Publications by authors named "Xingyu Lu"

88 Publications

Association Between Efficacy of Immune Checkpoint Inhibitors and Sex: An Updated Meta-Analysis on 21 Trials and 12,675 Non-Small Cell Lung Cancer Patients.

Front Oncol 2021 26;11:627016. Epub 2021 Aug 26.

Department of Integrative Oncology, China-Japan Friendship Hospital, Beijing, China.

Background: Mounting randomized clinical trials have proved that immune checkpoint inhibitors (ICIs) achieved better overall survival (OS) and progression-free survival (PFS) than chemotherapy drugs for advanced non-small cell lung cancer (NSCLC) patients. However, some literatures have indicated that different sexes might not have equal immune response. Also, no agreement reached on the issue whether therapeutic benefit of ICIs is related to sex.

Objectives: To explore the association between efficacy of ICIs for NSCLC patients and their sexes and summarize overall treatment-related adverse events (TRAEs) in an exploratory manner.

Methods: We performed this systematic review and meta-analysis of all potentially relevant studies retrieved from PubMed, EMBASE, and the Cochrane Library until June 2021, for eligible randomized controlled trials (RCTs) comparing immunotherapy with chemotherapy in advanced NSCLC patients. Literature screening, summary data extraction was performed independently and in duplicate. The pooled hazard ratio (HR) and 95% confidence interval (CI) of OS, PFS and TRAEs were calculated, applying STATA software and random-effects models. This study was registered in international prospective register of systematic reviews (PROSPERO), number CRD42020210797.

Results: Twenty-one trials involving 12,675 NSCLC patients were included. For patients with advanced NSCLC, ICIs significantly prolonged the OS (males: HR 0.73, 95%CI 0.67-0.79; females: HR 0.73, 95%CI 0.61-0.85) and PFS (males: HR 0.62, 95%CI 0.55-0.70; females: HR 0.68, 95%CI 0.55-0.81) chemotherapy. Overall, there was no statistical difference between their sexes (OS: P = 0.97; PFS: P = 0.43), respectively. Owing to insufficient TRAEs data of different sexes, we only found immunotherapy for NSCLC patients had more all-grades (RR 0.88; 95%CI 0.82-0.95) and 3-5 grades (RR 0.60; 95%CI 0.47-0.75) AEs compared with chemotherapy.

Conclusion: Our findings indicated that the interaction between immunotherapy efficacy and different sexes was equally evident. Overall, patients with NSCLC could obtain more benefits from ICIs than chemotherapy regimen regardless of their sexes.

Systematic Review Registration: PROSPERO (https://www.crd.york.ac.uk/prospero/), identifier CRD42020210797.
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http://dx.doi.org/10.3389/fonc.2021.627016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427763PMC
August 2021

An integrative analysis of genome-wide 5-hydroxymethylcytosines in circulating cell-free DNA detects noninvasive diagnostic markers for gliomas.

Neurooncol Adv 2021 Jan-Dec;3(1):vdab049. Epub 2021 Apr 16.

State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, and The Collaborative Innovation Centre for Brain Science, Fudan University, Shanghai, China.

Background: Gliomas, especially the high-grade glioblastomas (GBM), are highly aggressive tumors in the central nervous system (CNS) with dismal clinical outcomes. Effective biomarkers, which are not currently available, may improve clinical outcomes through early detection. We sought to develop a noninvasive diagnostic approach for gliomas based on 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA).

Methods: We obtained genome-wide 5hmC profiles using the 5hmC-Seal technique in cfDNA samples from 111 prospectively enrolled patients with gliomas and 111 age-, gender-matched healthy individuals, which were split into a training set and a validation set. Integrated models comprised 5hmC levels summarized for gene bodies, long noncoding RNAs (lncRNAs), -regulatory elements, and repetitive elements were developed using the elastic net regularization under a case-control design.

Results: The integrated 5hmC-based models differentiated healthy individuals from gliomas (area under the curve [AUC] = 84%; 95% confidence interval [CI], 74-93%), GBM patients (AUC = 84%; 95% CI, 74-94%), WHO II-III glioma patients (AUC = 86%; 95% CI, 76-96%), regardless of (encoding isocitrate dehydrogenase) mutation status or other glioma-related pathological features such as TERT, TP53 in the validation set. Furthermore, the 5hmC biomarkers in cfDNA showed the potential as an independent indicator from mutation status and worked in synergy with mutation to distinguish GBM from WHO II-III gliomas. Exploration of the 5hmC biomarkers for gliomas revealed relevance to glioma biology.

Conclusions: The 5hmC-Seal in cfDNA offers the promise as a noninvasive approach for effective detection of gliomas in a screening program.
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http://dx.doi.org/10.1093/noajnl/vdab049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209591PMC
April 2021

Enhanced electrochemical performance of MnO nanoparticles: graphene aerogels as conductive substrates and capacitance contributors.

Dalton Trans 2021 Jun;50(25):8776-8784

Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China.

An N-doped graphene aerogel (NGA) is used as a reactive container for growing MnO2 nanoparticles via a soaking-hydrothermal strategy. MnO2 nanoparticles pile up on the surface of reduced graphene oxide sheets with crosslinked structures serving as electrical conductors. Importantly, the NGA generates extra capacitance during the electrochemical process, which accomplishes a satisfactory compensation in the physical-chemical properties of MnO2. As a cathodic electrode for Zn-ion batteries, the MnO2/NGA exhibits a specific capacity of 275.8 mA h g-1 and pre-eminent cycling stability with a retention of 93.6% at 3 A g-1 after 1000 cycles. Meanwhile, the proposed electrode also shows a relatively high specific capacitance of 341 F g-1 for a supercapacitor in 1 M Na2SO4. Meanwhile, the long-term cycling stability shows only a slight decrease by 5.1% of the initial capacitance after 5000 continuous cycles at 3 A g-1, which indicates its superior electrochemical stability. Additionally, the assembled asymmetric supercapacitor also shows good electrochemical performance. This work highlights the extensive function of an N-doped graphene aerogel as a promising substrate for enhancing the electrochemical performance of MnO2, which opens the gate for wide potential applications of graphene aerogel composites emphasizing their complementary effects.
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http://dx.doi.org/10.1039/d1dt00404bDOI Listing
June 2021

Highly Efficient Electro-reforming of 5-Hydroxymethylfurfural on Vertically Oriented Nickel Nanosheet/Carbon Hybrid Catalysts: Structure-Function Relationships.

