Structure-Guided In Vitro to In Vivo Pharmacokinetic Optimization of Propargyl-Linked Antifolates.

Pharmacokinetic/pharmacodynamic properties are strongly correlated with the in vivo efficacy of antibiotics. Propargyl-linked antifolates, a novel class of antibiotics, demonstrate potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, including multidrug-resistant Staphylococcus aureus Here, we report our efforts to optimize the pharmacokinetic profile of this class to best match the established pharmacodynamic properties. High-resolution crystal structures were used in combination with in vitro pharmacokinetic models to design compounds that not only are metabolically stable in vivo but also retain potent antibacterial activity. The initial lead compound was prone to both N-oxidation and demethylation, which resulted in an abbreviated in vivo half-life (∼20 minutes) in mice. Stability of leads toward mouse liver microsomes was primarily used to guide medicinal chemistry efforts so robust efficacy could be demonstrated in a mouse disease model. Structure-based drug design guided mitigation of N-oxide formation through substitutions of sterically demanding groups adjacent to the pyridyl nitrogen. Additionally, deuterium and fluorine substitutions were evaluated for their effect on the rate of oxidative demethylation. The resulting compound was characterized and demonstrated to have a low projected clearance in humans with limited potential for drug-drug interactions as predicted by cytochrome P450 inhibition as well as an in vivo exposure profile that optimizes the potential for bactericidal activity, highlighting how structural data, merged with substitutions to introduce metabolic stability, are a powerful approach to drug design.

[Development of Lung Compression Degree Measurement Software of Pneumothorax and Its Application in Forensic Medicine].

OBJECTIVES: To develop a measurement software of lung compression degree to calculate the lung compression ratio in pneumothorax patients accurately and quickly, and then provide an objective assessment of damage degree in forensic clinical identification. METHODS: A volume calculation software was established according to the working principle of the CT instrument. CT data of 15 pneumothorax patients were selected as research objects. The lung compression ratio of pneumothorax patient was calculated by the lung compression volume calculation software of the CT instrument. Meanwhile, the lung compression ratio was also calculated by the developed volume calculation software. The lung compression ratio and operation time calculated by the two methods were analyzed statistically. Scatter plot graphs were draw based on related data, and the developed volume calculation software was verified. RESULTS: The difference between the lung compression ratios calculated by the two methods was not statistically significant, but showed a linear correlation （P<0.05）. The operation time of the developed volume calculation software was obviously shorter. CONCLUSIONS: The volume calculation software developed in this study can calculate the lung compression degree of pneumothorax more conveniently and rapidly with easy accessibility, which shows an application value in the forensic practice.

Role of the dorsal medial habenula in the regulation of voluntary activity, motor function, hedonic state, and primary reinforcement.

The habenular complex in the epithalamus consists of distinct regions with diverse neuronal populations. Past studies have suggested a role for the habenula in voluntary exercise motivation and reinforcement of intracranial self-stimulation but have not assigned these effects to specific habenula subnuclei. Here, we have developed a genetic model in which neurons of the dorsal medial habenula (dMHb) are developmentally eliminated, via tissue-specific deletion of the transcription factor Pou4f1 (Brn3a). Mice with dMHb lesions perform poorly in motivation-based locomotor behaviors, such as voluntary wheel running and the accelerating rotarod, but show only minor abnormalities in gait and balance and exhibit normal levels of basal locomotion. These mice also show deficits in sucrose preference, but not in the forced swim test, two measures of depression-related phenotypes in rodents. We have also used Cre recombinase-mediated expression of channelrhodopsin-2 and halorhodopsin to activate dMHb neurons or silence their output in freely moving mice, respectively. Optical activation of the dMHb in vivo supports intracranial self-stimulation, showing that dMHb activity is intrinsically reinforcing, whereas optical silencing of dMHb outputs is aversive. Together, our findings demonstrate that the dMHb is involved in exercise motivation and the regulation of hedonic state, and is part of an intrinsic reinforcement circuit.

A morphometric study of the thoracolumbar spine spinous process and lamina space in the Chinese.

