Qiguiyin Decoction Improves Multidrug-Resistant Pseudomonas aeruginosa Infection in Rats by Regulating Inflammatory Cytokines and the TLR4/MyD88/NF-κB Signaling Pathway.

Pseudomonas aeruginosa (PA), a Gram-negative bacterium, has a high detection rate in hospital-acquired infections. Recently, the frequent appearance of multidrug-resistant (MDR) PA strain with high morbidity and mortality rates has aggravated the difficulty in treating infectious diseases. Due to its multiple resistance mechanisms, the commonly used antibiotics have gradually become less effective. Qiguiyin decoction (QGYD) is a clinically experienced prescription of Chinese herbal medicine, and its combined application with antibiotics has been confirmed to be effective in the clinical treatment of MDR PA infection, which could be a promising strategy for the treatment of drug-resistant bacterial infections. However, the mechanism of QGYD restoring antibiotics susceptibility to MDR PA remains unclear. In the present study, we investigated the effects of QGYD and levofloxacin (LEV) singly or in combination on MDR PA-induced pneumonia rat models. Further analysis was carried out in the serum differential expression profiles of inflammatory cytokines by cytokine antibody array. Besides, the lung TLR4/MyD88/NF-κB signaling pathway was detected by RT-qPCR. Our results showed that based on the treatment of MDR PA-infected rat model with LEV, the combination of QGYD improved the general state and immune organ index. Furthermore, it moderately increased the expressions of proinflammatory cytokines including IL-1ß, IL-6, and TNF-α in the early stage of infection and decreased their release rapidly in the later stage, while regulated the same phase change of anti-inflammatory cytokine IL-10. In addition, the adhesion molecule ICAM-1 was significantly downregulated after QGYD combined with LEV treatment. Moreover, the mRNA expressions of TLR4, MyD88, NF-κB, and ICAM-1 were significantly downregulated. These results indicated that the mechanism of QGYD restoring LEV susceptibility to MDR PA was related to its regulation of inflammatory cytokines and the TLR4/MyD88/NF-κB signaling pathway, which provides theoretical support for the clinical application of QGYD combined with LEV therapy to MDR PA infection.

Functional characterization of a starch synthesis-related gene AmAGP in Amorphophallus muelleri.

AMORPHOPHALLUS: has attracted tremendous interest because of its high contents of glucomannan and starch. Very few genes regulating glucomannan and starch were reported in Amorphophallus. In this study, an ADP-glucose pyrophosphorylase (AGP) gene that plays a significant role in plant starch synthesis was cloned from Amorphophallus muelleri. It was shown that it encoded a predicted protein containing a conserved plant ADP-Glucose-PP repeat domain and seven potential ligand-binding sites. The real-time quantitative PCR showed that AmAGP was most abundant in tubers, and it was positively correlated with starch content. Additionally, its influencers about temperature and exogenous plant hormone were also discussed, showing that AmAGP expressed highly in tubers under treatments using 25°C and IAA. Furthermore, starch content was closely related to AmAGP expression level, suggesting that AmAGP was involved in the regulation of starch synthesis in A. muelleri. Therefore, identifying the sequence of AmAGP and its expression pattern during tuber enlarging and the changes of its transcript levels in response to temperature and plant hormones would contribute to a better understanding of starch synthesis, and also providing a reference information for future preferable breeding for obtaining more starch or more glucomannan in Amorphophallus.

Gene-Focused Networks Underlying Phenotypic Convergence in a Systematically Phenotyped Cohort With Heterogeneous Intellectual Disability.