Angew Chem Int Ed Engl 2021 Jun 20;60(26):14528-14535. Epub 2021 May 20.

Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning, 110016, China.

Ni-promoted electrocatalytic biomass reforming has shown promising prospect in enabling high value-added product synthesis. Here, we developed a novel hybrid catalyst with Ni nanosheet forests anchored on carbon paper. The hybrid catalyst exhibits high efficiency in electrooxidation of HMF to FDCA coupling with H production in high purity. The Ni nanosheets have small crystal grain sizes with abundant edges, which is able to deliver an efficient HMF oxidation to FDCA (selectivity >99 %) at low potential of 1.36 V with high stability. The post-reaction structure analysis reveals the Ni nanosheets would transfer electrons to carbon and readily turn into NiO and Ni(OH) during the reaction. DFT results suggest high valence Ni species would facilitate the chemical adsorption (activation) of HMF revealing the reaction pathway. This work emphasizes the importance of the precise control of Ni activity via atomic structure engineering.
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http://dx.doi.org/10.1002/anie.202102359DOI Listing
June 2021

Molecular Cages Self-Assembled by Imine Condensation in Water.

Angew Chem Int Ed Engl 2021 02 12;60(9):4705-4711. Epub 2021 Jan 12.

Department of Chemistry, Zhejiang University, Hangzhou, 310027, China.

Self-assembly by imine condensation in aqueous media is a formidable task because of the labile nature of imines in the presence of water. Here, by taking advantage of multivalence and ligand preorganization, basket-shaped triscationic cage molecules are self-assembled in high yields in both water and organic solvent, by condensing a hexaformyl and bisamine. These cages, especially the chiral ones, are stable or inert in aqueous solution, that is, no decomposition was observed upon dilution, precipitation, or exposure to competitive amines or aldehydes. Such water-compatibility allows the hosts to take advantage of the hydrophobic effect to accommodate hydrophobic guests. The chiral cage S-2 selectively binds and distinguishes one of two enantiomers, opening up opportunities for applications such as chiral compound separation. Chiral narcissistic self-sorting and sergeants-and-soldiers effects occur during cage formation when two amino precursors are involved in self-assembly.
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http://dx.doi.org/10.1002/anie.202013045DOI Listing
February 2021

Probing Microenvironmental Acidity in Lyophilized Protein and Vaccine Formulations Using Solid-state NMR Spectroscopy.

J Pharm Sci 2021 03 26;110(3):1292-1301. Epub 2020 Nov 26.

Pharmaceutical Sciences, Merck & Co., Inc, Kenilworth, NJ 07033, USA; Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, IN 47907, USA; Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA. Electronic address:

Biophysical and biochemical instability of therapeutic proteins in the solution state may necessitate the development of products in the solid form, due to their enhanced stability. Lyophilization is a widely used method to ensure dry state stabilization of biological products. A commonly encountered issue is the pH shifts that can occur due to undesired crystallization of a buffer component, resulting in loss of protein activities. However, it is technically challenging to noninvasively investigate the physicochemical environment in the lyophile matrix. In this work, we demonstrate an approach based on solid-state NMR to investigate the microenvironmental acidity in lyophilized protein formulations, using histidine, a commonly used buffer agent, as a molecular probe. The solid-state acidity in the lyophilized matrix can be assessed by monitoring the chemical shift changes of histidine. The protonation and tautomeric states of histidine lyophilized at a range of pH values from 4.5 to 11.0 were identified from full C and N resonance assignments in one-dimensional and two-dimensional NMR experiments. The results demonstrated a pH-dependence of histidine chemical shift in the amorphous state. Moreover, we successfully applied this protocol to investigate the microenvironmental pH in lyophilized formulations of the HPV vaccine and lactate dehydrogenase protein.
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http://dx.doi.org/10.1016/j.xphs.2020.11.017DOI Listing
March 2021

Alterations of 5-hydroxymethylcytosines in circulating cell-free DNA reflect retinopathy in type 2 diabetes.

Genomics 2021 01 20;113(1 Pt 1):79-87. Epub 2020 Nov 20.

Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, Ningbo University, Ningbo, Zhejiang, China. Electronic address:

Diabetic retinopathy (DR) is a common microvascular complication that may cause severe visual impairment and blindness in patients with type 2 diabetes mellitus (T2DM). Early detection of DR will expand the range of potential treatment options and enable better control of disease progression. Epigenetic dysregulation has been implicated in the pathogenesis of microvascular complications in patients with T2DM. We sought to explore the diagnostic value of 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA) for DR, taking advantage of a highly sensitive technique, the 5hmC-Seal. The genome-wide 5hmC profiles in cfDNA samples from 35 patients diagnosed with DR and 35 age-, gender-, diabetic duration-matched T2DM controls were obtained using the 5hmC-Seal, followed by a case-control analysis and external validation. The genomic distribution of 5hmC in cfDNA from patients with DR reflected potential gene regulatory relevance, showing co-localization with histone modification marks for active expression (e.g., H3K4me1). A three-gene signature (MESP1, LY6G6D, LINC01556) associated with DR was detected using the elastic net regularization on the multivariable logistic regression model, showing high accuracy to distinguish patients with DR from T2DM controls (AUC [area under curve] = 91.4%; 95% CI [confidence interval], 84.3- 98.5%), achieving a sensitivity of 88.6% and a specificity of 91.4%. In an external testing set, the 5hmC model detected 5 out of 6 DR patients and predicted 7 out of 8 non-DR patients with other microvascular complications. Circulating cfDNA from patients with DR contained 5hmC information that could be exploited for DR detection. As a novel non-invasive approach, the 5hmC-Seal holds the promise to be an integrated part of patient care and surveillance tool for T2DM patients.
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http://dx.doi.org/10.1016/j.ygeno.2020.11.014DOI Listing
January 2021

Toward Developing Discriminating Dissolution Methods for Formulations Containing Nanoparticulates in Solution: The Impact of Particle Drift and Drug Activity in Solution.

Mol Pharm 2020 11 21;17(11):4125-4140. Epub 2020 Oct 21.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States.

