Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

Nickel-Catalyzed Enantioselective C(sp3)-C(sp3) Cross-Electrophile Coupling of N-Sulfonyl Styrenyl Aziridines with Alkyl Bromides.

Herein, we report the first example of a highly enantioselective alkylative aziridine ring opening. Under the catalysis of a chiral nickel/pyridine-imidazoline complex, asymmetric C(sp3)-C(sp3) cross-electrophile coupling between racemic N-sulfonyl styrenyl aziridines and readily available primary alkyl bromides furnishes a variety of highly enantioenriched phenethylamine derivatives with complete regiocontrol and good functional group tolerance. Preliminary mechanistic studies support a reaction pathway consisting of regioselective iodolysis of aziridines in situ and subsequent enantioconvergent coupling of the generated ß-amino benzyl iodides with alkyl bromides.

Syntheses of Cannabinoid Metabolites: Ajulemic Acid and HU-210.

Cannabinoid metabolites have been reported to be more potent than their parent compounds. Among them, ajulemic acid (AJA) is a side-chain analog of Δ9-THC-11-oic acid, which would be a good template structure for the discovery of more potent analogues. Herein, we optimized the key allylic oxidation step to introduce the C-11 hydroxy group with a high yield. A series of compounds was prepared with this condition applied including HU-210, 11-nor-Δ8-tetrahydrocannabinol (THC)-carboxylic acid and Δ9-THC-carboxylic acid.

Docking cyclic peptides formed by a disulfide bond through a hierarchical strategy.

MOTIVATION: Cyclization is a common strategy to enhance the therapeutic potential of peptides. Many cyclic peptide drugs have been approved for clinical use, in which the disulfide-driven cyclic peptide is one of the most prevalent categories. Molecular docking is a powerful computational method to predict the binding modes of molecules. For protein-cyclic peptide docking, a big challenge is considering the flexibility of peptides with conformers constrained by cyclization. RESULTS: Integrating our efficient peptide 3D conformation sampling algorithm MODPEP2.0 and knowledge-based scoring function ITScorePP, we have proposed an extended version of our hierarchical peptide docking algorithm, named HPEPDOCK2.0, to predict the binding modes of the peptide cyclized through a disulfide against a protein. Our HPEPDOCK2.0 approach was extensively evaluated on diverse test sets and compared with the state-of-the-art cyclic peptide docking program AutoDock CrankPep (ADCP). On a benchmark dataset of 18 cyclic peptide-protein complexes, HPEPDOCK2.0 obtained a native contact fraction of above 0.5 for 61% of the cases when the top prediction was considered, compared with 39% for ADCP. On a larger test set of 25 cyclic peptide-protein complexes, HPEPDOCK2.0 yielded a success rate of 44% for the top prediction, compared with 20% for ADCP. In addition, HPEPDOCK2.0 was also validated on two other test sets of 10 and 11 complexes with apo and predicted receptor structures, respectively. HPEPDOCK2.0 is computationally efficient and the average running time for docking a cyclic peptide is about 34 min on a single CPU core, compared with 496 min for ADCP. HPEPDOCK2.0 will facilitate the study of the interaction between cyclic peptides and proteins and the development of therapeutic cyclic peptide drugs. AVAILABILITY AND IMPLEMENTATION: http://huanglab.phys.hust.edu.cn/hpepdock/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Heptanoic and medium branched-chain fatty acids as anaplerotic treatment for medium chain acyl-CoA dehydrogenase deficiency.

Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Synthetic mRNA rescues very long-chain acyl-CoA dehydrogenase deficiency in patient fibroblasts and a murine model.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid ß-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl-/-) mice, and Acadvl-/- mice to reverse the metabolic effects of the deficiency. Transfection of all cell types with hVLCAD mRNA resulted in high level expression of protein that localized to mitochondria with increased enzyme activity. Intravenous administration of LNP-VLCAD to Acadvl-/- mice produced a significant amount of VLCAD protein in liver, which declined over a week. Treated Acadvl-/- mice showed reduced hepatic steatosis, were more resistant to cold stress, and accumulated less toxic metabolites in blood than untreated animals. Results from this study support the potential for hVLCAD mRNA for treatment of VLCAD deficiency.