The broad spectrum of intellectual disability (ID) patients' clinical manifestations, the heterogeneity of ID genetic variation, and the diversity of the phenotypic variation represent major challenges for ID diagnosis. By exploiting a manually curated systematic phenotyping cohort of 3803 patients harboring ID, we identified 704 pathogenic genes, 3848 pathogenic sites, and 2075 standard phenotypes for underlying molecular perturbations and their phenotypic impact. We found the positive correlation between the number of phenotypes and that of patients that revealed their extreme heterogeneities, and the relative contribution of multiple determinants to the heterogeneity of ID phenotypes. Nevertheless, despite the extreme heterogeneity in phenotypes, the ID genes had a specific bias of mutation types, and the top 44 genes that ranked by the number of patients accounted for 39.9% of total patients. More interesting, enriched co-occurrent phenotypes and co-occurrent phenotype networks for each gene had the potential for prioritizing ID genes, further exhibited the convergences of ID phenotypes. Then we established a predictor called IDpred using machine learning methods for ID pathogenic genes prediction. Using10-fold cross-validation, our evaluation shows remarkable AUC values for IDpred (auc = 0.978), demonstrating the robustness and reliability of our tool. Besides, we built the most comprehensive database of ID phenotyped cohort to date: IDminer http://218.4.234.74:3100/IDminer/, which included the curated ID data and integrated IDpred tool for both clinical and experimental researchers. The IDminer serves as an important resource and user-friendly interface to help researchers investigate ID data, and provide important implications for the diagnosis and pathogenesis of developmental disorders of cognition.

A 7-Year Report of Spectrum of Inborn Errors of Metabolism on Full-Term and Premature Infants in a Chinese Neonatal Intensive Care Unit.

Inborn errors of metabolism (IEMs) have great repercussions in neonatal intensive care units (NICUs). However, the integrative analysis of the incidence for full-term and premature neonates of IEMs in NICUs have not been reported. In this study, we aimed to estimate the incidence of IEMs in the NICU population so as to better evaluate the impact of IEMs on Chinese NICUs. A total of 42,257 newborns (proportion of premature as 36.7%) enrolled to the largest Chinese NICU center for a sequential 7 years screen, and 66 were diagnosed with IEMs. The prevalence of IEMs in total, full-term, and premature infants was 1:640, 1:446, and 1:2,584, respectively. In spectrum of our NICU, diseases that cause endogenous intoxication like methylmalonic acidemia accounted for 93.9% (62/66), and this ratio was higher in full-term infants with 98.3% (59/60), while the most prevalent disease in premature newborn was hyperphenylalaninemia (50%, 3/6), respectively. The genetic analysis of 49 cases revealed 62 potentially pathogenic mutations in 10 well-documented pathogenic genes of IEMs, among which 21 were novel. In conclusion, differences in incidence and spectrum of full-term and premature births we obtained in NICU will provide diagnostic guidelines and therapeutic clues of neonatal IEMs for pediatricians.

Treatment of Hyperoxia-Induced Lung Injury with Lung Mesenchymal Stem Cells in Mice.

OBJECTIVE: Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm neonates and has no effective treatment. This study aimed to investigate the therapeutic effects of neonatal mouse lung resident mesenchymal stem cells (L-MSCs) on the hyperoxia-induced lung injury. METHODS: L-MSCs were separated and identified according to the MSC criterions. Hyperoxia-Induced Lung Injury (HILI) of neonatal KM mice was induced with hyperoxia (FiO2 = 60%) and investigated with pathological methods. Neonatal KM mice were divided into 3 groups (hyperoxia + L-MSC group, hyperoxia + PBS group, and air control group). Mice in the hyperoxia + L-MSC group were treated with L-MSCs at 3, 7, and 14 days after birth. After hyperoxia exposure for 21 days, the lung pathology, Radial Alveolar Count (RAC), CD31 expression, and vascular endothelial growth factor (VEGF) expression were investigated. RESULTS: After hyperoxia exposure, the body weight, RAC, CD31 expression, and VEGF expression in the hyperoxia + L-MSC group were significantly better than those in the hyperoxia + PBS group but inferior to those in the air control group significantly. These indicate L-MSCs are partially protective on the lung injury of mice with hyperoxia-induced BPD. CONCLUSION: L-MSCs are helpful for the prevention and treatment of BPD, and endogenous L-MSCs may play a role in the postinjury repair of the lung.

