Correspondence of CT perfusion imaging to pathological manifestations in rabbit models of hyperacute cerebral infarction

BACKGROUND： Could the infarction be diagnosed quickly and accurately at the acute stage by CT perfusion imaging （CTPI） technology？ Whether the images of CTPI will correspond with the pathological changes or not？ All the questions need to be solved by experimental and clinical studies. OBJECTIVE： To reveal the rules of perfusion map changes and guide the early diagnosis of hyperacute cerebral infarction by analyzing the correlation of CTPI with pathological manifestations for hyperacute cerebral infarction. DESIGN： A randomized controlled animal experiment. SETTING： Experimental Center of Medical Radiology, Longgang Central Hospital of Shenzhen City. MATERIALS： Forty-two adult New Zealand rabbits of （2.6±0.5） kg, either male or female, were randomly divided into experimental group （n =36） and control group （n =6）. Six rabbits in the experimental group were observed after ischemia for 0.5, 1, 2, 3, 4 and 6 hours respectively, and 1 rabbit in the control group was observed at each corresponding time point. METHODS： The experiments were carried out in the Experimental Center of Medical Radiology, Longgang Central Hospital of Shenzhen City from March 2003 to July 2004. Rabbit models of cerebral infarction were established by modified O＇Brein method. （1） The rabbits in the experimental group were scanned at 0.5, 1, 2, 3, 4 and 6 hours after ischemia respectively. The dynamic CT scan slice was 13 mm from the anterior edge of the frontal cortex, and six fake color functional images were obtained, including cerebral blood flow map （CBF map）, cerebral blood volume map （CBV map）, peak to enhancement map （PE map）, flow without vessels map, time to peak map （TP map）, time to start map （TS map）. The manifestations and changes of the functional maps in different interval were observed. （2） Bilateral symmetric ranges of interest （ROI） were drawn separately on the CBF map, CBV map, TP map and TS map. The blood flow parameters of focal and contralateral cerebral tissues could be obtained to calculate relative cerebral blood flow （rCBF, rCBF=focal CBF/contralateral CBF）, relative cerebral blood volume （rCBV, rCBV= focal CBV/contralateral CBV）, a relative time to peak （rTP, rTP= focal TP - contralateral TP）, a relative time to start （rTS, rTS= focal TP - contralateral TP）. （3） The perfusion maps were input into AutoCAD software. The percents of ischemic cores and peri-ischemic areas accounting for contralateral cerebral hemisphere were calculated. （4） The animals were anesthetized and killed, then the cerebellum and low brain stem were taken out. The brain tissues were cut on coronal plane at 14 mm from the anterior edge of the frontal cortex, a 2-mm piece anterior to the incision, and a 3-mm piece posterior to the incision. The anterior piece was fixed, stained and observed. A 1-mm slice was cut from the front of the posterior piece tissues as electron microscope sample, the remnant was fixed and then taken out, and the location and size of stained ＂white＂ areas were observed as the reference for electron microscope sample. （5） The correlation between CTPI and pathological manifestations was observed. MAIN OUTCOME MEASURES： （1） Laws of time and spatial changes of ischemic areas; （2） Pathological changes of the ischemic tissues; （3） Correspondency between CTPI and pathological manifestations. RESULTS： （1） Laws of time and spatial changes of ischemic areas： Relative ischemic-core areas were consistent in each perfusion map, increased incessantly along with the ischemic times. Relative peri-ischemic areas were inconsistent in each perfusion map, on CBF map from 1 to 6 hours after ischemia, the area of ischemic core increased from （1.503±0.523）% to （7.125± 1.054）%, the ascending trend occurred. But the peri-ischemic areas showed a descending trend on CBF map, the areas decreased from （8.960±0.719）% to （5.445 ± 0.884）% from 0.5 to 6 hours; The relative areas were the largest one on TP maps, the average value was （32.796±3.029）% at 0.5 hour after ischemia happening （60.540±1.683）% at 6 hours. The trend of ischemic areas was increased. No obvious change was observed on TS maps. （2） Pathological changes of the ischemic tissues： Under light microscope, there was no obvious change at 0.5- 2 hours after ischemia, edema at 3 hours, karyopycnosis at 4 hours and eosinophilous changes at 6 hours; Under electron microscope, there was edema in ischemic cores within 4 hours after ischemia, whereas karyopycnosis or structure vanished after 4 hours; Edema was observed in peri-ischemic areas. （3） Correlation between CTPI and pathological manifestations： On CTPI maps, the ischemic core was blue on CBF and CBV maps, black on TP and TS maps. Along with the ischemic times, the rCBF and rCBV decreased, whereas the rTP and rTS prolonged. Hemodynamic parameters were not significantly different within 2 hours of ischemia and 2 hours after ischemia. The rTP and rTS became 0 after 1 and 2 hours respectively. On CTPI maps the peri-ischemic area was red on CBF and CBV maps, red and yellow on TS maps, red on TP maps. Along with the ischemic times, the rCBF decreased, and the lowest level was always at about 20%, whereas the rTP and rTS prolonged. CONCLUSION： （1） CTPI manifestations corresponded well with pathological findings, and it is a sensitive, stable and reliable technique to diagnose hyperacute cerebral infarction. （2） TP map was more sensitive than CBF map and TS map in exhibiting the peri-ischemic areas, thus TP maps could be a good choice for observing peri-ischemic areas.