The Synthesis of Biphasic Metabolites of Carfentanil.

Carfentanil is an ultra-potent synthetic opioid. The Russian police force used both carfentanil and remifentanil to resolve a hostage incident in Moscow. This reported use sparked an interest in the pharmacology and toxicology of carfentanil in the human body, and data on its metabolites were later published. However, there have been few studies on the synthesis of carfentanil metabolites, and biological extraction has also put forward large uncertainty in subsequent studies. The aim of the present study is to investigate the synthesis of biphasic metabolites that are unique to carfentanil. The purpose was to produce corresponding metabolites conveniently, quickly, and at low cost that can be used for comparison with published structures and to confirm the administration of carfentanil.

Synthesis and biological evaluation of novel all-hydrocarbon cross-linked aza-stapled peptides.

Novel all-hydrocarbon cross-linked aza-stapled peptides were designed and synthesized for the first time by ring-closing metathesis between two aza-alkenylglycine residues. Three aza-stapled peptidic analogues based on the peptide dual inhibitor of p53-MDM2/MDMX interactions were synthesized and screened for biological activities. Among the three aza-stapled peptides, aSPDI-411 displayed increased anti-tumor activity, binding affinities to both MDM2 and MDMX, and cell membrane permeability compared to its linear peptide counterpart.

Visible light-promoted intermolecular cyclization/aromatization of chalcones and 2-mercaptobenzimidazoles via an EDA complex and a mechanism study.

Visible-light-promoted cyclization and aromatization of chalcones with 2-mercaptobenzimidazoles have been successfully developed to obtain diverse imidazo[2,1-b]thiazoles, and C-S and C-N bonds were constructed in one step. The reaction uses oxygen in the air as an oxidant, and the method does not need an external photocatalyst or a transition metal catalyst. The strategy features mild conditions, a simple system, readily accessible feedstocks, and a friendly environment. UV absorption spectroscopy and control experiments have shown that the reaction mechanism involves the formation of an electron-donor-acceptor (EDA) complex from thiolate anions and chalcones. In order to verify the mechanism, we studied the structure and HOMO/LUMO of the EDA complex by density functional theory (DFT) calculations. The results show that the π-π stacking between chalcones and 2-mercaptobenzimidazoles will cause a red shift of the UV absorption wavelength in the presence of Cs2CO3, and also provide a theoretical basis for the electron transfer of EDA complexes.

Holo Protein Conformation Generation from Apo Structures by Ligand Binding Site Refinement.

An important part in structure-based drug design is the selection of an appropriate protein structure. It has been revealed that a holo protein structure that contains a well-defined binding site is a much better choice than an apo structure in structure-based drug discovery. Therefore, it is valuable to obtain a holo-like protein conformation from apo structures in the case where no holo structure is available. Meeting the need, we present a robust approach to generate reliable holo-like structures from apo structures by ligand binding site refinement with restraints derived from holo templates with low homology. Our method was tested on a test set of 32 proteins from the DUD-E data set and compared with other approaches. It was shown that our method successfully refined the apo structures toward the corresponding holo conformations for 23 of 32 proteins, reducing the average all-heavy-atom RMSD of binding site residues by 0.48 Å. In addition, when evaluated against all the holo structures in the protein data bank, our method can improve the binding site RMSD for 14 of 19 cases that experience significant conformational changes. Furthermore, our refined structures also demonstrate their advantages over the apo structures in ligand binding mode predictions by both rigid docking and flexible docking and in virtual screening on the database of active and decoy ligands from the DUD-E. These results indicate that our method is effective in recovering holo-like conformations and will be valuable in structure-based drug discovery.

Inclusion of Desolvation Energy into Protein-Protein Docking through Atomic Contact Potentials.