Two novel L2HGDH mutations identified in a rare Chinese family with L-2-hydroxyglutaric aciduria.

BACKGROUND: L-2-Hydroxyglutaric aciduria (L-2-HGA) is a rare organic aciduria neurometabolic disease that is inherited as an autosomal recessive mode and have a variety of symptoms, such as psychomotor developmental retardation, epilepsy, cerebral symptoms as well as increased concentrations of 2-hydroxyglutarate (2-HG) in the plasma, urine and cerebrospinal fluid. The causative gene of L-2-HGA is L-2-hydroxyglutarate dehydrogenase gene (L2HGDH), which consists of 10 exons. CASE PRESENTATION: We presented a rare patient primary diagnosis of L-2-HGA based on the clinical symptoms, magnetic resonance imaging (MRI), and gas chromatography-mass spectrometry (GC-MS) results. Mutational analysis of the L2HGDH gene was performed on the L-2-HGA patient and his parents, which revealed two novel mutations in exon 3: a homozygous missense mutation (c.407 A > G, p.K136R) in both the maternal and paternal allele, and a heterozygous frameshift mutation [c.407 A > G, c.408 del G], (p.K136SfsX3) in the paternal allele. The mutation site p.K136R of the protein was located in the pocket of the FAD/NAD(P)-binding domain and predicted to be pathogenic. CONCLUSION: We predicted the homozygous missense mutation (c.407 A > G, p.K136R) was considered as the pathogenic mutation of the patient. The study highlights the power of pedigree analysis in order to interpret novel mutations.

Mechanistic Insights into the Nickel-Catalyzed Cross-Coupling Reaction of Benzaldehyde with Benzyl Alcohol via C-H Activation: A Theoretical Investigation.

With the aid of density functional theory (DFT) calculations, mechanistic investigations have been carried out for the nickel-catalyzed dehydrogenative cross-coupling reaction of benzaldehyde with benzyl alcohol in the presence of N-heterocyclic carbene (NHC) ligand. The overall Ni(0)/Ni(II) catalytic cycle consists of four basic steps: ligand exchange, oxidative addition, hydrogen transfer, and reductive elimination. Considerable interests are paid on detecting the transition state of the rate-determining step, with particular emphasis on the structural and electronic properties, together with clarifying the important roles of external oxidant and hydrogen acceptor. The hydrogen transfer process in the oxidative addition step is rate-determining in the whole catalytic cycle, which is accomplished by C-Ha (active Ha) activation without generating the high energy nickel hydride intermediate. Such process could be understood as the direct hydrogen transfer, instead of general concerted oxidative addition to low valent transition metal. The analysis of the bond distances, electron distributions, and orbital interactions highlights the direct hydrogen transfer mechanism. Furthermore, by exploring the influences from the electronic effect of different substrates on the reaction energy barriers, the  a,a,a-trifluoroacetophenone could accelerate the direct hydrogen transfer with low activate energy.

Next-generation sequencing-based molecular diagnosis of neonatal hypotonia in Chinese Population.

Neonatal hypotonia is extremely challenging to diagnose because numerous disorders present similar clinical manifestations. Two panels for diagnosing neonatal hypotonia were developed, which enriches 35 genes corresponding to 61 neonatal hypotonia-related disorders. A cohort of 214 neonates with hypotonia was recruited from 2012 to 2014 in China for this study. Of these subjects, twenty-eight neonates with hypotonia were eliminated according to exclusion criteria and 97 were confirmed using traditional detection methods. The clinical diagnoses of the remaining 89 neonates with hypotonia were approached by targeted next-generation sequencing (NGS). Among the 89 tested neonates, 25 potentially pathogenic variants in nine genes (RYR1, MECP2, MUT, CDKL5, MPZ, PMM2, MTM1, LAMA2 and DMPK) were identified in 22 patients. Six of these pathogenic variants were novel. Of the 186 neonates with hypotonia, we identified the genetic causes for 117 neonates by the traditional detection methods and targeted NGS, achieving a high solving rate of 62.9%. In addition, we found seven neonates with RETT syndrome carrying five mutations, thus expanding the mutation profiles in Chinese neonates with hypotonia. Our study highlights the utility of comprehensive molecular genetic testing, which provides the advantage of speed and diagnostic specificity without invasive procedures.