BACKGROUND: The aim of the study was to analyse the anatomical parameters of the thoracolumbar spine spinous process and lamina space for Chinese anatomic study, and provide an anatomical reference for its clinical operation. MATERIALS AND METHODS: Samples from 24 adult autopsy subjects were obtained from the thoracolumbar spine spinous process and lamina space at levels T1 through L5. Direct measurements were made for the spinous process spacing distance, spinous process length, width, thickness and inclination angle, and the lamina space width and height. RESULTS: 1. Distance of the spine spinous process spacing: Thoracic part: The maximum tip distance was observed at T4~T5 level, and the minimum tip distance was observed at T9~T10 level. The maximum centre distance and root distance were observed at T11~T12 level, and the minimum were observed at T5~T6 level separately. Lumbar part: distance of spinous process spacing in lumbar part showed a decreasing pattern from L1~L2 to L5~S1. 2. Length, width, thickness of the spine spinous process: 1) The length of the spinous process: The upper border gradually increased from T1 to T6 and then decreased till T12 region. The centre region is T8 maximum, T11 minimum. The lower border length showed a decreasing trend from T1 to T12. Lumbar part: The length increased from L1 and reached maximum value at L3. Then, the length decreased gradually to reach minimum value at L5. 2) The width of the spinous process: The width showed an increasing trend from T1 to T12. Lumbar part: Maximum width was seen at L3 and a minimum L5. 3) The thickness of the spinous process: Tip thickness > Centre thickness > Root thickness in each thoracic and lumbar vertebra. Thoracic part: the maximum tip thickness is T1, T7 minimum, The maximum centre thickness is T12, T7 minimum. The maximum root height is T6, T9 minimum. Lumbar part: Maximum tip thickness was seen at L1, and a minimum L3. Maximum centre thickness was seen at L5, and a minimum L2. Maximum root thickness was seen at L2, and a minimum L1. 3. Inclination angle of the spine spinous process: The inclination angle gradually decreased from T1 to T7 to minimum value at T7 and then increased till T12 region. 4. Width and height of lamina space: 1) The width of lamina space: For thoracic part, the data became shorter gradually from T1~T2 to T5~T6, and then increased till to T11~T12. For lumbar part, the width of lamina space increased from T12~L1 to L5~S1. 2) The height of lamina space: In the thoracic vertebrae, the maximum height of centre region was observed at T11~T12 and the minimum mean value was observed at T3~T4. In the lumbar vertebrae, the height of the lamina space was gradually increased from T12~L1 to L5~S1. CONCLUSIONS: This study reports morphometric data of the thoracolumbar spine spinous process and lamina space in the Chinese population, which provides an anatomic basis for thoracolumbar spine design of internal fixation, posterior surgery, puncture and epidural anaesthesia.

MiR-5195-3p inhibits the proliferation of glioma cells by targeting BIRC2.

OBJECTIVE: The aim of this study was to investigate the effects of microRNA-5195-3p (miR-5195-3p) on the proliferation of glioma cells and to explore its related mechanisms. PATIENTS AND METHODS: The expression level of miR-5195-3p in glioma tissues and cell lines was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Online prediction websites (TargetScan and miRanda) were used to screen the potential targets of miR-5195-3p. Luciferase reporter gene assay and Western blot were performed to confirm the targets of miR-5195-3p. Furthermore, the effects of miR-5195-3p on cell proliferation were detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), colony formation assay, and flow cytometry, respectively. RESULTS: MiR-5195-3p was lowly expressed in both glioma tissues and cells. Baculoviral IAP repeat-containing 2 (BIRC2) was identified as a direct target of miR-5195-3p. Over-expression of miR-5195-3p in glioma cells significantly decreased the protein expression of BIRC2. Besides, the proliferative capacity and colony formation ability were significantly inhibited after transfection of miR-5195-3p in vitro. In addition, flow cytometry indicated that an evident G1 phase arrest occurred in miR-5195-3p over-expressed group. CONCLUSIONS: In this work, we emphasized the suppressor function of miR-5195-3p in the proliferation of glioma cells. Furthermore, our findings provided an experimental basis for the research and treatment of glioma.

Impact of participation in a surgical site infection surveillance network: results from a large international cohort study.

