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The field of therapeutic peptides is experiencing a surge, fueled by their advantageous features. These include predictable metabolism, enhanced safety profile, high selectivity, and reduced off-target effects compared with small-molecule drugs. Despite progress in addressing limitations associated with peptide drugs, a significant bottleneck remains: the absence of a large-scale in silico screening method for a given protein target structure. Such methods have proven invaluable in accelerating small-molecule drug discovery. The high flexibility of peptide structures and the large diversity of peptide sequences greatly hinder the development of urgently needed computational methods. Here, we report a method called MDockPeP2_VS to address these challenges. It integrates molecular docking with structural conservation between protein folding and protein-peptide binding. Briefly, we discovered that when the interfacial residues are conserved, a sequence fragment derived from a monomeric protein exhibits a high propensity to bind a target protein with a similar conformation. This valuable insight significantly reduces the search space for peptide conformations, resulting in a substantial reduction in computational time and making in silico peptide screening practical. We applied MDockPeP2_VS to develop peptide inhibitors targeting the TEM-1 β-lactamase of Escherichia coli , a key mechanism behind antibiotic resistance in gram-negative bacteria. Among the top 10 peptides selected from in silico screening, TF7 (KTYLAQAAATG) showed significant inhibition of β-lactamase activity with a K i value of 1.37 ± 0.37 µM. This fully automated, large-scale structure-based in silico peptide screening software is available for free download at https://zougrouptoolkit.missouri.edu/mdockpep2_vs/download.html., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA), a known modulator of eIF4A1 function, as an inhibitor of HIV-1 replication in primary CD4 + T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather, the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA, but together, they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together, several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions.

The HIV-1 capsid protein (CA) assumes distinct structural forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, functional contributions of individual CA structures remain unclear, as evaluation of CA presents several technical challenges. To address this knowledge gap, we identified CA-targeting aptamers with different structural specificities, which emerged through a branched SELEX approach using an aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for the CA lattice or bound both the CA lattice and CA hexamer. We then evaluated four representatives to reveal aptamer regions required for binding, highlighting interesting structural features and challenges in aptamer structure determination. Further, we demonstrate binding to biologically relevant CA structural forms and aptamer-mediated affinity purification of CA from cell lysates without virus or host modification, supporting the development of structural form-specific aptamers as exciting new tools for the study of CA.

An essential regulatory hub for retroviral replication events, the 5' untranslated region (UTR) encodes an ensemble of cis-acting replication elements that overlap in a logical manner to carry out divergent RNA activities in cells and in virions. The primer binding site (PBS) and primer activation sequence initiate the reverse transcription process in virions, yet overlap with structural elements that regulate expression of the complex viral proteome. PBS-segment also encompasses the attachment site for Integrase to cut and paste the 3' long terminal repeat into the host chromosome to form the provirus and purine residues necessary to execute the precise stoichiometry of genome-length transcripts and spliced viral RNAs. Recent genetic mapping, cofactor affinity experiments, NMR and SAXS have elucidated that the HIV-1 PBS-segment folds into a three-way junction structure. The three-way junction structure is recognized by the host's nuclear RNA helicase A/DHX9 (RHA). RHA tethers host trimethyl guanosine synthase 1 to the Rev/Rev responsive element (RRE)-containing RNAs for m7-guanosine Cap hyper methylation that bolsters virion infectivity significantly. The HIV-1 trimethylated (TMG) Cap licenses specialized translation of virion proteins under conditions that repress translation of the regulatory proteins. Clearly host-adaption and RNA shapeshifting comprise the fundamental basis for PBS-segment orchestrating both reverse transcription of virion RNA and the nuclear modification of m7G-Cap for biphasic translation of the complex viral proteome. These recent observations, which have exposed even greater complexity of retroviral RNA biology than previously established, are the impetus for this article. Basic research to fully comprehend the marriage of PBS-segment structures and host RNA binding proteins that carry out retroviral early and late replication events is likely to expose an immutable virus-specific therapeutic target to attenuate retrovirus proliferation., (© 2024. The Author(s).)