Metabolomic Research on Newborn Infants With Intrauterine Growth Restriction.

To compare differences in metabolites between newborns with intrauterine growth restriction (IUGR) and those who are appropriate for gestational age (AGA) in order to understand the changes in metabolites of newborns with IUGR and to explore the possible metabolic mechanism of tissue and organ damages in patients with IUGR, with the ultimate goal of providing the basis for clinical intervention.A total of 60 newborns with IUGR and 60 AGA newborns who were hospitalized in the neonatal intensive care unit of our hospital between January 2011 and December 2015 and who underwent metabolic disease screening were enrolled in this study. The differences in 21 amino acids and 55 carnitines in peripheral blood, as well as changes in the ratios of free carnitine and acylcarnitine to total carnitine, were compared.Metabolites, particularly alanine, homocysteine, leucine, methionine, ornithine, serine, tyrosine, isovaleryl carnitine, and eicosenoyl carnitine, differed according to newborns' birth weight (<3rd percentile, 3rd-5th percentiles, 5th-10th percentiles, and 10th-90th percentiles), with those with lower birth weight showing the greater difference (P < 0.05). Metabolites also differed by gestational age, and the differences observed were mainly as follows: preterm and full-term newborns showed differences in metabolites, mainly in alanine, proline, cerotoyl carnitine, and tetradecanedioyl carnitine (P < 0.05); preterm and full-term AGA newborns showed differences in metabolites, mainly in alanine, glutamine, homocysteine, pipecolic acid, proline, heptanoyl carnitine, and sebacoyl carnitine (P < 0.05); and preterm and full-term newborns with IUGR showed differences in metabolites, mainly in arginine, glutamic acid, homocysteine, histidine, leucine, isoleucine, ornithine, serine, threonine, tryptophan, valine, heptanoyl carnitine, decanoyl carnitine, linoleyl carnitine, methylmalonyl carnitine, glutarylcarnitine, sebacoyl carnitine, hydroxyacetyl carnitine, and hydroxyhexadecancenyl carnitine (P < 0.05). Among newborns with IUGR, metabolites differed among males and females, mainly in aspartic acid, glutamic acid, and hexacosenoic acid (P < 0.05). Birth weight had no significant effects on free carnitine concentration or on the ratios of free carnitine and acylcarnitine to total carnitine (P < 0.05).IUGR infants exhibit significant abnormalities in amino acid and acylcarnitine metabolism, especially those with birth weight below the third percentile. With increasing birth weight, amino acids and acylcarnitines showed compensatory increases or reductions, and when birth weight reached the 10th percentile, the newborns with IUGR resembled the AGA newborns.

Mechanistic and kinetic study on the reaction of ozone and trans-2-chlorovinyldichloroarsine.

