RNA-based translation activators for targeted gene upregulation.

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.

Trapping of Nicotinic Acetylcholine Receptor Ligands Assayed by In Vitro Cellular Studies and In Vivo PET Imaging.

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high affinity for α4ß2-type nicotinic receptors (α4ß2Rs) are trapped in intracellular acidic vesicles containing α4ß2Rs in vitro Nicotine, with lower pKa and α4ß2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in male and female mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labeled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4ß2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to α4ß2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in ß2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on ß2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4ß2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.SIGNIFICANCE STATEMENT Mechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4ß2R-containing acidic vesicles. Using a fluorescent-labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4ß2R-containing Golgi satellites (GSats). In vivo PET imaging with F-18-labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

Performance & clinical utility of oropharyngeal versus nasopharyngeal swabs in COVID-19.

Background & objectives: The oropharyngeal (OP) and nasopharyngeal (NP) swab samples are the most recommended clinical specimens for detecting SARS-CoV-2 in an individual through the quantitative real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) method. The primary objective of this study was to compare the performance of NP and OP swabs for the diagnosis of COVID-19 among 2250 concomitant samples (1125 NP + 1125 OP) using rRT-PCR test. Methods: This study was conducted at a tertiary care hospital in southern India. The study compared the specificity and efficacy of the two samples (NP & OP swabs) in 1125 individuals suspected having COVID-19 infection. The rRT-PCR values from all the samples were compared based on gender, age group and viral load. The differences between unmatched proportion and matched proportion were analysed. Agreement between the two methods was assessed using Kappa statistic. Absolute sensitivity, specificity, positive and negative predictive values (PPV and NPV) for OP and NP swabs were analysed. Results: The study identified a fair degree of agreement between OP and NP swabs in diagnosis of COVID-19 (kappa = 0.275, P <0.001). There was also a fair degree of agreement between NP and OP swabs irrespective of gender, age or duration of symptoms. NP swabs had better sensitivity and NPV as compared to OP swabs, however, specificity and PPV were 100 per cent for both. Interpretation & conclusions: The present study showed that both OP and NP swabs had similar sensitivity and specificity for predicting the presence of SARS-CoV-2.

Gastrointestinal Tract Disease Classification from Wireless Endoscopy Images Using Pretrained Deep Learning Model.

Wireless capsule endoscopy is a noninvasive wireless imaging technology that becomes increasingly popular in recent years. One of the major drawbacks of this technology is that it generates a large number of photos that must be analyzed by medical personnel, which takes time. Various research groups have proposed different image processing and machine learning techniques to classify gastrointestinal tract diseases in recent years. Traditional image processing algorithms and a data augmentation technique are combined with an adjusted pretrained deep convolutional neural network to classify diseases in the gastrointestinal tract from wireless endoscopy images in this research. We take advantage of pretrained models VGG16, ResNet-18, and GoogLeNet, a convolutional neural network (CNN) model with adjusted fully connected and output layers. The proposed models are validated with a dataset consisting of 6702 images of 8 classes. The VGG16 model achieved the highest results with 96.33% accuracy, 96.37% recall, 96.5% precision, and 96.5% F1-measure. Compared to other state-of-the-art models, the VGG16 model has the highest Matthews Correlation Coefficient value of 0.95 and Cohen's kappa score of 0.96.

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite's satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.

Phenomics, genomics of oil palm (Elaeis guineensis Jacq.): way forward for making sustainable and high yielding quality oil palm.

Oil palm (Elaeis guineensis Jacq.) is a heterogeneous, perennial crop having long breeding cycle with a genome size of 1.8 Gb. The demand for vegetable oil is steadily increasing, and expected that nearly 240-250 million tons of vegetable oil may be required by 2050. Genomics and next generation technologies plays crucial role in achieving the sustainable availability of oil palm with good yield and high quality. A successful breeding programme in oil palm depends on the availability of diverse gene pool, ex-situ conservation and their proper utilization for generating elite planting material. The major breeding methods adopted in oil palm are either modified recurrent selection or the modified reciprocal recurrent selection method. The QTLs of yield and related traits are chiefly located on chromosome 4, 10, 12 and 15 which is discussed in the current review. The probable chromosomal regions influencing the less height increment is observed to be on chromosomes 4, 10, 14 and 15. Advanced genomic approaches together with bioinformatics tools were discussed thoroughly for achieving sustainable oil palm where more efforts are needed. Major emphasis is given on oil palm crop improvement using holistic approaches of various genomic tools. Also a road map given on the milestones in the genomics and way forward for making oil palm to high yielding quality oil palm.