Protein-protein interactions are crucial in many biological processes. Therefore, determining the structure of a protein-protein complex is valuable for understanding its molecular mechanisms and developing drugs. Molecular docking is a powerful computational tool in the prediction of protein-protein complex structures, in which a scoring function with good performance is very important. In this study, we have proposed a hybrid scoring function of atomic contact-based desolvation energies and distance-dependent interatomic potentials for protein-protein interactions, named HITScorePP, where the atomic contact desolvation energies were derived using an iterative method and the distance-dependent potentials were directly taken from our ITScorePP scoring function. Integrating the hybrid scoring function into our fast Fourier transform (FFT) based HDOCK docking scheme, the updated docking program, named HDOCK2.0, significantly improved the docking performance on the 55 newly added complexes in the protein docking benchmark 5.0 and a data set of 19 antibacterial protein complexes. HDOCK2.0 was also compared with four other state-of-the-art docking programs including Rosetta, ZDOCK3.0.2, FRODOCK3.0, ATTRACT, and PatchDock and obtained the overall best performance in binding mode predictions. These results demonstrated the accuracy of our hybrid scoring function and the necessity of included desolvation effects in protein-protein docking.

miR-182-5p attenuates Schistosoma japonicum-induced hepatic fibrosis by targeting tristetraprolin.

Egg granuloma formation in the liver is the main pathological lesion caused by Schistosoma japonicum infection, which generally results in liver fibrosis and may lead to death in advanced patients. MicroRNAs (miRNAs) regulate the process of liver fibrosis, but the putative function of miRNAs in liver fibrosis induced by S. japonicum infection is largely unclear. Here, we detect a new miRNA, miR-182-5p, which shows significantly decreased expression in mouse livers after stimulation by soluble egg antigen (SEA) of S. japonicum or S. japonicum infection. Knockdown or overexpression of miR-182-5p in vitro causes the increased or decreased expression of tristetraprolin (TTP), an important immunosuppressive protein in the process of liver fibrosis. Furthermore, knockdown of miR-182-5p in vivo upregulates TTP expression and significantly alleviates S. japonicum-induced hepatic fibrosis. Our data demonstrate that downregulation of miR-182-5p increases the expression of TTP in mouse livers following schistosome infection, which leads to destabilization of inflammatory factor mRNAs and attenuates liver fibrosis. Our results uncover fine-tuning of liver inflammatory reactions related to liver fibrosis caused by S. japonicum infection and provide new insights into the regulation of schistosomiasis-induced hepatic fibrosis.

Adaptive Contrastive Learning with Label Consistency for Source Data Free Unsupervised Domain Adaptation.

Unsupervised domain adaptation, which aims to alleviate the domain shift between source domain and target domain, has attracted extensive research interest; however, this is unlikely in practical application scenarios, which may be due to privacy issues and intellectual rights. In this paper, we discuss a more challenging and practical source-free unsupervised domain adaptation, which needs to adapt the source domain model to the target domain without the aid of source domain data. We propose label consistent contrastive learning (LCCL), an adaptive contrastive learning framework for source-free unsupervised domain adaptation, which encourages target domain samples to learn class-level discriminative features. Considering that the data in the source domain are unavailable, we introduce the memory bank to store the samples with the same pseudo label output and the samples obtained by clustering, and the trusted historical samples are involved in contrastive learning. In addition, we demonstrate that LCCL is a general framework that can be applied to unsupervised domain adaptation. Extensive experiments on digit recognition and image classification benchmark datasets demonstrate the effectiveness of the proposed method.

Upregulation of KSRP by miR-27b attenuates schistosomiasis-induced hepatic fibrosis by targeting TGF-ß1.

Hepatic stellate cells (HSCs) are the main effectors for various types of hepatic fibrosis, including Schistosome-induced hepatic fibrosis. Multiple inflammatory cytokines/chemokines, such as transforming growth factor-ß1 (TGF-ß1), activate HSCs, and contribute to the development of hepatic fibrosis. MicroRNAs regulate gene expression at the posttranscriptional level and are involved in regulation of inflammatory cytokine/chemokine synthesis. In this study, we showed that soluble egg antigen (SEA) stimulation and Schistosoma japonicum infection downregulate miR-27b expression and increase KH-type splicing regulatory protein (KSRP) mRNA and protein levels in vitro and in vivo. miR-27b regulates the stabilization of TGF-ß1 mRNA through targeting KSRP by interacting with their AU-rich elements in hepatocytes and non-parenchymal cells, which has an effect on the activation of HSCs. Importantly, our results have shown that either knockdown miR-27b or overexpression of KSRP attenuates S. japonicum-induced hepatic fibrosis in vivo. Therefore, our study highlights the crucial role of miR-27b and KSRP in the negative regulation of immune reactions in hepatocyte and non-parenchymal cells in response to SEA stimulation and S. japonicum infection. It reveals that manipulation of miR-27b or KSRP might be a useful strategy not only for treating Schistosome-induced hepatic fibrosis but also for curing hepatic fibrosis in general.