Singlet and triplet potential energy surfaces for the atmospheric ozonation of trans-2-chlorovnyldichloroarsine (lewisite) are investigated theoretically. Optimizations of the reactants, products, intermediates and transition states are carried out at the BHandHLYP/6-311+G(d,p) level. Single point energy calculations are performed at the CCSD(T)/6-311+G(d,p) level based on the optimized structures. The detailed mechanism is presented and discussed. Various possible H (or Cl)-abstraction and C (or As)-addition/elimination pathways are considered. The results show that the As-addition/elimination is more energetically favorable than the other mechanisms. Rice-Ramsperger-Kassel-Marcus (RRKM) theory is used to compute the rate constants over the possible atmospheric temperature range of 200-3000 K and the pressure range of 10(-8)-10(9) Torr. The calculated rate constant is in good agreement with the available experimental data. The total rate coefficient shows positive temperature dependence and pressure independence. The modified three-parameter Arrhenius expressions for the total rate coefficient and individual rate coefficients are represented. Calculation results show that major product is CHClCHAs(OOO)Cl2 (s-IM3) at the temperature below 600 K and O2 + CHClCHAsOCl2 (s-P9) play an important role at the temperature between 600 and 3000 K. Time-dependent DFT (TD-DFT) calculations indicate that CHCl(OOO)CHAsCl2 (s-IM3) and CHOAsCl2 (s-P5) can take photolysis easily in the sunlight. Due to the absence of spectral information for arsenide, computational vibrational spectra of the important intermediates and products are also analyzed to provide valuable evidence for subsequent experimental identification.

Theoretical design and simulation of supramolecular polymer unit based on multiple hydrogen bonds.

The heterocyclic urea of deazapterin (DeAPa) and its protomeric conformers (b, c) with different substituents are selected as the building block for a series of dimers in different configurations. The stabilities of all dimers in various conditions have been investigated by density functional theory. Homodimer of b has more stability than other dimers. Topological analyses certify the coexistence of intermolecular with intramolecular H-bonds. Investigations into frequency demonstrate that all H-bonds show an evident red shift in their stretching vibrational frequencies. Electron donating substituents can provide favorable free energies of the dimer. Solvent effect computations suggest that the dimerization can be favored in weakly polar solvents, such as toluene and chloroform. UV-visible spectra exhibit obvious difference of maximum absorption wavelengths between monomers and dimers, thus may have potential applications for identifying intermolecular H-bonds and calculating association constant of DeAP equilibrium systems in experiments.

Theoretical study on the gas phase reaction of propargyl alcohol with hydroxyl radical.

The reaction of propargyl alcohol with hydroxyl radical has been studied extensively at CCSD(T)/aug-cc-pVTZ//MP2/cc-pVTZ level. This is the first time to gain a conclusive insight into the reaction mechanism and kinetics for this important reaction in detail. Two reaction mechanisms were revealed, namely addition/elimination and hydrogen abstraction mechanism. The reaction mechanism confirms that OH addition to C≡C triple bond forms the chemically activated adducts, IM1 (·CHCOHCH2OH) and IM2 (CHOH·CCH2OH), and the hydrogen abstraction pathways (-CH2OH bonded to the carbon atom and alcohol hydrogen) may occur via low barriers. Harmonic model of Rice-Ramsperger-Kassel-Marcus theory and variational transition state theory are used to calculate the overall and individual rate constants over a wide range of temperatures and pressures. The calculated rate constants are in good agreement with the experimental data. At atmospheric pressure with Ar as bath gas, IM1 (·CHCOHCH2OH) and IM2 (CHOH·CCH2OH) formed by collisional stabilization are dominant in the low temperature range. The production of CHCCHOH + H2O via hydrogen abstraction becomes dominate at higher temperature. The fraction of IM3 (CH2COHCH2·O) is very significant over the moderate temperature range.

The mechanistic study of the hydroxyl radical reaction with trans-2-chlorovinyldichloroarsine.