The Trans-Activator Receptor (TAR) RNA, located at the 5'-end untranslated region (5' UTR) of the human immunodeficiency virus type 1 (HIV-1), is pivotal in the virus's life cycle. As the initial functional domain, it folds during the transcription of viral mRNA. Although TAR's role in recruiting the Tat protein for trans-activation is established, the detailed kinetic mechanisms at play during early transcription, especially at points of temporary transcriptional pausing, remain elusive. Moreover, the precise physical processes of transcriptional pause and subsequent escape are not fully elucidated. This study focuses on the folding kinetics of TAR and the biological implications by integrating computer simulations of RNA folding during transcription with nuclear magnetic resonance (NMR) spectroscopy data. The findings reveal insights into the folding mechanism of a non-native intermediate that triggers transcriptional pause, along with different folding pathways leading to transcriptional pause and readthrough. The profiling of the cotranscriptional folding pathway and identification of kinetic structural intermediates reveal a novel mechanism for viral transcriptional regulation, which could pave the way for new antiviral drug designs targeting kinetic cotranscriptional folding pathways in viral RNAs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

One of the earliest living systems was likely based on RNA ("the RNA world"). Mineral surfaces have been postulated to be an important environment for the prebiotic chemistry of RNA. In addition to adsorbing RNA and thus potentially reducing the chance of parasitic takeover through limited diffusion, minerals have been shown to promote a range of processes related to the emergence of life, including RNA polymerization, peptide bond formation, and self-assembly of vesicles. In addition, self-cleaving ribozymes have been shown to retain activity when adsorbed to the clay mineral montmorillonite. However, simulation studies suggest that adsorption to minerals is likely to interfere with RNA folding and, thus, function. To further evaluate the plausibility of a mineral-adsorbed RNA world, here we studied the effect of the synthetic clay montmorillonite K10 on the malachite green RNA aptamer, including binding of the clay to malachite green and RNA, as well as on the formation of secondary structures in model RNA and DNA oligonucleotides. We evaluated the fluorescence of the aptamer complex, adsorption to the mineral, melting curves, Förster resonance energy transfer interactions, and 1 H-NMR signals to study the folding and functionality of these nucleic acids. Our results indicate that while some base pairings are unperturbed, the overall folding and binding of the malachite green aptamer are substantially disrupted by montmorillonite. These findings suggest that minerals would constrain the structures, and possibly the functions, available to an adsorbed RNA world., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The discovery of ribozymes has inspired exploration of RNA's potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme's catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by -40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π-π and donor atom-π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms., (© 2022. The Author(s).)

How to hide messages in digital images so that messages cannot be discovered and tampered with is a compelling topic in the research area of cybersecurity. The interpolation-based reversible data hiding (RDH) scheme is especially useful for the application of medical image management. The biometric information of patients acquired by biosensors is embedded into an interpolated medical image for the purpose of authentication. The proposed scheme classifies pixel blocks into complex and smooth ones according to each block's dynamic range of pixel values. For a complex block, the minimum-neighbor (MN) interpolation followed by DIM embedding is applied, where DIM denotes the difference between the block's interpolated pixel values and the maximum pixel values. For a smooth block, the block mean (BM) interpolation is followed by a prediction error histogram (PEH) embedding and a difference expansion (DE) embedding is applied. Compared with previous methods, this adaptive strategy ensures low distortion due to embedding for smooth blocks while it provides a good payload for complex blocks. Our scheme is suitable for both medical and general images. Experimental results confirm the effectiveness of the proposed scheme. Performance comparisons with state-of-the-art schemes are also given. The peak signal to noise ratio (PSNR) of the proposed scheme is 10.32 dB higher than the relevant works in the best case.