Enantioseparation and stereoselective dissipation of the novel chiral fungicide pydiflumetofen by ultra-high-performance liquid chromatography tandem mass spectrometry.

Pydiflumetofen is a novel and efficient broad-spectrum chiral fungicide consisting of a pair of enantiomers. A simple and sensitive chiral analytical method was established to determine the enantiomers of this chiral fungicide in food and environmental samples by ultra-high-performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) using QuEChERS method coupled with octadecylsilane-dispersive solid-phase extraction (C18-dSPE) as extraction procedure. The specific optical rotation and the absolute configuration of the enantiomers were identified by polarimetry and electronic circular dichroism (ECD). The elution order of the pydiflumetofen enantiomers on Lux Cellulose-2 was S-(-)-pydiflumetofen and R-(+)-pydiflumetofen. The average recoveries of eleven matrices ranged from 71.3% to 107.4%. The intraday relative standard deviations (RSDs) were less than 11.8%, and the interday RSDs were less than 12.6% for the two enantiomers. Stereoselective dissipation in pakchoi and soil were observed: S-(-)-pydiflumetofen was degraded faster than R-(+)-pydiflumetofen in pakchoi, causing the enantiomer fraction (EF) of the enantiomers to change from 0.50 to 0.42 in 7 days. However, R-(+)-pydiflumetofen was degraded faster than S-(-)-pydiflumetofen in soil, causing the EF of the enantiomers to change from 0.49 to 0.52 in 21 days. This study provides a method for monitoring pydiflumetofen enantiomer residues, which is crucial for improving risk assessments and the development of chiral pesticides.

Longitudinal Changes in Crude Protein and Amino Acids in Human Milk in Chinese Population: A Systematic Review.

OBJECTIVE: More than 20% of the world population live in China, which has made significant achievement in human milk research. Part of the data that were published in Chinese were, however, unavailable to non-Chinese speakers. There was also no comprehensive overview of crude protein and amino acid levels in human milk in Chinese population. This systematic review aimed to compile the data on human milk crude protein and amino acid levels in Chinese population. METHODS: After searching for and screening original research articles in both English and Chinese, 23 published from 1987 to 2019 were identified (18 in Chinese and 5 in English). The data were pooled into 9 defined lactation stages. RESULTS: Crude protein and amino acids (protein bound plus nonprotein bound) concentrations gradually decreased during the first 60 postpartum days and remained relatively static thereafter. The concentrations and dynamic change of crude protein and amino acids were similar to those in other populations. By contrast, the longitudinal changes in free amino acids (nonprotein bound) were less clear due to the limited data available. Several common weaknesses were identified in these studies. CONCLUSIONS: Our study represented the most comprehensive overview on crude protein and amino acid concentrations in human milk in Chinese population, and enhanced the knowledge of protein and amino acid intakes and requirements by Chinese infants.

Rolling Element Bearing Fault Diagnosis under Impulsive Noise Environment Based on Cyclic Correntropy Spectrum.

Rolling element bearings are widely used in various industrial machines. Fault diagnosis of rolling element bearings is a necessary tool to prevent any unexpected accidents and improve industrial efficiency. Although proved to be a powerful method in detecting the resonance band excited by faults, the spectral kurtosis (SK) exposes an obvious weakness in the case of impulsive background noise. To well process the bearing fault signal in the presence of impulsive noise, this paper proposes a fault diagnosis method based on the cyclic correntropy (CCE) function and its spectrum. Furthermore, an important parameter of CCE function, namely kernel size, is analyzed to emphasize its critical influence on the fault diagnosis performance. Finally, comparisons with the SK-based Fast Kurtogram are conducted to highlight the superiority of the proposed method. The experimental results show that the proposed method not only largely suppresses the impulsive noise, but also has a robust self-adaptation ability. The application of the proposed method is validated on a simulated signal and real data, including rolling element bearing data of a train axle.