The trans-2-chlorovinyldichloroarsine (Lewisite) was produced and handled during WWI and WWII as chemical warfare agents. It was very difficult to explore its chemical characterization by experiments ways. The quantum chemical calculations proved to be a precise and harmless method for the toxicological system. In this paper, the gas phase reaction mechanisms of OH radical with trans-2-chlorovinyldichloroarsine (lewisite) were studied by second-order Møller-Plesset perturbation theory (MP2) method. The geometries of reactants, products, complexes, and transition states were optimized at the MP2/6-311++G(d,p) level. To gain more accurate mechanistic knowledge, the single-point energies were calculated using G3 and CCSD(T) method. This reaction exhibited three mechanisms, namely, direct hydrogen abstraction, direct chlorine abstraction, and addition/elimination. Multichannel Rice-Ramsperger-Kassel-Marcus theory and transition-state theory have been carried out for overall and individual rate constants over a wide range of temperatures and pressures. The computational results indicated that addition/elimination reaction is more favorable than direct hydrogen abstraction and direct chlorine abstraction. The major products for the total reaction are AsCl2 and CHClCH2O generated via C(2)-addition/elimination.

Theoretical study on the gas phase reaction of allyl chloride with hydroxyl radical.

The reaction of allyl chloride with the hydroxyl radical has been investigated on a sound theoretical basis. This is the first time to gain a conclusive insight into the reaction mechanism and kinetics for important pathways in detail. The reaction mechanism confirms that OH addition to the C=C double bond forms the chemically activated adducts, IM1 (CH2CHOHCH2Cl) and IM2 (CH2OHCHCH2Cl) via low barriers, and direct H-abstraction paths may also occur. Variational transition state model and multichannel RRKM theory are employed to calculate the temperature-, pressure-dependent rate constants. The calculated rate constants are in good agreement with the experimental data. At 100 Torr with He as bath gas, IM6 formed by collisional stabilization is the major products in the temperature range 200-600 K; the production of CH2CHCHCl via hydrogen abstractions becomes dominant at high temperatures (600-3000 K).

Role of bone marrow-derived mesenchymal stem cells in the prevention of hyperoxia-induced lung injury in newborn mice.

BPD (bronchopulmonary dysplasia) is predominantly characterized by persistent abnormalities in lung structure and arrested lung development, but therapy can be palliative. While promising, the use of BMSC (bone marrow-derived mesenchymal stem cell) in the treatment of lung diseases remains controversial. We have assessed the therapeutic effects of BMSC in vitro and in vivo. In vitro co-culturing with injured lung tissue increased the migration-potential of BMSC; and SP-C (surfactant protein-C), a specific marker of AEC2 (type II alveolar epithelial cells), was expressed. Following intraperitoneal injection of BMSC into experimental BPD mice on post-natal day 7, it was found that BMSC can home to the injured lung, express SP-C, improve pulmonary architecture, attenuate pulmonary fibrosis and increase the survival rate of BPD mice. This work supports the notion that BMSC are of therapeutic benefit through the production of soluble factors at bioactive levels that regulate the pathogenesis of inflammation and fibrosis following hyperoxia.

Toward an understanding of the protein interaction network of the human liver.

Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26.

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2-p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26-HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.

The scaffold protein TANK/I-TRAF inhibits NF-kappaB activation by recruiting polo-like kinase 1.

TANK/I-TRAF is a TRAF-binding protein that negatively regulates NF-kappaB activation. The underlying mechanism of this activity remains unclear. Here we show that TANK directly interacts with PLK1, a conserved cell cycle-regulated kinase. PLK1 inhibits NF-kappaB transcriptional activation induced by TNF-alpha, IL-1beta, or several activators, but not by nuclear transcription factor p65. PLK1 expression reduces the DNA-binding activity of NF-kappaB induced by TNF-alpha. Moreover, endogenous activation of PLK1 reduces the TNF-induced phosphorylation of endogenous IkappaBalpha. PLK1 is bound to NEMO (IKKgamma) through TANK to form a ternary complex in vivo. We describe a new regulatory mechanism for PLK1: PLK1 negatively regulates TNF-induced IKK activation by inhibiting the ubiquitination of NEMO. These findings reveal that the scaffold protein TANK recruits PLK1 to negatively regulate NF-kappaB activation and provide direct evidence that PLK1 is required for the repression function of TANK.