The acquisition of m 7 G-cap-binding proteins is now recognized as a major variable driving the form and function of host RNAs. This manuscript compares the 5'-cap-RNA binding proteins that engage HIV-1 precursor RNAs, host mRNAs, small nuclear (sn)- and small nucleolar (sno) RNAs and sort into disparate RNA-fate pathways. Before completion of the transcription cycle, the transcription start site of nascent class II RNAs is appended to a non-templated guanosine that is methylated (m 7 G-cap) and bound by hetero-dimeric CBP80-CBP20 cap binding complex (CBC). The CBC is a nexus for the co-transcriptional processing of precursor RNAs to mRNAs and the snRNA and snoRNA of spliceosomal and ribosomal ribonucleoproteins (RNPs). Just as sn/sno-RNAs experience hyper-methylation of m 7 G-cap to trimethylguanosine (TMG)-cap, so do select HIV RNAs and an emerging cohort of mRNAs. TMG-cap is blocked from Watson:Crick base pairing and disqualified from participating in secondary structure. The HIV TMG-cap has been shown to license select viral transcripts for specialized cap-dependent translation initiation without eIF4E that is dependent upon CBP80/NCBP3. The exceptional activity of HIV precursor RNAs secures their access to maturation pathways of sn/snoRNAs, canonical and non-canonical host mRNAs in proper stoichiometry to execute the retroviral replication cycle.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m 7 G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m 7 G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Enzymes can aid in optimal feed stock utilization when used as feed additives. A range of toxicological studies were performed to evaluate the safety profile of a novel phytase (phytase HM) from Citrobacter b raakii produced in Aspergillus oryzae . Phytase HM was found to be non-mutagenic and non-clastogenic in in vitro tests. Further, the phytase HM preparation did not exhibit irritative potential to the eye and skin when applied in in vitro models. A 13-week subchronic toxicity study with oral administration of phytase HM to rats did not show any adverse effects. Efficacy studies showed that the dietary supplementation of this phytase significantly improved growth performance and bone mineralization in broiler chickens and piglets fed P-deficient diets, and increased retention of phosphorus (P) and calcium (Ca), and phytate-P degradation in excreta of broiler chickens in a dose-dependent manner. In conclusion, there are no safety concerns using phytase HM as a feed additive and the phytase is well tolerated by broiler chickens and pigs. Further, phytase HM improves with high efficacy the growth performance in both broiler chickens and pigs., Competing Interests: The authors have affiliated with companies with commercial interest in animal feed but none of the authors has any conflicts of interest concerning this paper., (© 2021 The Author(s).)

HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNALys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A total of 480 male Cobb 500 broiler chicks were assigned to one of 6 dietary treatments to explore the energy equivalence of myo-inositol compared with dextrose. The 6 dietary treatments included a corn and soy-based control ration formulated with 5% anhydrous dextrose and 5 further diets that were generated by the sequential displacement of increments of 1% dextrose with myo-inositol. Each diet was fed to 8 replicate cages of 10 chicks per cage from day 8 to day 18 after hatch. The BW gain, feed intake, and feed conversion ratio (FCR) were measured, and on day 15 to day 17, excreta were collected to estimate the total tract nutrient retention. Ileal digestibility of nutrients and tibia mineral content was assessed on day 18. The displacement of dextrose with myo-inositol generated a significant linear reduction in the FCR that did not reach a plateau at 5% dietary inclusion of myo-inositol. There was no effect of the displacement of dextrose with myo-inositol on bone mineral concentration. However, supplemental myo-inositol linearly reduced ileal digestibility of DM, calcium, and ileal digestible energy. Myo-inositol addition resulted in a significant linear increase in the total tract retention of CP. It can be concluded that myo-inositol has an energy equivalence equal to approximately 78% of that of dextrose for young broiler chicks but exerts a range of extra caloric effects that improve feed efficiency and may influence nitrogen (N) retention and the uric acid cycle. Future work should focus on the role of phytase and myo-inositol on uric acid, creatine kinase, and other metabolites involved in renal function and biochemical flows of N in urine and feces in nonruminants., (Copyright © 2020. Published by Elsevier Inc.)

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3'-18-nt of tRNA Lys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNA Lys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNA Lys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNA Lys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNA Lys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors., Competing Interests: K.S. is consultant for the Shift Pharmaceuticals, Overland Park, KS 66211, USA. Other authors declare no conflict of interest.