Enabling formulations are an attractive approach to increase the dissolution rate, solubility, and oral bioavailability of poorly soluble compounds. With the growing prevalence of poorly soluble drug compounds in the pharmaceutical pipeline, supersaturating drug delivery systems (SDDS), a subset of enabling formulations, have grown in popularity due to their properties allowing for drug concentrations greater than the corresponding crystalline solubility. However, the extent of supersaturation generated as the enabling formulation traverses the gastrointestinal (GI) tract is dynamic and poorly understood. The dynamic nature of supersaturation is a result of several competing kinetic processes such as dissolution, solubilization by formulation and endogenous surfactants, crystallization, and absorption. Ultimately, the free drug concentration, which is equivalent to the drug's inherent thermodynamic activity amid these kinetic processes, defines the true driving force for drug absorption. However, in cases where solubilizing agents are present (i.e., surfactants and bile salts), drug molecules may associate with colloidal nanoscale species, complicating drug activity determination. These nanoscale species can drift into the aqueous boundary layer (ABL), increasing the local API activity at the membrane surface, resulting in increased bioavailability. Herein, a novel approach was developed to accurately measure thermodynamic drug activity in complex media containing drug distributed in nanoparticulate species. This approach captures the influence of the ABL on the observed flux and, ultimately, the predicted unbound drug concentration. The results demonstrate that this approach can help to (1) measure the true extent of local supersaturation in complex systems containing solubilizing excipients and (2) elucidate the mechanisms by which colloidal aggregates can modulate the drug activity in solution and potentially enhance the flux observed across a membrane. The utilization of these techniques may provide development scientists with a strategy to evaluate formulation sensitivity to nanospeciation and allow formulators to maximize the driving force for absorption in a complex environment, perhaps enabling the development of dissolution methods with greater discrimination and correlation to pre-clinical and clinical data sets.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00599DOI Listing
November 2020

Quantifying Pharmaceutical Formulations from Proton Detected Solid-State NMR under Ultrafast Magic Angle Spinning.

J Pharm Sci 2020 10 15;109(10):3045-3053. Epub 2020 Jul 15.

Pharmaceutical Sciences, Merck & Co., Inc., Kenilworth, NJ 07033, USA; Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, IN 47907, USA; Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA. Electronic address:

Probing form conversions of active pharmaceutical ingredients in solid dosages is critical for understanding the physicochemical stability of drug substances in formulations. The multicomponent and low drug loading nature of drug products often results in challenges to quantify the phase stability, at a low detection limit and with the chemical resolution that differentiate drug molecules and excipients, for routine laboratory techniques. Recent advancement of ultrafast magic angle spinning (UF-MAS) enables proton-detected solid-state nuclear magnetic resonance (ssNMR) techniques to characterize pharmaceutical materials with enhanced resolution and sensitivity. This study demonstrates one of the first documented cases implementing 60 kHz UF-MAS techniques to quantify the minor content of pioglitazone free base (PIO-FB) in a binary system with its hydrochloride salt (PIO-HCl) and a multicomponent formulation with typical excipients. One-dimensional H methods can unambiguously differentiate the two forms and exhibit a limit of detection at 1.77% (w/w). Moreover, we extended it to a two-dimensional H-H correlation for minimizing peak overlap and successfully quantifying approximately 2.0% (w/w) PIO-FB in a multicomponent formulation. These results have demonstrated that H ssNMR as a novel method to quantify solid dosages at a higher resolution and faster acquisition than conventional C techniques.
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http://dx.doi.org/10.1016/j.xphs.2020.06.026DOI Listing
October 2020

Molecular packing of pharmaceuticals analyzed with paramagnetic relaxation enhancement and ultrafast magic angle pinning NMR.

Phys Chem Chem Phys 2020 Jun 4;22(23):13160-13170. Epub 2020 Jun 4.

MRL, Merck & Co., Inc., Kenilworth, New Jersey 07033, USA.

Understanding the relationship between the structure and the physicochemical attributes of crystalline pharmaceuticals requires high-resolution molecular details. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy is an indispensable tool for analyzing molecular structures, but often experiences challenges of low spectral resolution and sensitivity, particularly in the characterization of unlabeled pharmaceutical materials. Besides, the relatively long spin-lattice relaxation times in pharmaceutical crystals result in time-consuming data collections. In this study, we utilize ultrafast magic angle spinning (UF-MAS) of the sample at 60 and 110 kHz to enable proton and fluorine spectroscopies for probing the structural details of crystalline posaconazole. Paramagnetic relaxation enhancement (PRE), obtained by doping Cu(ii) ions into the crystalline lattice and coating on particle surface, is implemented to shorten the spin-lattice relaxation time for speeding up the ssNMR acquisition. Our results demonstrate a remarkably improved H and F resolution and sensitivity, which enables multi-dimensional H-H and heteronuclear H-F correlations. In combination with density functional theory (DFT) calculations of chemical shifts, molecular details of posaconazole are established in terms of H and F networks for identifying "head-to-tail" and "head-to-head" intermolecular packings, with presumably critical contacts that stabilize the crystalline structure. The PRE and UF-MAS techniques enable the high-resolution structure characterization of fluorinated drug molecules in pharmaceutical formulations at natural abundance.
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http://dx.doi.org/10.1039/d0cp02049dDOI Listing
June 2020

Atomic-Level Drug Substance and Polymer Interaction in Posaconazole Amorphous Solid Dispersion from Solid-State NMR.

Mol Pharm 2020 07 28;17(7):2585-2598. Epub 2020 May 28.

Pharmaceutical Sciences, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States.

Despite the wide utilization of amorphous solid dispersions (ASDs) for formulating poorly water-soluble drugs, fundamental understanding of the structural basis behind their stability and dissolution behavior is limited. This is largely due to the lack of high-resolution structural tools for investigating multicomponent and amorphous systems in the solid state. In this study, we present what is likely the first publication quantifying the molecular interaction between the drug and polymer in ASDs at an angstrom level by utilizing F magic angle spinning (MAS) nuclear magnetic resonance (NMR) techniques. A variant of the F-C rotational-echo and double-resonance (REDOR) technique was developed to quantify interatomic distances by implementing a supercycled symmetry-based recoupling schedule and synchronized simultaneous detection. We successfully deployed the technique to identify "head-to-head" and "head-to-tail" packing of crystalline posaconazole (POSA). To probe molecular interactions between POSA and hypromellose acetate succinate (HPMCAS) in the dispersion, as a major goal of this study, two-dimensional (2D) H-F correlation experiments were performed. The approach facilitated observation of intermolecular hydrogen-to-fluorine contacts between the hydroxyl group of the polymer and the difluorophenyl group of the drug substance. Atomic distance measurement, utilizing the developed F-C REDOR technique, revealed the close proximity of C-F at 4.3 Å. Numerical modeling analysis suggested a possible hydrogen bonding interaction between the polymer O-H group as an acceptor and POSA fluorine (O-H···F) or difluorophenyl ring (O-H···Ph) as a donor. These F MAS NMR techniques, including 2D F-H heteronuclear correlation and F-C atomic distance measurement, may shed light on the nature (i.e., type and strength) of drug-polymer interactions in ASDs and offer a new high-resolution analytical protocol for probing the microstructure of amorphous pharmaceutical materials.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00268DOI Listing
July 2020

Understanding Molecular Interactions in Rafoxanide-Povidone Amorphous Solid Dispersions from Ultrafast Magic Angle Spinning NMR.

