations based on phosphorimidazolide chemistry afford various CF3-substituted mono- and dinucleoside oligophosphates in good yields. The utility of the trifluoromethylated nucleotides as probes for 19F NMR-based real-time enzymatic reaction monitoring is demonstrated with three different human nucleotide hydrolases (Fhit, DcpS, and cNIIIB). Substrate and product(s) resonances were sufficiently separated to enable effective tracking of each enzymatic activity of interest.

Imagechain-Application of Blockchain Technology for Images.

Imagechain is a cryptographic structure that chain digital images with hash links. The most important feature, which differentiates it from blockchain, is that the pictures are not stored inside the blocks. Instead, the block and the image are combined together in the embedding process. Therefore, the imagechain is built from standard graphic files that may be used in the same way as any other image, but additionally, each of them contains a data block that links it to a previous element of the chain. The presented solution does not require any additional files except the images themselves. It supports multiple file formats and embedding methods, which makes it portable and user-friendly. At the same time, the scheme provides a high level of security and resistance to forgery. This is achieved by hashing the whole file with embedded data, so the image cannot be altered or removed from the chain without losing integrity. This article describes the basic concept of an imagechain together with building blocks and applications. The two most important issues are embedding methods and block structure.

Intelligent Data Management and Security in Cloud Computing.

This paper will present the authors' own techniques of secret data management and protection, with particular attention paid to techniques securing data services. Among the solutions discussed, there will be information-sharing protocols dedicated to the tasks of secret (confidential) data sharing. Such solutions will be presented in an algorithmic form, aimed at solving the tasks of protecting and securing data against unauthorized acquisition. Data-sharing protocols will execute the tasks of securing a special type of information, i.e., data services. The area of data protection will be defined for various levels, within which will be executed the tasks of data management and protection. The authors' solution concerning securing data with the use of cryptographic threshold techniques used to split the secret among a specified group of secret trustees, simultaneously enhanced by the application of linguistic methods of description of the shared secret, forms a new class of protocols, i.e., intelligent linguistic threshold schemes. The solutions presented in this paper referring to the service management and securing will be dedicated to various levels of data management. These levels could be differentiated both in the structure of a given entity and in its environment. There is a special example thereof, i.e., the cloud management processes. These will also be subject to the assessment of feasibility of application of the discussed protocols in these areas. Presented solutions will be based on the application of an innovative approach, in which we can use a special formal graph for the creation of a secret representation, which can then be divided and transmitted over a distributed network.

Distributed Steganography in PDF Files-Secrets Hidden in Modified Pages.

This paper shows how to diffuse a message and hide it in multiple PDF files. Presented method uses dereferenced objects and secret splitting or sharing algorithms. It is applicable to various types of PDF files, including text documents, presentations, scanned images etc. Because hiding process is based on structure manipulation, the solution may be easily combined with content-dependent steganographic techniques. The hidden pages are not visible in typical application usage, which was tested with seven different programs.

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Analogues of the mRNA 5'-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.

Medical ethical moral and legal aspects of Jehovah´s Witnesses operations.

OBJECTIVE: To point out principles of blood sparing surgery. Medical ethical moral and legal aspects of operations on Jehovahs Witnesses. DESIGN: Retrospective clinical study. Review of articles. SETTING: Dept. of Gynecology and Obstetrics, University Hospital, Olomouc; Dept. of Health Care Sciences, Faculty of Humanities, Tomas Bata University Zlín. MATERIALS AND METHODS: 72 Jehovahs Witnesses patients were operated on for various benign and malignant gynecological diseases since 2007-2017. All patiens were operated according to the rules of blood sparing surgery. RESULTS: There were no excesive blood loss at any of the operations. The estimated blood loss was between 10 to 550 ml. CONCLUSIONS: The main principles of blood sparing surgery should be applied not only for Jehovahs Witnesses but for all patients. Even if the blood transfusion is the last resort for excessive blood loss during complicated operations it always carries some health risks. There are also the economical aspects. Blood transfusions should be therefore used only at very rare occasions. Jehovahs Witnesses refuse blood transfusions at all even if it is the only life saving resort. Our legislation deal with this problem but there are also moral and ethical aspects. The attitude of gynecological surgeons how to solve this problem differ a great deal.

Robotic paraaortic lymphadenectomy in oncogynecology. Double side docking of daVinci S system increases the success rates of high paraaortic lymph node dissection in endometrial cancer.

OBJECTIVE: To present an overview of minimally invasive approaches to suprapelvic lymphadenectomy and compare two different methods of staging robotic transperitoneal paraaortic lymphadenectomies in patients with early stages of endometrial cancer. DESIGN: Retrospective study and literature review. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine and Dentistry, Palacky University Olomouc, University Hospital Olomouc. METHODS: In this retrospective study we enrolled 70 patients with early stages of endometrial cancer undergoing staging robotic surgery at the Department of Obstetrics and Gynecology, University Hospital Olomouc from January 2016 to March 2018. Primary systematic pelvic and paraaortic lymphadenectomy was suggested in all patients. In 39 out of 70 patients single docking was used for robotic staging surgery, whereas in 28 patients the procedure was done using double side docking approach. Number of patients with total and infra-renal suprapelvic lymphadenectomy, number of para-aortic lymphonodes retrieved and the rate of lymphadenectomy complications were compared. RESULTS: Robotic surgery was performed in 67 (96%) out of 70 patients. In three cases (0,4%) laparoscopy was converted to laparotomy. Single side docking was used in 39 cases (58%), whereas in 28 patients (42%) double side docking was used. Paraaortic lymhadenectomy was performed in 45 cases (67%). In 16 patients (24%) the upper limit of the left renal wein was reached. Upper limit of paraaortic lymphadenectomy was above inferior mesenteric artery but did not reach left renal vein in 19 cases (28%). Inferior mesenteric represented upper limit of paraaortic lymphadenectomy in 10 patients (15%). Number of paraaortic lymphonodes retrived (4,9 ± 3,3 vs 3,7 ± 4,9, p = 0,028) as well as number of paraaortic lymphadenectomies with upper limit at the left renal vein (p < 0,0001) was higher in double side docking cases. Complication rates were low in both groups and the differences were not significant. CONCLUSION: Number of lymphonodes retrieved as well as the number of paraaortic lymphadenectomy cases with upper limit at the left renal vein was higher in double side docking group. Operating time, complication and conversion rates were low without differences between both groups.