Background: Tamarix chinensis Lour (TCL) is a shrub that usually grows in arid or semiarid desert areas and saline-alkali fields. It is a traditional Chinese herbal medicine with hepatoprotective, antioxidant, antibacterial, and antitumor activities., Aim: To investigate the possible protective effects of TCL against liver injury induced by chronic ethanol intake., Methods: C57BL/6J male mice were fed a Lieber-DeCarli lipid diet containing alcohol and received (by gavage) a water-alcohol extract (80%) of TCL (100 and 200 mg/kg BW) or distilled water for 4 wk. After euthanasia, liver tissues were observed histologically with hematoxylin and eosin staining and Oil red O staining, and the levels of alanine aminotransferase, aspartate transaminase, hepatic lipids, reactive oxygen species, malondialdehyde, and superoxide dismutase were measured. In addition, expression of the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and downstream proinflammatory cytokines were determined., Results: Compared with the ethanol group, mice in the TCL-treated group (200 mg/kg) had significantly lower serum levels of alanine aminotransferase (mean, 34.1 IU/L vs 45.3 IU/L, P < 0.01) and aspartate transaminase (mean, 89.6 IU/L vs 115.7 IU/L, P < 0.01), as well as marked reduction of hepatic tissue reactive oxygen species (decreased by 27.5%, P < 0.01) and malondialdehyde (decreased by 76.6%, P < 0.01) levels, with a significant increase of superoxide dismutase (Increased by 73.2%, P < 0.01). Expression of the NLRP3 inflammasome and its downstream cytokines [interleukin (IL)-1β, tumor necrosis factor-α, and IL-6], and recruitment of natural killer T cells to the liver, were reduced in the TCL-treated incubation with a Lieber-DeCaril ethanol lipid diet group., Conclusion: These findings suggest that a TCL extract (200 mg/kg) protects against chronic ethanol-induced liver injury, probably by inhibiting the NLRP3-caspase-1-IL-1β signaling pathway and suppressing oxidative stress., Competing Interests: Conflict-of-interest statement: We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit RT in enzymatic and viral replication assays. Some aptamers inhibit RT from only a few viral clades, while others show broad-spectrum inhibition. Biophysical determinants of recognition specificity are poorly understood. We investigated the interface between HIV-1 RT and a broad-spectrum UCAA-family aptamer. SAR and hydroxyl radical probing identified aptamer structural elements critical for inhibition and established the role of signature UCAA bulge motif in RT-aptamer interaction. HDX footprinting on RT ± aptamer shows strong contacts with both subunits, especially near the C-terminus of p51. Alanine scanning revealed decreased inhibition by the aptamer for mutants P420A, L422A and K424A. 2D proton nuclear magnetic resonance and SAXS data provided constraints on the solution structure of the aptamer and enable computational modeling of the docked complex with RT. Surprisingly, the aptamer enhanced proteolytic cleavage of precursor p66/p66 by HIV-1 protease, suggesting that it stabilizes the productive conformation to allow maturation. These results illuminate features at the RT-aptamer interface that govern recognition specificity by a broad-spectrum antiviral aptamer, and they open new possibilities for accelerating RT maturation and interfering with viral replication., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The safety of a novel microbial muramidase (Muramidase 007) as a feed additive for swine was evaluated in a target animal safety study (Experiment 1). Forty weanling pigs were allotted to 4 dietary treatments: T1 control group, and 3 groups receiving Muramidase 007 in increasing doses: T2 65,000 (1X), T3 325,000 (5X) and T4 650,000 (10X) LSU(F)/kg feed. The efficacy of Muramidase 007 on growth performance was evaluated in a feeding experiment (Experiment 2). A total of 288 piglets were allotted to two groups: T1 control group and T2 receiving Muramidase 007 at 50,000 (LSU(F)/kg feed. In Experiment 1, no growth depression of pigs was observed. No adverse effects of Muramidase 007 were observed for any of the hematology and serum chemistry parameters measured or on pig health status. Post-mortem evaluation showed no adverse effects due to Muramidase 007 supplementation in the gross pathology or in the histological examination. In Experiment 2, Muramidase 007 significantly increased overall (d 0-42) average daily gain (ADG) and tended to improve overall average daily feed intake (ADFI) and day 42 body weight of nursery pigs and had no effect on feed conversion ratio (FCR). Overall, results of these studies show that there were no adverse effects of Muramidase 007 compared to the control group., (© 2019 The Authors.)

DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNA Lys3 -viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT., (© 2019 Brady et al.)

Anti-HIV-1 drug design has been notably challenging due to the virus' ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS] 2 ; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2 H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The long-range base pairing between the 5BSL3. 2 and 3'X domains in hepatitis C virus (HCV) genomic RNA is essential for viral replication. Experimental evidence points to the critical role of metal ions, especially Mg 2+ ions, in the formation of the 5BSL3.2:3'X kissing complex. Furthermore, NMR studies suggested an important ion-dependent conformational switch in the kissing process. However, for a long time, mechanistic understanding of the ion effects for the process has been unclear. Recently, computational modeling based on the Vfold RNA folding model and the partial charge-based tightly bound ion (PCTBI) model, in combination with the NMR data, revealed novel physical insights into the role of metal ions in the 5BSL3.2-3'X system. The use of the PCTBI model, which accounts for the ion correlation and fluctuation, gives reliable predictions for the ion-dependent electrostatic free energy landscape and ion-induced population shift of the 5BSL3.2:3'X kissing complex. Furthermore, the predicted ion binding sites offer insights about how ion-RNA interactions shift the conformational equilibrium. The integrated theory-experiment study shows that Mg 2+ ions may be essential for HCV viral replication. Moreover, the observed Mg 2+ -dependent conformational equilibrium may be an adaptive property of the HCV genomic RNA such that the equilibrium is optimized to the intracellular Mg 2+ concentration in liver cells for efficient viral replication.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat ( TR ) to a variable repeat ( VR ) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR -independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation., Competing Interests: Conflict of interest statement: J.F.M. is a founder of AvidBiotics Corporation and a member of its scientific advisory board. H.G. is a former consultant for AvidBiotics Corporation. The remaining authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

The 3'-end of the genomic RNA of the hepatitis C virus (HCV) embeds conserved elements that regulate viral RNA synthesis and protein translation by mechanisms that have yet to be elucidated. Previous studies with oligo-RNA fragments have led to multiple, mutually exclusive secondary structure predictions, indicating that HCV RNA structure may be context-dependent. Here we employed a nuclear magnetic resonance (NMR) approach that involves long-range adenosine interaction detection, coupled with site-specific 2 H labeling, to probe the structure of the intact 3'-end of the HCV genome (385 nucleotides). Our data reveal that the 3'-end exists as an equilibrium mixture of two conformations: an open conformation in which the 98 nucleotides of the 3'-tail (3'X) form a two-stem-loop structure with the kissing-loop residues sequestered and a closed conformation in which the 3'X rearranges its structure and forms a long-range kissing-loop interaction with an upstream cis-acting element 5BSL3.2. The long-range kissing species is favored under high-Mg 2+ conditions, and the intervening sequences do not affect the equilibrium as their secondary structures remain unchanged. The open and closed conformations are consistent with the reported function regulation of viral RNA synthesis and protein translation, respectively. Our NMR detection of these RNA conformations and the structural equilibrium in the 3'-end of the HCV genome support its roles in coordinating various steps of HCV replication.

We develop a partial charge-based tightly bound ion (PCTBI) model for the ion effects in RNA folding. On the basis of the Monte Carlo tightly bound ion (MCTBI) approach, the model can account for ion fluctuation and correlation effects, and can predict the ion distribution around the RNA. Furthermore, unlike the previous coarse-grained RNA charge models, where negative charges are placed on the phosphates only, the current new model considers the detailed all-atom partial charge distribution on the RNA. Thus, the model not only keeps the advantage of the MCTBI model, but also has the potential to provide important detailed information unattainable by the previous MCTBI models. For example, the model predicts the reduction in ion binding upon protein binding and ion-induced conformational switches. For hepatitis C virus genomic RNA, the model predicts a Mg 2+ -induced stabilization of a kissing motif for a cis-acting regulatory element in the genomic RNA. Extensive theory-experiment comparisons support the reliability of the theoretical predictions. Therefore, the model may serve as a robust starting point for further development of an accurate method for ion effects in an RNA conformational equilibrium and RNA-cofactor interactions.

HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5'-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2 H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5'-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5' (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2 H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones., Competing Interests: The authors declare no conflict of interest.

Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5'-untranslated region (5'-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5'-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5'-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5'-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5'-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS-segment of the HIV-1 5'-UTR during virus assembly., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The burden of care for frail elderly persons on families and the society is ever real as our population ages. Given the dual-income nature of many working families, day care centers offer a strong alternative to nursing homes for families wishing to provide custodial care and meaningful engagement for seniors while continuing to uphold their filial duties. Recognizing this, several initiatives, such as SPICE (Singapore Programme for Integrated Care for the Elderly) and Weekend Respite Care, have been launched to enhance the services of Singapore's day care centers. This article traces the evolution of this process, distills current challenges, and offers policy recommendations to improve Singapore's day care services for seniors., (Copyright © 2015 AMDA - The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

The 5' leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a (2)H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; Ψ(CES)). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging., (Copyright © 2015, American Association for the Advancement of Science.)

Lung cancer is a leading cause of death world-wide and the long-term survival rate for patients with lung cancer is one of the lowest for any cancer. Toll-like receptors (TLRs), evolutionarily conserved innate, are expressed in a wide variety of tissues and cell types, and they play key role in the innate immune system. TLRs have been found to be expressed by some kinds of tumor cells. However, what is the biological function of TLRs on tumor cells and whether human lung cancer cells can express TLRs remain to be fully understood. This review was performed to sum up the role of TLRs in lung cancer.

The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the (1)H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson-Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 4(3) possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA (1)H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.

This study was aimed to investigate the effect of baicalin on proliferation and apoptosis of HL-60 cells and its mechanism. Cell proliferation was assayed by using Cell Counting Kit-8. The morphological changes of HL-60 cells were examined by light microscopy and nucleolus morphological changes were observed by fluorescent microscopy after Hoechst 33342 staining. The early cell apoptosis was detected by using flow cytometry with Annexin V-FITC/PI double staining. The expression of caspase-3, caspase-9, Bcl-2 and Bax mRNA was detected by RT-PCR and Western blot assay was carried out to examine Bax, Bcl-2, caspase-8 and cleaved caspase-3 expression. The results showed that Baicalin inhibited the proliferation of HL-60 cells in a time- and concentration-dependent manner. HL-60 cells exhibited typical morphological features (for example, cell shrinkage, membrane blebbing and formation of apoptotic bodies). Cell apoptosis in early stage could be detected, the expression of caspase-3, caspase-9 and Bax mRNA was obviously up-regulated, while the Bcl-2 expression down-regulated, and accordingly Bcl-2/Bax ratio decreased. Such results were consistent with the expression of these proteins. In addition, the expression of cleaved caspase-8 protein was induced significantly after treated with baicalin. It is concluded that baicalin can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through decreasing Bcl-2/Bax ratio by intrinsic pathway and through extrinsic pathway. It suggests that baicalin may be a promising drug for the therapy of acute myeloid leukemia.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5'-leader (5'-L) of the viral RNA. Recent studies revealed that the native 5'-L exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5'-L that are important for packaging, we generated HIV-1 5'-L RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding, and packaging. Our findings identify a 159-residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5'-L and contains elements required for efficient RNA packaging., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