Mol Pharm 2020 06 21;17(6):2196-2207. Epub 2020 May 21.

Pharmaceutical Sciences, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States.

In solid dosage formulations, probing intermolecular interactions between active pharmaceutical ingredients (APIs) and polymeric excipients, which have a mechanistic impact on physical stability as well as bioavailability, remains a challenge. In recent years, solid-state NMR spectroscopy has been demonstrated to be a powerful tool to provide structural details with an atomic resolution of therapeutic organic compounds and formulation products. However, conventional C-detected techniques often suffer from poor resolution and low sensitivity due to the disordered structure of certain materials such as amorphous pharmaceuticals and C natural abundance, hindering in-depth investigations. In this study, we utilize the magic angle spinning (MAS) technique with ultrafast speeds (UF-MAS: ν = 60 and 110 kHz) and demonstrate the enabled methods with H detection to study the amorphous molecular complex of rafoxanide and povidone in the solid state. The downfield shift of the RAF amide proton, resolved under UF-MAS, and its correlations with aliphatic protons of PVP, serve as strong evidence of the existence of intermolecular hydrogen bonding. Two-dimensional (2D) H-detected H{C} and H-H correlation experiments, interestingly, exhibit distinct API-polymer interactions in the spray-dried amorphous solid dispersions (ASDs), utilizing aqueous and organic cosolvents and organic solvents mixtures. The rich intermolecular interactions in the aqueously prepared ASDs presumably contribute to the physical stability, and the interactions are retained in the solution state to maintain supersaturation for an enhanced dissolution profile. This study presents the first application of UF-MAS NMR characterization of therapeutic solid dosages at a spinning frequency of 110 kHz and uncovers the molecular mechanisms of solvent-mediated pharmaceutical dispersions.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00317DOI Listing
June 2020

Molecular Mechanism of Crystalline-to-Amorphous Conversion of Pharmaceutical Solids from F Magic Angle Spinning NMR.

J Phys Chem B 2020 06 19;124(25):5271-5283. Epub 2020 May 19.

Pharmaceutical Sciences, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States.

Crystalline and amorphous materials usually possess distinct physicochemical properties due to major variations in long-range and local molecular packings. Enhanced fundamental knowledge of the molecular details of crystalline-to-amorphous interconversions is necessary to correlate the intermolecular structure to material properties and functions. While crystal structures can be readily obtained by X-ray crystallography, the microstructure of amorphous materials has rarely been explored due to a lack of high-resolution techniques capable of probing local molecular structures. Moreover, there is increasing interest in understanding the molecular nature of amorphous solids in pharmaceutical sciences due to the widespread utilization of amorphous active pharmaceutical ingredients (APIs) in pharmaceutical development for solubility and bioavailability enhancement. In this study, we explore multidimensional C and F magic angle spinning (MAS) NMR spectroscopy to study the molecular packing of amorphous posaconazole (POSA) in conjunction with the crystalline counterpart. Utilizing methods integrating homonuclear and heteronuclear H, C, and F correlation spectroscopy and atomic F-to-C distance measurements, we identified the major differences in molecular packing between crystalline and amorphous POSA. The intermolecular "head-to-head" interaction along the molecule's major axis, as well as the "head-to-tail" molecular packing perpendicular to the major axis in POSA crystals, was recapitulated by MAS NMR. Furthermore, critical intermolecular distances in the crystal lattice were determined. Most importantly, the head-to-tail contact of two neighboring molecules was found to be preserved in amorphous POSA, suggesting localized molecular order, whereas crucial interactions for head-to-head packing are absent in the amorphous form resulting in long-range disorder. Our study, likely one of the first documented examples, provides molecular-level structural details to understand the molecular mechanism of crystalline-to-amorphous conversion of fluorine-containing drug substances occurring in drug processing and development and establish a high-resolution experimental protocol for investigating amorphous materials.
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http://dx.doi.org/10.1021/acs.jpcb.0c02131DOI Listing
June 2020

Oxygen reduction to hydrogen peroxide on oxidized nanocarbon: Identification and quantification of active sites.

J Colloid Interface Sci 2020 Aug 7;573:376-383. Epub 2020 Apr 7.

School of Chemistry and Materials Science, Ludong University, Yantai, China; Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning, China. Electronic address:

Two-electron electrochemical oxygen reduction reaction (ORR) on non-metallic nanocarbon catalysts yielding hydrogen peroxide (HO) has attracted considerable research attentions in the field of electrochemistry, catalysis and material science. Here, oxidized carbon black (OCB) with different amounts of surface oxygen functional groups were synthesized for ORR. OCB catalysts exhibited a significant activity improvement for the two-electron pathway comparing with non-oxidized ones. Identification and quantification of active sites for two-electron pathway of ORR process were further realized via linking the activity to structural compositions of OCB, and both carboxyl (-COOH) and carbonyl (-CO) groups were proved responsible for two-electron pathway ORR to produce HO. The intrinsic activity of -COOH (k = 1.34 × 10 mol) was found higher than that of -CO (k = 0.25 × 10 mol). The present work provides new physical-chemical insights on the mechanism of oxygen reduction reactions on carbon-based catalysts, paving the way for the establishment of structure-function relations and further development of novel non-metallic catalytic systems.
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http://dx.doi.org/10.1016/j.jcis.2020.04.030DOI Listing
August 2020

Genome-Wide Transcriptomic Analysis Reveals a Regulatory Network of Oxidative Stress-Induced Flowering Signals Produced in Litchi Leaves.

Genes (Basel) 2020 03 18;11(3). Epub 2020 Mar 18.