Formulation and design optimization of nano-transferosomes using pioglitazone and eprosartan mesylate for concomitant therapy against diabetes and hypertension.

Hypertension, a form of cardiovascular diseases, is considered a major risk factor associated with deaths in type 2 diabetes patients. The current medication systems for treating such chronic coexisting diseases are limited and challenging due to the difficulties in overcoming the side effects from complex therapeutic and treatment regimen. The objective of the present study is to design and optimize pioglitazone (PIO) and eprosartan mesylate (EM)-loaded nano-transferosomes (NTs) using Design-Expert software, aiming its transdermal delivery as a novel combination therapy for concomitant treatment of hypertensive diabetic patients. The developed formulations were characterized for various parameters, including in-vitro skin permeation, skin irritation, in-vivo antidiabetic, and antihypertensive activities. NTs were prepared using PIO and EM as the two model drugs and optimized using Box-Behnken design by considering phospholipid (X1), surfactant (X2), ratio of solvents (X3), and sonication time (X4), as independent variables, each at three levels. Entrapment efficiency (Y1 and Y2) and flux (Y3 and Y4) of PIO and EM, respectively, were selected as dependent variables. Among all the prepared formulations, one optimized formulation was chosen by the point prediction method and evaluated for drug-polymer compatibility, particle size, and surface charge analysis, followed by skin permeation and pharmacodynamic studies. The optimized nano-transferosomal gel (ONTF) showed all responses which confirm with the values predicted by the design. Pharmacodynamic studies showed improved and prolonged management of diabetes and hypertension in Wistar rats after the ONTF was applied, compared to oral and drug-loaded NT formulations. Results of the current study suggest that the development of such combinational delivery system can result in a rational therapeutic regimen for effective treatment of concomitant disease conditions of diabetic hypertensive patients.

Factors Associated With Mortality in Toxic Encephalopathy Due to Shigellosis in Children.

OBJECTIVE: To study the clinical characteristics and factors associated with mortality among children with Shigella encephalopathy. METHODS: The data collection was done prospectively from January, 2018 to May, 2019 with retrospective data from June, 2016 to December, 2017. The study cohort consisted of 58 children <12 years of age with Shigella encephalopathy admitted to the pediatric intensive care unit. Shigella encephalopathy was confirmed if culture or real time polymerase chain reaction (PCR) of a stool sample or rectal swab was positive, with temporal association of diarrhea with seizures, altered sensorium or both. Association of mortality with risk factors was tested using chi square test, and the strength of association was estimated in terms of relative risk (RR) and 95% CI. RESULTS: Seizures and altered sensorium were the predominant neurological symptoms. Shock occurred in 32 (55%) children, while blood in stools was a feature in only 6 (10%) children. S. sonnei was the commonest species identified on stool culture (19;33%). On univariate analysis, prolonged seizures, shock, prolonged altered sensorium, multi-organ dysfunction, lymphocytopenia at admission and need for mechanical ventilation were significantly associated with mortality. On multivariate regression, delayed presentation (presentation to the hospital 48 hours after the onset of symptoms) and prolonged altered sensorium (>12 hours) were found to be independently associated with mortality. CONCLUSION: Recognition of factors associated with mortality in Shigella encephalopathy may assist in better monitoring of sicker children and improved outcomes.

Revealing architectural order with quantitative label-free imaging and deep learning.

We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue slices. We combine QLIPP with deep neural networks to predict fluorescence images of diverse cell and tissue structures. QLIPP images reveal anatomical regions and axon tract orientation in prenatal human brain tissue sections that are not visible using brightfield imaging. We report a variant of U-Net architecture, multi-channel 2.5D U-Net, for computationally efficient prediction of fluorescence images in three dimensions and over large fields of view. Further, we develop data normalization methods for accurate prediction of myelin distribution over large brain regions. We show that experimental defects in labeling the human tissue can be rescued with quantitative label-free imaging and neural network model. We anticipate that the proposed method will enable new studies of architectural order at spatial scales ranging from organelles to tissue.