BACKGROUND: Surveillance of surgical site infections (SSIs) is a core component of effective infection control practices, though its impact has not been quantified on a large scale. AIM: To determine the time-trend of SSI rates in surveillance networks. METHODS: SSI surveillance networks provided procedure-specific data on numbers of SSIs and operations, stratified by hospitals' year of participation in the surveillance, to capture length of participation as an exposure. Pooled and procedure-specific random-effects Poisson regression was performed to obtain yearly rate ratios (RRs) with 95% confidence intervals (CIs), and including surveillance network as random intercept. FINDINGS: Of 36 invited networks, 17 networks from 15 high-income countries across Asia, Australia and Europe participated in the study. Aggregated data on 17 surgical procedures (cardiovascular, digestive, gynaecological-obstetrical, neurosurgical, and orthopaedic) were collected, resulting in data concerning 5,831,737 operations and 113,166 SSIs. There was a significant decrease in overall SSI rates over surveillance time, resulting in a 35% reduction at the ninth (final) included year of surveillance (RR: 0.65; 95% CI: 0.63-0.67). There were large variations across procedure-specific trends, but strong consistent decreases were observed for colorectal surgery, herniorrhaphy, caesarean section, hip prosthesis, and knee prosthesis. CONCLUSION: In this large, international cohort study, pooled SSI rates were associated with a stable and sustainable decrease after joining an SSI surveillance network; a causal relationship is possible, although unproven. There was heterogeneity in procedure-specific trends. These findings support the pivotal role of surveillance in reducing infection rates and call for widespread implementation of hospital-based SSI surveillance in high-income countries.

On the human CYP2C9*13 variant activity reduction: a molecular dynamics simulation and docking study.

Cytochrome P450 2C9 (CYP2C9) plays a key role in the metabolism of clinical drugs. CYP2C9 is a genetically polymorphic enzyme and some of its allelic variants have less activity compared to the wild-type form. Drugs with a narrow therapeutic index may cause serious toxicity to the individuals who carry such allele. CYP2C9*13, firstly identified by some of the present authors in a Chinese poor metabolizer of lornoxicam, is characterized by mutation encoding Leu90Pro substitution. Kinetic experiments show that CYP2C9*13 has less catalytic activity in elimination of diclofenac and lornoxicam in vitro. In order to explore the structure-activity relationship of CYP2C9*13, the three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 are constructed on the basis of the X-ray crystal structure of human CYP2C9*1 (PDB code 1R9O) by molecular dynamics simulations. The structure change caused by Leu90Pro replacement is revealed and used to explain the dramatic decrease of the enzymatic activity in clearance of the two CYP2C9 substrates: diclofenac and lornoxicam. The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified. The backbone of residues 106-108 in CYP2C9*13 turns over and their side chains block the entrance for substrates accessing so that the entrance of *13 shrinks greatly than that in the wild-type, which is believed to be the dominant mechanism of the catalytic activity reduction. Consequent docking study which is consistent with the results of the kinetic experiments by Guo et al. identifies the most important residues for enzyme-substrate complexes.

Dorsal Medial Habenula Regulation of Mood-Related Behaviors and Primary Reinforcement by Tachykinin-Expressing Habenula Neurons.

Animal models have been developed to investigate aspects of stress, anxiety, and depression, but our understanding of the circuitry underlying these models remains incomplete. Prior studies of the habenula, a poorly understood nucleus in the dorsal diencephalon, suggest that projections to the medial habenula (MHb) regulate fear and anxiety responses, whereas the lateral habenula (LHb) is involved in the expression of learned helplessness, a model of depression. Tissue-specific deletion of the transcription factor Pou4f1 in the dorsal MHb (dMHb) results in a developmental lesion of this subnucleus. These dMHb-ablated mice show deficits in voluntary exercise, a possible correlate of depression. Here we explore the role of the dMHb in mood-related behaviors and intrinsic reinforcement. Lesions of the dMHb do not elicit changes in contextual conditioned fear. However, dMHb-lesioned mice exhibit shorter immobility time in the tail suspension test, another model of depression. dMHb-lesioned mice also display increased vulnerability to the induction of learned helplessness. However, this effect is not due specifically to the dMHb lesion, but appears to result from Pou4f1 haploinsufficiency elsewhere in the nervous system. Pou4f1 haploinsufficiency does not produce the other phenotypes associated with dMHb lesions. Using optogenetic intracranial self-stimulation, intrinsic reinforcement by the dMHb can be mapped to a specific population of neurokinin-expressing habenula neurons. Together, our data show that the dMHb is involved in the regulation of multiple mood-related behaviors, but also support the idea that these behaviors do not reflect a single functional pathway.