Guangdong Litchi Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou 510642, China.

Litchi is an important subtropical fruit tree that requires an appropriately low temperature to trigger floral initiation. Our previous studies have shown that reactive oxygen species (ROS) are involved in litchi flowering. To identify oxidative stress-induced flowering related genes in leaves, 'Nuomici' potted trees were grown at medium low-temperature conditions (18/13 °C for day/night, medium-temperature). The trees were treated with the ROS generator methyl viologen dichloride hydrate (MV) as the MV-generated ROS treatment (MM, medium-temperature plus MV) and water as the control treatment (M, medium-temperature plus water). Sixteen RNA-sequencing libraries were constructed, and each library generated more than 5,000,000 clean reads. A total of 517 differentially expressed genes (DEGs) were obtained. Among those DEGs, plant hormone biosynthesis and signal transduction genes, ROS-specific transcription factors, such as AP2/ERF and WRKY genes, stress response genes, and flowering-related genes () and () were significantly enriched. Then, as a confirmatory experiment, the potted trees were uniformly sprayed with MV, N,N'-dimethylthiourea (DMTU, ROS scavenger) plus MV, and water at medium-temperature. The results showed that the MV-generated ROS promoted flowering and changed related gene expression, but these effects were repressed by DMTU treatment. The results of our studies indicate that ROS could promote flowering and partly bypass chilling for litchi flowering.
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http://dx.doi.org/10.3390/genes11030324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140818PMC
March 2020

Dosiomics improves prediction of locoregional recurrence for intensity modulated radiotherapy treated head and neck cancer cases.

Oral Oncol 2020 05 6;104:104625. Epub 2020 Mar 6.

Department of Biomedical Engineering, Southern Medical University, Guangzhou, Guangdong 510515, China. Electronic address:

Objectives: To investigate whether dosiomics can benefit to IMRT treated patient's locoregional recurrences (LR) prediction through a comparative study on prediction performance inspection between radiomics methods and that integrating dosiomics in head and neck cancer cases.

Materials And Methods: A cohort of 237 patients with head and neck cancer from four different institutions was obtained from The Cancer Imaging Archive and utilized to train and validate the radiomics-only prognostic model and integrate the dosiomics prognostic model. For radiomics, the radiomics features were initially extracted from images, including CTs and PETs, and selected on the basis of their concordance index (CI) values, then condensed via principle component analysis. Lastly, multivariate Cox proportional hazards regression models were constructed with class-imbalance adjustment as the LR prediction models by inputting those condensed features. For dosiomics integration model establishment, the initial features were similar, but with additional 3-dimensional dose distribution from radiation treatment plans. The CI and the Kaplan-Meier curves with log-rank analysis were used to assess and compare these models.

Results: Observed from the independent validation dataset, the CI of the model for dosiomics integration (0.66) was significantly different from that for radiomics (0.59) (Wilcoxon test, p=5.9×10). The integrated model successfully classified the patients into high- and low-risk groups (log-rank test, p=2.5×10), whereas the radiomics model was not able to provide such classification (log-rank test, p=0.37).

Conclusion: Dosiomics can benefit in predicting the LR in IMRT-treated patients and should not be neglected for related investigations.
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http://dx.doi.org/10.1016/j.oraloncology.2020.104625DOI Listing
May 2020

Multi-sequence MR image-based synthetic CT generation using a generative adversarial network for head and neck MRI-only radiotherapy.

Med Phys 2020 Apr 26;47(4):1880-1894. Epub 2020 Feb 26.

Department of Biomedical Engineering, Southern Medical University, Guangzhou, 510515, Guangdong, China.

Purpose: The purpose of this study is to investigate the effect of different magnetic resonance (MR) sequences on the accuracy of deep learning-based synthetic computed tomography (sCT) generation in the complex head and neck region.

Methods: Four sequences of MR images (T1, T2, T1C, and T1DixonC-water) were collected from 45 patients with nasopharyngeal carcinoma. Seven conditional generative adversarial network (cGAN) models were trained with different sequences (single channel) and different combinations (multi-channel) as inputs. To further verify the cGAN performance, we also used a U-net network as a comparison. Mean absolute error, structural similarity index, peak signal-to-noise ratio, dice similarity coefficient, and dose distribution were evaluated between the actual CTs and sCTs generated from different models.

Results: The results show that the cGAN model with multi-channel (i.e., T1 + T2 + T1C + T1DixonC-water) as input to predict sCT achieves higher accuracy than any single MR sequence model. The T1-weighted MR model achieves better results than T2, T1C, and T1DixonC-water models. The comparison between cGAN and U-net shows that the sCTs predicted by cGAN retains additional image details are less blurred and more similar to the actual CT.

Conclusions: Conditional generative adversarial network with multiple MR sequences as model input shows the best accuracy. The T1-weighted MR images provide sufficient image information and are suitable for sCT prediction in clinical scenarios with limited acquisition sequences or limited acquisition time.
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http://dx.doi.org/10.1002/mp.14075DOI Listing
April 2020

Immunosuppressive therapy for elderly-acquired pure red cell aplasia: cyclosporine A may be more effective.

Ann Hematol 2020 Mar 22;99(3):443-449. Epub 2020 Jan 22.

Department of Hematology, the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Collaborative Innovation Center for Cancer Personalized Medicine, 300# Guangzhou Road, Nanjing, 210029, Jiangsu Province, China.

This current study retrospectively analyzed the clinical characteristics of 69 adult patients with acquired pure red cell aplasia (PRCA) including 40 elderly and 29 non-elderly patients from September 2009 to June 2019. The remission induction therapy regimens included cyclosporine A (CsA), corticosteroids (CS), or other immunosuppressive agents. The overall response rate was 55% (22/40) in the elderly group compared with 75.9% (22/29) in non-elderly patients (P = 0.075). In elderly patients, the best remission was achieved in the group treated with CsA than those treated with CS or other immunosuppressive agents (83.3% vs 26.7% vs 42.9%%, P = 0.004). However, outcomes of remission were similar among different treatment groups (P = 0.458) in non-elderly patients. CS induced a higher response rate in the non-elderly than that in the elderly (88.9% vs 26.7%, P = 0.009). By univariate and multivariate analysis, the clinical efficacy of elderly patients with acquired PRCA was closely associated with an induction regimen of CsA (P = 0.009; P = 0.017). In conclusion, CsA might produce higher response rate than CS and other drugs in elderly patients with acquired PRCA.
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http://dx.doi.org/10.1007/s00277-020-03926-6DOI Listing
March 2020

Clay-Polymer Nanocomposites Prepared by Reactive Melt Extrusion for Sustained Drug Release.