Microscopy is central to biological research and has enabled scientist to study the structure and dynamics of cells and their components within. Often, fluorescent dyes or trackers are used that can be detected under the microscope. However, this procedure can sometimes interfere with the biological processes being studied. Now, Guo, Yeh, Folkesson et al. have developed a new approach to examine structures within tissues and cells without the need for a fluorescent label. The technique, called QLIPP, uses the phase and polarization of the light passing through the sample to get information about its makeup. A computational model was used to decode the characteristics of the light and to provide information about the density and orientation of molecules in live cells and brain tissue samples of mice and human. This way, Guo et al. were able to reveal details that conventional microscopy would have missed. Then, a type of machine learning, known as 'deep learning', was used to translate the density and orientation images into fluorescence images, which enabled the researchers to predict specific structures in human brain tissue sections. QLIPP can be added as a module to a microscope and its software is available open source. Guo et al. hope that this approach can be used across many fields of biology, for example, to map the connectivity of nerve cells in the human brain or to identify how cells respond to infection. However, further work in automating other aspects, such as sample preparation and analysis, will be needed to realize the full benefits.

Molecular docking analysis of phytochemicals from ethanolic extract of crescentia cujete with the auto inhibited parkin catalytic domain.

The autoinhibited Parkin catalytic domain (PDB ID: 4BM9) receptor has been described to have a role in the ubiquitination of α-syn in Parkinson's disease. Therefore, it is of interest to discuss the molecular docking analysis data of phytochemicals from ethanolic extract of Crescentia cujete with the auto inhibited Parkin catalytic domain. We report the docking features of the phytochemical named 1, 2-Ethanediamine, N-(2-aminoethyl) with the target protein for further consideration towards the design and development of anti-Parkinson agents.

Anticorrosion Behavior of ZnO Nanoparticles Coated on Mild Steel in NaCl Solution.

This work focuses on the environment protected, ecological procedure by the combination of ZnO nanoparticles utilizing the extraction of Ocimum sanctum. The prepared nanoparticles are examined by different methods like Fourier-transform infrared spectroscopy (FTIR), X-ray Diffraction (XRD), Field emission scanning electron microscopy (FE-SEM), Energy Dispersive X-ray Analysis (EDAX). A systematic study has been made on the result of ZnO nano-coating for the corrosion behavior of mild steel. The ZnO nanoparticles of average diameter in the range 18-22 nm were coated on mild steel in nickel bath solution. The anticorrosion properties on the coated mild steel was carefully tested in 3.5% NaCl solution by performing potentio-dynamic polarization measurement and electrochemical impedance spectroscopy. Surface morphology of the coated mild steel immersed in corrosive solution was judged by using SEM with EDAX. The ZnO nano coating has shown a perfect protection against corrosion and the shielding capability is in the range between 86-95%. The incorporation of ZnO nanoparticles has upgraded the process of mild steel in all corrosion media are subjected to investigation.

Genome-wide association study for leaf area, rachis length and total dry weight in oil palm (Eleaeisguineensis) using genotyping by sequencing.

The marker-trait association for complex traits using genotyping by sequencing (GBS) method is being widely spread in plants. The study aimed to identify significant single nucleotide polymorphism (SNP) associations for rachis length (RL), leaf area (LA) and total dry weight (TrDW) in oil palm among diverse African germplasm. The Illumina NextSeq platform has been used for SNP genotyping and retained 4031 fully informative SNPs after applying the filter criterion. These 4031 SNPs were used for genome wide association study for the above three traits. The LD decay rates of the African germplasm using GBS data of SNP is observed to be 25 Kb at 0.45 of average pair wise correlation coefficient (r2). Association mapping led to the identification of seven significant associations for three traits using MLM approach at a P value of ≤ 0.001. Three associations were identified for total dry weight, two each for leaf area index and rachis length. The qtlLA1 was found to be highly significant at a P value of 7.39E-05 (18.4% phenotypic variance) which is located on chromosome 4. Two QTLs (qtlLA2 and qtlRL1) were located on chromosome 1, which explained 11.9% and 12.4% of phenotypic variance respectively. Three QTLs for total dry weight were located on chromosome 2, 14 and 16, all-together explained 40% phenotypic variance. The results showed that the SNP-trait associations identified in the present study could be used in selection of elite oil palm germplasm for higher yields.

Wound Healing Activity of Plants from the Convolvulaceae Family.