Biflavonoids from the aerial part of Stephania tetrandra.

Investigation of the aerial part of Stephania tetrandra led to the isolation of two biflavonoids, stephaflavone A and stephaflavone B, with a 3-6" linkage pattern, together with beta-sitosterol. Their structures were established on the basis of their spectroscopic data and their physicochemical properties.

Two butylated aminooligosaccharides isolated from the culture filtrate of Streptomyces luteogriseus.

Two novel aminooligosaccharides, butytatins M03 and M13 were isolated and purified from the culture filtrate of Streptomyces luteogriseus. Analysis by liquid chromatography coupled to electrospray ionization mass spectrometry indicated their resemblance to isovalertatin, with a four-carbon acyl group. Their structures were established by NMR as aminooligosaccharide derivatives possessing a butylated side chain.

Structural revision of isovalertatins M03, M13, and M23 isolated from the culture of Streptomyces luteogriseus.

Three aminooligosaccharides, isovalertatins M03 (1), M13 (2), and M23 (3) were isolated and purified from the culture filtrate of Streptomyces luteogriseus. Their physicochemical properties, liquid chromatographic behavior, and spectroscopic data were in full accordance with the reported compounds [Xu, Q.; Wang, Q.; Lu, D. CN Patent 1100756, 1995; Chem. Abstr. 1995, 123, 110278n], but their structures were reinvestigated and revised by spectroscopic methods, including ESI multistage mass spectrometry and 2-dimensional NMR techniques.

[Trend and causes of injury deaths among children in Macheng city of Hubei province during 1969 through 1998].

OBJECTIVES: To describe time trend of mortality and proportional mortality of injury and to determine the characteristics of distribution of injury deaths in children aged 0 - 14 in Macheng city during 1969 to 1998. METHODS: Data were collected from Notification System for Deaths of Macheng City during 1969 to 1998. Injury claimed 14,510 deaths caused by injury in children in total during these years. Changes in mortality and proportional mortality for injury were fitted with log-linear and simple linear regression models to compare their difference in children with different gender and ages. RESULTS: Both injury mortality and proportional mortality in infants declined during the past 30 years, and injury mortality dropped but proportional mortality due to injury increased gradually in children aged 1 - 14 years old. From 1984 to 1998, the average injury mortality was 81.36 per 100,000 children of 0 - 14 of age, with a potential years of life lost rate was 57.92 per 1,000 children. Boys had higher injury mortality than girls. Injury mortality in infants reached 560.15 per 100,000, and mechanical suffocation was the leading cause of injury deaths in infants. Injury mortality was 95.48 per 100,000 in children aged 1 - 4 years. Injury deaths in children aged 0 - 4 accounted for 73.2 percent of total injury deaths. And, drowning was the leading cause of injury deaths in children aged 1 - 14 years. CONCLUSION: Children under five years old were the vulnerable population for injury death needed for special care and strategy and measures should be taken to control injury in children in the rural areas.

[Germination temperature of seeds of medicinal plants].

According to our experiments in the last ten years, the germination behaviour of the seeds of medicinal plants may be classified into five types by different temperature requirements for germination: (1) low temperature type; (2) middle temperature type; (3) high temperature type; (4) alternating temperature type; (5) wide ranging temperature type. This classification is helpful for cultivators to determine the time of sowing.

Preparation of evodiamine solid dispersions and its pharmacokinetics.

In order to increase the dissolution rate and bioavailability, solid dispersions of evodiamine in PVP K(30) with different enriched samples of evodiamine to PVP K(30) ratios were prepared by solvent method. Our studies showed that the dissolution rate of evodiamine was significantly higher in the solid dispersion system in comparison with that in enriched samples of evodiamine or physical mixtures. The increase of the dissolution rate was evidently related to the ratio of evodiamine to PVP K(30). The solid dispersion system (enriched samples of evodiamine/PVP K(30)= 1/6, w/w) gave the highest dissolution rate: about 27.7-fold higher than that of enriched samples of evodiamine in hard capsules. Powder X-ray diffraction studies showed that enriched samples of evodiamine presented a total chemical stability after its preparation as solid dispersions. In vivo administration studies indicated that solid dispersions of evodiamine in hard capsules had a higher C(max) and a shorter T(max) than those of physical mixture in hard capsules, and the differences of C(max) and T(max) between them were significant. These results suggest that solid dispersions of evodiamine in hard capsules has a notably faster and greater absorption rate than enriched samples of evodiamine in physical mixture hard capsule and corresponds with the in vitro dissolution.