Pharmaceutics 2020 Jan 7;12(1). Epub 2020 Jan 7.

College of Pharmacy, the University of Texas at Austin, 2409 University Avenue, A1920, Austin, TX 78712, USA.

Clay-polymer nanocomposites have exhibited a great potential as carriers for controlled release drug delivery. This study aims to prepare exfoliated montmorillonite-Eudragit RS nanocomposites using reactive melt extrusion and investigate the influence of claying loading, clay types (sodium montmorillonite (Cloisite Na) vs. organomodified montmorillonite (Cloisite 20)) on clay-polymer interactions and drug release properties. The clays were used as the filler material at various levels in Eudragit RS and theophylline was used as the active pharmaceutical ingredient. The resulting structure of the nanocomposites was characterized using TEM (transmission electron microscopy) and XRPD (X-ray powder diffraction). The hygroscopicity of the nanocomposites was investigated using DVS (dynamic vapor sorption). The effect of the interfacial interaction between the polymer and clay sheet, the clay loading as well as the clay type on the drug release behavior were further studied by dissolution testing. TEM and XRPD data show that when the clay content is increased from 5% to 15% by weight, the nanocomposite's structure switches from a fully exfoliated state to intercalated structures or partial exfoliation with stacked clay layers. FT-IR (fourier transform infrared spectroscopy) and ssNMR (solid-state NMR) results suggest that Cloisite Na and Cloisite 20 layers exhibit different interaction strengths with polymer networks by creating compacted complex structures. The addition of nanoclay in the formulation could robustly adjust drug release profiles, and the clay concentration and type are important factors that affect the crossing-linking density of the nanocomposites by adjusting the drug release properties. This study indicates that the clay-Eudragit RS nanocomposites provide an improved oral controlled drug delivery system that minimizes the drug dosing frequency, potentially leading to improved patient compliance.
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http://dx.doi.org/10.3390/pharmaceutics12010051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022276PMC
January 2020

Se NMR Probes the Protein Environment of Selenomethionine.

J Phys Chem B 2020 01 7;124(4):601-616. Epub 2020 Jan 7.

Department of Chemistry and Biochemistry , University of Delaware , Newark , Delaware 19716 , United States.

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (Se). Here we exploit the sensitivity of Se NMR to the nucleus' chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic Se chemical shifts and relaxation times for six GB1 variants by solution-state Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of Se NMR experiments that exploit selenomethionine to probe proteins.
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http://dx.doi.org/10.1021/acs.jpcb.9b07466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088340PMC
January 2020

Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate.

Int J Biol Macromol 2020 Jan 28;143:41-48. Epub 2019 Nov 28.

College of Biological and Food Engineering, Changshu Institute of Technology, 99 South Third Ring Road, Changshu 215500, China; College of Food Science and Light Industry, Nanjing Tech University, 30 Puzhu South Road, Nanjing 211816, China. Electronic address:

Chondroitinase ABC I (ChSase ABC I) is a key enzyme of chondroitin sulfate (CS) degradation and widely used for CS detection in the medicine filed. However, the recombinant ChSase ABC I was weakly expressed in Escherichia coli because the forms of it were mostly inclusion bodies. In this study, a signal peptide (pelB) was used for the soluble form expression of ChSase ABC I in E. coli. Then the culture condition for ChSase ABC I expression was optimized through response surface methodology. Results revealed that the expression level of ChSase ABC I in a 7.5 L fermentor (29.03 mL) was approximately 1.65-fold higher than that of the shake flask level (17.55 mL). The enzymatic properties and kinetic constants of recombinant ChSase ABC I were also studied. Recombinant ChSase ABC I was also used to detect the specific disaccharides content of CS from different sources. This study not only eliminates the problem of the enzyme expressed as an inclusion body, but also solves the current problem of expensive ChSase ABC. In a word, it would be an ideal strategy for ChSase ABC high-efficiency expression and a great method to detect specific disaccharides of CS in biomedical field.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.11.215DOI Listing
January 2020

Identification and characterization of IRF9 from black carp Mylopharyngodon piceus.

Dev Comp Immunol 2020 02 22;103:103528. Epub 2019 Oct 22.

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China. Electronic address:

Interferon regulatory factor 9 (IRF9) plays a crucial role in JAK-STAT signaling in human and mammal. However, the relationship between IRF9 and STAT1 in teleost fish remains largely unknown. The previous study has elucidated that two STAT1 isoforms (bcSTAT1a and bcSTAT1b) of black carp (Mylopharyngodon piceus) play an important role during the innate immune activation initiated by grass carp reovirus (GCRV). In this paper, black carp IRF9 (bcIRF9) has been identified and characterized. bcIRF9 was distributed majorly in the nucleus and the linker domain (LD) of bcIRF9 was vital for its nuclear localization. bcIRF9 showed ISRE-inducing activity in reporter assay and presented antiviral activity against GCRV in plaque assay, in which both DNA binding domain (DBD) and LD of bcIRF9 were essential for its antiviral signaling. bcIRF9 was identified to interact with both bcSTAT1a and bcSTAT1b in the co-immunoprecipitation assay. It was interesting that bcIRF9-mediated antiviral signaling was up-regulated by bcSTAT1a; however, down-regulated by bcSTAT1b. Thus, our data support the conclusion that bcIRF9 plays an important role in the innate immune defense against GCRV, in which two STAT1 proteins function differently.
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http://dx.doi.org/10.1016/j.dci.2019.103528DOI Listing
February 2020

Induced complete remission faster in adult patients with acquired pure red cell aplasia by combining cyclosporine A with corticosteroids.

Medicine (Baltimore) 2019 Oct;98(41):e17425

Department of Hematology, Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Hematology of Nanjing Medical University, Collaborative Innovation Center for Cancer Personalized Medicine Nanjing, Jiangsu Province.