Significance: Compounds derived from plants are gaining importance for the treatment of several diseases. Many plants from the Convolvulaceae family contain compounds that have demonstrated wound healing and antidiabetic activity. Such compounds can be effectively used as a part of treatments to promote wound healing in diabetics and used in combination with antimicrobial therapy to reduce the likelihood of drug resistance and allergic reactions. Novel strategies for developing herbal formulations such as nanoparticles and adhesive patches can improve the delivery of plant-based therapeutic agents. Recent Advances: Studies have confirmed the antidiabetic and wound healing activities of Merremia tridentata, Argyreia speciosa, and Ipomoea batatas, whereas Evolvulus alsinoides, Evolvulus nummularius, Argyreia cuneata, and Ipomoea carnea have wound healing activity. Critical Issues: Drug resistance is a major problem associated with antimicrobial therapy and can affect wound healing processes. Phytoconstituents can facilitate healing processes and reduce reliance on antibiotics. Future Directions: Plants from the Convolvulaceae family have had frequent traditional uses, and all plants selected for this study have antimicrobial, antidiabetic, and wound healing properties. Detailed phytochemical studies of these plants can help develop novel wound healing therapies.

Development of fluorescence imaging probes for nicotinic acetylcholine α4ß2∗ receptors.

Nicotinic acetylcholine α4ß2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4ß2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4ß2∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4ß2∗ nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4ß2∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4ß2∗ nAChRs, labeled with antibodies specific for a ß2 subunit extracellular epitope indicating that nifrofam labels α4ß2∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4ß2∗ nAChRs in brain regions.

Selective and regulated trapping of nicotinic receptor weak base ligands and relevance to smoking cessation.

To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4ß2-type nicotinic receptors (α4ß2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing α4ß2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of α4ß2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing α4ß2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in α4ß2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed.

Identifying core competency areas to assess communication skills among interns at a tertiary teaching hospital in southern India.

BACKGROUND.: We aimed to develop a teaching-learning and evaluation programme on communication skills for interns. Core competency areas for focused communication skills training and assessment were identified to achieve the obective. We then assessed the identified competencies among interns using objective structured clinical examination (OSCE), before the start of internship. METHODS.: Five core areas for focused training and evaluation were identified on the basis of responses of practising physicians in local settings. OSCE stations were developed for evaluation based on the identified competency areas. A pre-test OSCE was administered to 30 interns. RESULTS.: Five core areas selected for training and evaluation were: (i) communicating with a parent resistant to immunization; (ii) interacting with a patient who has psychosomatic complaints; (iii) explaining risks and procedures; (iv) breaking bad news; and (v) communicating with patients and bystanders in a casualty setting. Thirty of 160 interns were selected to participate in the OSCE before the training (pre-test). The lowest score was for breaking bad news. Scores indicated that explaining risks and procedures, communicating in a busy casualty setting and dealing with psychosomatic complaints were areas that required extensive training and practice. CONCLUSIONS.: We were able to identify core competency areas for focused training and evaluation of communication skills suited to the local context and used OSCE to evaluate the skills before the start of internship.

Restriction spectrum imaging predicts response to bevacizumab in patients with high-grade glioma.

BACKGROUND: Diffusion-weighted imaging has shown initial promise for evaluating response to bevacizumab in patients with high-grade glioma (HGG). However, it is well recognized that the apparent diffusion coefficient (ADC) is influenced by bevacizumab-induced reductions in edema, which may limit its prognostic value. We demonstrate that an advanced diffusion-weighted imaging technique, restriction spectrum imaging (RSI), improves the evaluation of response to bevacizumab because unlike ADC, RSI is not affected by resolution of edema. METHODS: RSI and ADC maps were analyzed for 40 patients with HGG prior to and following initiation of bevacizumab. Volumes of interest were drawn for regions of contrast enhancement (CE) and fluid attenuated inversion recovery (FLAIR) hyperintensity and histogram percentiles within volumes of interest were calculated for ADC 10th percentile (ADC-CE10%, ADC-FLAIR10%) and for RSI 90th percentile (RSI-CE90%, RSI-FLAIR90%). Cox proportional hazard models were used to evaluate the relationship between imaging parameters, progression-free survival (PFS), and overall survival (OS). RESULTS: An increase in RSI-FLAIR90% following bevacizumab was the strongest predictor of poor PFS (P= .016) and OS (P= .004), whereas decreases in ADC-FLAIR10% showed a weaker association with OS only (P= .041). Within the CE region, increases in RSI-CE90% alone were associated with poorer OS. Correlational analysis revealed that decreases in FLAIR volume were associated with decreases in ADC-FLAIR10%, but not with changes in RSI-FLAIR90%. CONCLUSION: RSI is less influenced by changes in edema, conferring an advantage of RSI over ADC for evaluating response to anti-angiogenic therapy in patients with HGG.