Like all retroviruses, the human immunodeficiency virus selectively packages two copies of its unspliced RNA genome, both of which are utilized for strand-transfer-mediated recombination during reverse transcription-a process that enables rapid evolution under environmental and chemotherapeutic pressures. The viral RNA appears to be selected for packaging as a dimer, and there is evidence that dimerization and packaging are mechanistically coupled. Both processes are mediated by interactions between the nucleocapsid domains of a small number of assembling viral Gag polyproteins and RNA elements within the 5'-untranslated region of the genome. A number of secondary structures have been predicted for regions of the genome that are responsible for packaging, and high-resolution structures have been determined for a few small RNA fragments and protein-RNA complexes. However, major questions regarding the RNA structures (and potentially the structural changes) that are responsible for dimeric genome selection remain unanswered. Here, we review efforts that have been made to identify the molecular determinants and mechanism of human immunodeficiency virus type 1 genome packaging., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background and Aim: Mice with alymphoplasia (aly/aly) mutation characterized by a lack of lymph nodes, Peyer's patches, and well-defined lymphoid follicles in the spleen were found. In this study, we used splenectomized aly/aly mice to elucidate the effects of secondary lymphoid organs in the development of aly/aly autoimmune pancreatitis., Methods: Forty-eight 10-week-old aly/aly mice were divided into two groups for splenectomy and sham operation. Histological and immunohistochemical analyses of the pancreas were performed at the ages of 20, 30, and 40 weeks old after operation, respectively., Results: Our results showed that mononuclear cell infiltration was restricted to the interlobular connective tissues at the age of 20 weeks, and not increase obviously at the age of 30 and 40 weeks in splenectomized aly/aly mice. Furthermore, an apparent decrease in the expressions of CD4(+) T, CD8(+) T, and B cells was detected in the pancreatic tissues compared with sham aly/aly mice, however, no significant difference in macrophage expression between mice with and without a splenectomy., Conclusions: Inflammation infiltration and development of the pancreatitis in aly/aly mice were suppressed effectively after splenectomy, which was, at least partly, correlated to inhibition of the infiltration of T and B cells in pancreatic tissues but not to macrophages.

Objective: To investigate the distribution of ghrelin, an important appetite regulatory factor related to obesity, in the stomach of Suncus murinus, and attempted to elucidate the ghrelin-mediated regulatory effect in this animal., Methods: The stomachs of Suncus murinus were divided into 5 sections, cardia, fundus, greater curvature, lesser curvature, and pylorus, for investigating the ghrelin-producing cells by immunohistochemistry and Western blotting. Then Suncus murinus were randomized into two groups with ghrelin intraperitoneal injection (ghrelin-ip group) and saline intraperitoneal injection (control group), respectively. The effects of food intake and body weight were measured, and furthermore, the distribution of ghrelin in stomach was also investigated by immunohistochemistry and Western blotting., Results: The immunolocalization and protein levels of ghrelin differed significantly in different regions of the stomach of Suncus murinus. Furthermore, ghrelin administration did not change the rate of food intake, but resulted in an increase in body weight compared with the control group. In this study, we elucidated the distribution of ghrelin-producing cells in the stomach of Suncus murinus in detail for the first time. Ghrelin intraperitoneal administration was found to induce an increase in body weight without changing food intake in this species., Conclusion: Our study implied ghrelin showed a different regulatory function in Suncus murinus from other species. It is considered that ghrelin may be associated with obesity-resistance phenomenon in Suncus murinus.

Aim: To clarify the innervation of human gallbladder, with special reference to morphological understanding of gallstone formation after gastrectomy., Methods: The liver, gallbladder and surrounding structures were immersed in a 10 mg/L solution of alizarin red S in ethanol to stain the peripheral nerves in cadavers (n=10). Innervation in the areas was completely dissected under a binocular microscope. Similarly, innervation in the same areas of 10 Suncus murinus (S. murinus) was examined employing whole mount immunohistochemistry., Results: Innervation of the gallbladder occurred predominantly through two routes. One was from the anterior hepatic plexus, the innervation occurred along the cystic arteries and duct. Invariably this route passed through the hepatoduodenal ligament. The other route was from the posterior hepatic plexus, the innervation occurred along the cystic duct ventrally. This route also passed through the hepatoduodenal ligament dorsally. Similar results were obtained in S. murinus., Conclusion: The route from the anterior hepatic plexus via the cystic artery and/or duct is crucial for preserving gallbladder innervation. Lymph node dissection specifically in the hepatoduodenal ligament may affect the incidence of gallstones after gastrectomy. Furthermore, the route from the posterior hepatic plexus via the common bile duct and the cystic duct to the gallbladder should not be disregarded. Preservation of the plexus may attenuate the incidence of gallstone formation after gastrectomy.