To evaluate whether the adult patients with acquired pure red cell aplasia (PRCA) could benefit more from cyclosporine A (CsA) combined with corticosteroids (CS) than CsA or CS alone.Seventy-three patients were evaluated in 2 institutions (6 patients lost to follow-up).The induction therapy included CsA (n = 21), CS (n = 21), or CsA combined with CS (n = 31), and remission was achieved in 16/21 (76.2%), 10/21 (47.6%), and 21/31 (71.0%) patients, respectively. Higher complete remission (CR) rate was achieved in CsA combined with CS group than in CS group (61.3% vs 19.0%, P = .003). Patients achieved CR faster in CsA combined with CS group than in CS group or CsA group (median time, 1 month vs 2 month vs 3 month, P = .010). By multivariate analysis, CsA combined with CS therapy and primary PRCA were the influence factors for CR rate. Twenty-seven patients relapsed due to discontinuation or tapering therapy, and 19 patients regained response by increasing the dose of original regimens or changing to other immunosuppressive therapy. Complete remission to induction therapy was a correlative factor for death (P = .035).CsA combined with CS produced faster and higher CR rate in treating adult patients with PRCA than did CsA or CS alone.
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http://dx.doi.org/10.1097/MD.0000000000017425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6799648PMC
October 2019

5-Hydroxymethylcytosines in Circulating Cell-Free DNA Reveal Vascular Complications of Type 2 Diabetes.

Clin Chem 2019 11 1;65(11):1414-1425. Epub 2019 Oct 1.

Department of Clinical Laboratory, Center for Gene Diagnosis, and Program of Clinical Laboratory, Zhongnan Hospital of Wuhan University, Wuhan, China;

Background: Long-term complications of type 2 diabetes (T2D), such as macrovascular and microvascular events, are the major causes for T2D-related disability and mortality. A clinically convenient, noninvasive approach for monitoring the development of these complications would improve the overall life quality of patients with T2D and help reduce healthcare burden through preventive interventions.

Methods: A selective chemical labeling strategy for 5-hydroxymethylcytosines (5hmC-Seal) was used to profile genome-wide 5hmCs, an emerging class of epigenetic markers implicated in complex diseases including diabetes, in circulating cell-free DNA (cfDNA) from a collection of Chinese patients (n = 62). Differentially modified 5hmC markers between patients with T2D with and without macrovascular/microvascular complications were analyzed under a case-control design.

Results: Statistically significant changes in 5hmC markers were associated with T2D-related macrovascular/microvascular complications, involving genes and pathways relevant to vascular biology and diabetes, including insulin resistance and inflammation. A 16-gene 5hmC marker panel accurately distinguished patients with vascular complications from those without [testing set: area under the curve (AUC) = 0.85; 95% CI, 0.73-0.96], outperforming conventional clinical variables such as urinary albumin. In addition, a separate 13-gene 5hmC marker panel could distinguish patients with single complications from those with multiple complications (testing set: AUC = 0.84; 95% CI, 0.68-0.99), showing superiority over conventional clinical variables.

Conclusions: The 5hmC markers in cfDNA reflected the epigenetic changes in patients with T2D who developed macrovascular/microvascular complications. The 5hmC-Seal assay has the potential to be a clinically convenient, noninvasive approach that can be applied in the clinic to monitor the presence and severity of diabetic vascular complications.
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http://dx.doi.org/10.1373/clinchem.2019.305508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055673PMC
November 2019

Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma.

Gut 2019 12 29;68(12):2195-2205. Epub 2019 Jul 29.

Department of Public Health Sciences, University of Chicago, Chicago, Illinois, USA.

Objective: The lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma (HCC). We sought to develop a non-invasive diagnostic approach using circulating cell-free DNA (cfDNA) for the early detection of HCC.

Design: Applying the 5hmC-Seal technique, we obtained genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA samples from 2554 Chinese subjects: 1204 patients with HCC, 392 patients with chronic hepatitis B virus infection (CHB) or liver cirrhosis (LC) and 958 healthy individuals and patients with benign liver lesions. A diagnostic model for early HCC was developed through case-control analyses using the elastic net regularisation for feature selection.

Results: The 5hmC-Seal data from patients with HCC showed a genome-wide distribution enriched with liver-derived enhancer marks. We developed a 32-gene diagnostic model that accurately distinguished early HCC (stage 0/A) based on the Barcelona Clinic Liver Cancer staging system from non-HCC (validation set: area under curve (AUC)=88.4%; (95% CI 85.8% to 91.1%)), showing superior performance over α-fetoprotein (AFP). Besides detecting patients with early stage or small tumours (eg, ≤2.0 cm) from non-HCC, the 5hmC model showed high capacity for distinguishing early HCC from high risk subjects with CHB or LC history (validation set: AUC=84.6%; (95% CI 80.6% to 88.7%)), also significantly outperforming AFP. Furthermore, the 5hmC diagnostic model appeared to be independent from potential confounders (eg, smoking/alcohol intake history).

Conclusion: We have developed and validated a non-invasive approach with clinical application potential for the early detection of HCC that are still surgically resectable in high risk individuals.
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http://dx.doi.org/10.1136/gutjnl-2019-318882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872444PMC
December 2019

Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites.

J Am Chem Soc 2019 06 23;141(22):8694-8697. Epub 2019 May 23.

Department of Chemistry, Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics , The University of Chicago , Chicago , Illinois 60637 , United States.

5-Hydroxymethylcytosine (5hmC) arises from the oxidation of 5-methylcytosine (5mC) by Fe and 2-oxoglutarate-dependent 10-11 translocation (TET) family proteins. Substantial levels of 5hmC accumulate in many mammalian tissues, especially in neurons and embryonic stem cells, suggesting a potential active role for 5hmC in epigenetic regulation beyond being simply an intermediate of active DNA demethylation. 5mC and 5hmC undergo dynamic changes during embryogenesis, neurogenesis, hematopoietic development, and oncogenesis. While methods have been developed to map 5hmC, more efficient approaches to detect 5hmC at base resolution are still highly desirable. Herein, we present a new method, Jump-seq, to capture and amplify 5hmC in genomic DNA. The principle of this method is to label 5hmC by the 6- N3-glucose moiety and connect a hairpin DNA oligonucleotide carrying an alkyne group to the azide-modified 5hmC via Huisgen cycloaddition (click) chemistry. Primer extension starts from the hairpin motif to the modified 5hmC site and then continues to "land" on genomic DNA. 5hmC sites are inferred from genomic DNA sequences immediately spanning the 5-prime junction. This technology was validated, and its utility in 5hmC identification was confirmed.
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http://dx.doi.org/10.1021/jacs.9b02512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061342PMC
June 2019

Author Correction: 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers.