Selective phosphodiesterase-2A inhibitor alleviates radicular inflammation and mechanical allodynia in non-compressive lumbar disc herniation rats.

PURPOSE: Phosphodiesterase inhibitors possess anti-inflammatory properties. In addition, some studies report that phosphodiesterase 2A (PDE2A) are highly expressed in the dorsal horn of the spinal cord. The present study aimed to investigate whether intrathecal administration of Bay 60-7550, a specific PDE2A inhibitor, could alleviate mechanical allodynia in non-compressive lumbar disc herniation (NCLDH) rats. METHODS: Rat NCLDH models by autologous nucleus pulposus implantation to dorsal root ganglion were established. Vehicle or Bay 60-7550 (0.1, 1.0 mg/kg) was injected by intrathecal catheter at day 1 post-operation. The ipsilateral mechanical withdrawal thresholds were analyzed from the day before surgery to day 7 after surgery. At day 7 post-operation, the ipsilateral lumbar (L4-L6) segments of the spinal dorsal horns were removed, and tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) expressions were measured by ELISA. Furthermore, PDE2A mRNA and protein expressions in spinal cord were measured by Real-Time PCR and Western blot. RESULTS: Intrathecal administration of the PDE2A inhibitor Bay 60-7550, significantly attenuated mechanical allodynia, down-regulated spinal TNF-α, IL-1ß and IL-6 over-expressions, increased the expression of spinal cAMP, as well as cGMP in a more remarkable manner, and decreased the spinal PDE2A expression in NCLDH rats in a dose-dependent manner. CONCLUSIONS: Bay 60-7550 alleviated mechanical allodynia and inflammation in NCLDH rats, which might be associated with increased cAMP and especially cGMP increase. Thus, spinal PDE2A inhibition might represent a potential analgesic strategy for radiculopathy treatment in non-compressive lumbar disc herniation.

Characterization and Expression Pattern Analysis of the T-Complex Protein-1 Zeta Subunit in Musca domestica L (Diptera).

Chaperonins, belonging to the T-complex protein-1 (TCP-1) family, assist in the correct folding of nascent and misfolded proteins. It is well-known that in mammals, the zeta subunit of the TCP-1 complex (TCP-1ζ) plays a vital role in the folding and assembly of cytoskeleta proteins. This study reported for the first time the cloning, characterization and expression pattern analysis of the TCP-1ζ from Musca domestica, which was named as MdTCP-1ζ. The MdTCP-1ζ cDNA is 1,803 bp long with a 1,596 bp open reading frame that encodes a protein with 531 bp amino acids. The analysis of the transcriptional profile of MdTCP-1ζ using qRT-PCR revealed relatively high expression in the salivary glands and trachea at the tissues while among the developmental stages. The highest expression was observed only in the eggs suggesting that the MdTCP-1ζ may play a role in embryonic development. The expression of MdTCP-1ζ was also significantly induced after exposure to short-term heat shock and infection by Escherichia coli, Staphylococcus aureus, or Candida albicans. This suggested that MdTCP-1ζ may take part in the immune responses of housefly and perhaps contribute to the protection against cellular injury.

Haplotype diversity in mitochondrial genome in a Chinese Han population.

Investigations into the use of mitochondrial genome (mtGenome) typing by massively parallel sequencing technologies are well underway in many areas, including forensic genetics. Previous studies have demonstrated that mtGenome sequencing data generated from Ion torrent personal genome machine (PGM) system were highly viable and reliable in forensic research. In this study, 145 whole mtGenomes from unrelated Chinese Han population were sequenced using the Ion PGM system. Results showed that 145 distinct haplotypes were obtained at a relatively high coverage with limited strand bias. The distribution of variants across the entire mtGenomes was illustrated and 70.74% of the variants were observed outside of the control region. An overall increase in the number of unique haplotypes as well as haplotype diversity were observed by detection of mtGenome compared with hypervariable region I/II (HV I/II) and control region (CR). This study demonstrates the substantially higher degree of haplotype resolution with whole-mtGenome sequences in comparison to HV I/II or CR that historically targeted for forensic testing, which shows the potential value of mtGenome typing in forensic testing in the future.