Dexamethasone attenuates lipopolysaccharide-induced liver injury by downregulating glucocorticoid-induced tumor necrosis factor receptor ligand in Kupffer cells.

AIM: Glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) plays pro-inflammatory roles in immune response. Thus, our aim was to assess if dexamethasone attenuates lipopolysaccharide (LPS)-induced liver injury by affecting GITRL in Kupffer cells (KC). METHODS: A BALB/c mouse model of liver injury was established by i.p. injecting with LPS (10 mg/kg) co-treated with or without dexamethasone (3 mg/kg). Blood and liver samples were obtained for analysis of liver morphology, GITRL expression, hepatocellular function and cytokine levels at 24 h after injection. KC were isolated and challenged by LPS (1 µg/mL), with or without dexamethasone (10 µM) co-treatment, or with GITRL siRNA pre-transfection. The GITRL expression and cytokine levels were assayed at 24 h after challenge. RESULTS: Dexamethasone treatment significantly improved the survival rate of endotoxemic mice (P < 0.05), whereas serum alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor (TNF)-α, interleukin (IL)-6 and γ-interferon levels were significantly decreased (P < 0.05, respectively). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphological analysis; and expression of GITRL in liver tissue and KC was downregulated (P < 0.05). Consistent with these in vivo experiments, inhibited expression of GITRL, TNF-α and IL-6 caused by dexamethasone treatment were also observed in LPS-stimulated KC. The GITRL, TNF-α and IL-6 expression was also significantly inhibited by GITRL gene silencing. CONCLUSION: The TNF-α and IL-6 expression of LPS-stimulated KC was inhibited by GITRL gene silencing. Dexamethasone attenuates LPS-induced liver injury, at least proportionately, by downregulating GITRL in KC.

Catalytic activities of human cytochrome P450 2C9*1, 2C9*3 and 2C9*13.

Cytochrome P450 2C9 (CYP2C9) is a geneticly polymorphic enzyme responsible for the metabolism of some clinically important drugs. CYP2C9*13 is an allele identified in a Chinese poor metabolizer of lornoxicam which has a Leu90Pro amino acid substitution. This paper reports on a study aimed at comparing the catalytic properties of CYP2C9*13 with those of the wild-type CYP2C9*1 and mutant CYP2C9*3 (Ile359Leu) in the COS-7 expression system using various substrates. CYP2C9*3 and *13 produced far lower luminescence than CYP2C9*1 in luciferin H metabolism. CYP2C9*13 exhibited an 11-fold increase in Km but no change in Vmax with tolbutamide as the substrate, a five-fold increase in Km and an 88.8% reduction in Vmax with diclofenac. These data indicate that CYP2C9*13 exhibits reduced metabolic activity toward all studied CYP2C9 substrates. The magnitude of the CYP2C9*13-associated decrease in intrinsic clearance (Vmax/Km) is greater than that associated with CYP2C9*3.

Profiling of isovalertatin family aminooligosaccharides extracted from the culture of Streptomyces luteogriseus by using liquid chromatography/electrospray ionization mass spectrometry.

A rapid and novel procedure using the liquid chromatography coupled to an electrospray ionization ion trap mass spectrometry technique was applied for the profiling of isovalertatin-family aminooligosaccharides in the extract from the culture filtrate of Streptomyces luteogriseus. The aminooligosaccharides were separated on a C-8 reversed-phase column with an acetonitrile-alkaline water gradient. The desired homologues were detected using the multiple reaction monitoring mode, and the chemical structures were confirmed by analyzing the characteristic fragment ions in their collision-induced dissociation spectra. This facile procedure led to the identification of all the five known aminooligosaccharides, isovalertatins M03, M13, M23, D03, and D23, in addition to the characterization of at least 41 novel isovalertatins, the molecular weights of which ranged from 729 to 2,793. This kind of assay should be intended as a simple and convenient way for the high-throughput analysis of screening aminooligosaccharides and potentially other structural families of natural products.