Cell Res 2019 Jul;29(7):599

Department of Medicine, The University of Chicago, Chicago, IL, 60637, USA.

In the initial published version of this article, there was a mistake in the P value for the correlation between gene-expression changes and 5 hmC changes in tumors. The correct P value should be same as the P value shown in Fig. S6A: 9.8 × 10 (mistakenly shown as "9.8 × 10" in the main text). This correction does not affect the description of results or the conclusions of this study, since the range of P value is between 0 and 1.
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http://dx.doi.org/10.1038/s41422-019-0182-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796932PMC
July 2019

Molecular Interactions in Posaconazole Amorphous Solid Dispersions from Two-Dimensional Solid-State NMR Spectroscopy.

Mol Pharm 2019 06 9;16(6):2579-2589. Epub 2019 May 9.

Merck Research Laboratories (MRLs) , Merck & Co., Inc. , Kenilworth , New Jersey 07033 , United States.

Molecular interactions between the active pharmaceutical ingredient and polymer have potentially substantial impacts on the physical stability of amorphous solid dispersions (ASDs), presumably by manipulating molecular mobility and miscibility. However, structural details for understanding the nature of the molecular contacts and mechanistic roles in various physicochemical and thermodynamic events often remain unclear. This study provides a spectroscopic characterization of posaconazole (POSA) formulations, a second-generation triazole antifungal drug (Noxafil, Merck & Co., Inc., Kenilworth, NJ, USA), at molecular resolution. One- and two-dimensional (2D) solid-state NMR (ssNMR) techniques including spectral editing, heteronuclear H-C, F-C, N-C, and F-H polarization transfer, and spin correlation and ultrafast magic angle spinning, together with the isotopic labeling strategy, were utilized to uncover molecular details in POSA ASDs in a site-specific manner. Active groups in triazole and difluorophenyl rings exhibited rich but distinct categories of interactions with two polymers, hypromellose acetate succinate and hypromellose phthalate, including intermolecular O-H···O═C and O-H···F-C hydrogen bonding, π-π aromatic packing, and electrostatic interaction. Interestingly, the chlorine-to-fluorine substituent in POSA, one of the major structural differences from itraconazole that could facilitate binding to the biological target, offers an additional contact with the polymer. These findings exhibit 2D ssNMR as a sensitive technique for probing sub-nanometer structures of pharmaceutical materials and provide a structural basis for optimizing the type and strength of drug-polymer interactions in the design of amorphous formulations.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00174DOI Listing
June 2019

Three-Dimensional NMR Spectroscopy of Fluorinated Pharmaceutical Solids under Ultrafast Magic Angle Spinning.

Anal Chem 2019 05 24;91(9):6217-6224. Epub 2019 Apr 24.

Merck Research Laboratories (MRLs), Merck & Co., Inc. , Kenilworth , New Jersey 07033 , United States.

High-resolution solid-state analysis of multicomponent molecular systems, e.g., pharmaceutical formulations, is a great challenge. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy plays a critical role in the characterization of solid dosage forms due to its capabilities of chemical identification, quantification, and structural elucidation at a molecular level. However, the low NMR sensitivity as well as the high spectral complexity and low drug loading of multicomponent products hinder an in-depth investigation of the active pharmaceutical ingredient (API) at the natural isotopic abundance. Herein, we developed two new three-dimensional (3D) ssNMR methods, including H-F-H and F-F-H correlations and successfully applied them to characterize a fluorinated drug molecule, aprepitant, and its commercial nanoparticulate formulation EMEND (Merck & Co, Inc., Kenilworth, NJ, USA). These H-detection methods utilize the significantly enhanced sensitivity and resolution of H and F afforded by 60 kHz ultrafast magic angle spinning (MAS) and enable the analysis of milligram samples. The 3D techniques simultaneously provide homonuclear H-H and F-F, and heteronuclear H-F correlations of the crystalline aprepitant without interferences from other pharmaceutical components in the drug product. Moreover, our results demonstrate that F is a highly sensitive spin for probing molecular details of fluorinated drug substances in solid formulations, due to its high isotopic abundance, large gyromagnetic ratio, and absence of signal interference from pharmaceutical excipients, as well as for characterizing structural properties of a broad range of fluorine-containing materials.
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http://dx.doi.org/10.1021/acs.analchem.9b00884DOI Listing
May 2019

Enhanced Aerosolization of High Potency Nanoaggregates of Voriconazole by Dry Powder Inhalation.

Mol Pharm 2019 05 10;16(5):1799-1812. Epub 2019 Apr 10.

College of Pharmacy , The University of Texas at Austin , Austin , Texas 78712 , United States.

Invasive pulmonary aspergillosis is a deadly fungal infection with a high mortality rate, particularly in patients having undergone transplant surgery. Voriconazole, a triazole antifungal pharmaceutical product, is considered as a first-line therapy for invasive pulmonary aspergillosis, and exhibits efficacy even for patients who have failed other antifungal drug therapies. The objective of this study is to develop high potency nanoaggregates of crystalline voriconazole composition for dry powder inhalation using the particle engineering process, thin film freezing. In this study, mannitol at low concentrations acted as a surface texture-modifying agent, and we evaluated the physicochemical and aerodynamic properties of the voriconazole formulations containing different amounts of mannitol. In vitro aerosol performance data demonstrated that powder formulations consisting of 90 to 97% (w/w) voriconazole were the optimum for inhalation with a fine particle fraction (% of delivered dose) as high as 73.6 ± 3.2% and mass median aerodynamic diameter of 3.03 ± 0.17 μm when delivered by a commercially available device. The thin film freezing process enabled phase-separated submicron crystalline mannitol to be oriented such as to modify the surface texture of the crystalline voriconazole nanoaggregates, thus enhancing their aerosolization. Addition of as low as 3% (w/w) mannitol significantly increased the fine particle fraction (% of metered dose) of voriconazole nanoaggregates when compared to compositions without mannitol (40.8% vs 24.6%, respectively). The aerosol performance of the voriconazole nanoaggregates with 5% (w/w) mannitol was maintained for 13 months at 25 °C/60% RH. Therefore, voriconazole nanoaggregates having low amounts of surface texture-modifying mannitol made by thin film freezing are a feasible local treatment option for invasive pulmonary aspergillosis with high aerosolization efficiency and drug loading for dry powder inhalation.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00907DOI Listing
May 2019
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