QM/MM study on the O_(2)activation reaction of 4-hydroxylphenyl pyruvate dioxygenase reveals a common mechanism forα-ketoglutarate dependent dioxygenase

The dioxygen activation catalyzed by 4-hydorxylphenyl pyruvate dioxygenase(HPPD)were reinvestigated by using hybrid quantum mechanics/molecular mechanics(QM/MM)approaches at the B3LYP/6-311++G(d,p):AMBER level.These studies showed that this reaction consisted of two steps including the dioxygen addition/decarboxylation and hetero O-O bond cleavage,where the first step was found to be rate-determining.The former step initially runs on a septet potential energy surface(PES),then switches to a quintet PES after crossing a septet/quintet minimum energy crossing point(MECP)5-7M2,whereas the rest step runs on the quintet PES.The reliability of our theoretical predictions is supported by the excellent agreement of the calculated free-energy barrier value of 16.9 kcal/mol with available experimental value of 16-17 kcal/mol.The present study challenges the widely accepted view which holds that the O2activation catalyzed byα-keto glutamate(α-KG)dioxygenase mainly runs on the quintet PES and provides new insight into the catalytic mechanism ofα-KG dioxygenase and/or other related Fe(Ⅱ)-dependent oxygenase.

A Hg(Ⅱ)-specific probe for imaging application in living systems and quantitative analysis in environmental/food samples

Mercury ions are highly toxic and can accumulate along food chains in water,soil,crops and animals.Effective detection of mercury ions in various media is of great significance for maintaining the ecological environment and protecting people's health.In this work,a mercury ions specific fluorescent probe was developed by a simple one-step reaction of commercial substrates of 4-chloro-7-nitro-2,1,3-benzoxadiazole and 1-(2-aminoethyl)-4-methylpiperazine.Investigation on sensing behavior showed that this probe had high sensitivity and selectivity towards mercury ions.Furthermore,this probe could be used as a tool to track the level of mercury ions in living system.In living cells,the probe with green emission emitted a bright red fluorescence when it was bound to mercury ions.In Arabidopsis thaliana,similar red emission could be detected from the root tip and stalk when A.thaliana was grown in culture medium containing mercury ions.The imaging in zebrafish showed that mercury ions were mainly concentrated in the stomach and head of zebrafish.Especially,this probe could be applied in quantitative analysis of mercury ions in tap water,green tea,sea shrimp and soil.This work provided a practical tool for the detection of mercury ions in living systems and quantitative analysis in real samples.

G-quadruplexes in genomes of viruses infecting eukaryotes or prokaryotes are under different selection pressures from hosts

G-quadruplexes in viral genomes can be applied as the targets of antiviral therapies, which has attracted wide interest. However, it is still not clear whether the pervasive number of such elements in the viral world is the result of natural selection for functionality. In this study, we identified putative quadruplex-forming sequences(PQSs) across the known viral genomes and analyzed the abundance, structural stability, and conservation of viral PQSs. A Viral Putative G-quadruplex Database(http://jsjds.hzau.edu.cn/MBPC/Vi PGD/index.php/home/index) was constructed to collect the details of each viral PQS, which provides guidance for selecting the desirable PQS. The PQS with two putative G-tetrads(G2-PQS) was significantly enriched in both eukaryotic viruses and prokaryotic viruses, whereas the PQSs with three putative G-tetrads(G3-PQS) were only enriched in eukaryotic viruses and depleted in prokaryotic viruses. The structural stability of PQSs in prokaryotic viruses was significantly lower than that in eukaryotic viruses. Conservation analysis showed that the G2-PQS, instead of G3-PQS, was highly conserved within the genus. This suggested that the G2-quadruplex might play an important role in viral biology, and the difference in the occurrence of G-quadruplex between eukaryotic viruses and prokaryotic viruses may result from the different selection pressures from hosts.

The structure of 4-hydroxylphenylpyruvate dioxygenase complexed with 4-hydroxylphenylpyruvic acid reveals an unexpected inhibition mechanism

4-Hydroxyphenylpyruvate dioxygenase(HPPD)is an important target for both drug and pesticide discovery.As a typical Fe(II)-dependent dioxygenase,HPPD catalyzes the complicated transformation of 4-hydroxyphenylpyruvic acid(HPPA)to homogentisic acid(HGA).The binding mode of HPPA in the catalytic pocket of HPPD is a focus of research interests.Recently,we reported the crystal structure of Arabidopsis thaliana HPPD(At HPPD)complexed with HPPA and a cobalt ion,which was supposed to mimic the pre-reactive structure of At HPPD-HPPA-Fe(II).Unexpectedly,the present study shows that the restored At HPPD-HPPA-Fe(II)complex is still nonreactive toward the bound dioxygen.QM/MM and QM calculations reveal that the HPPA resists the electrophilic attacking of the bound dioxygen by the trim of its phenyl ring,and the residue Phe381 plays a key role in orienting the phenyl ring.Kinetic study on the F381 A mutant reveals that the HPPD-HPPA complex observed in the crystal structure should be an intermediate of the substrate transportation instead of the pre-reactive complex.More importantly,the binding mode of the HPPA in this complex is shared with several well-known HPPD inhibitors,suggesting that these inhibitors resist the association of dioxygen(and exert their inhibitory roles)in the same way as the HPPA.The present study provides insights into the inhibition mechanism of HPPD inhibitors.

Insight into the Structural Requirements of Protoporphyrino- gen Oxidase Inhibitors： Molecular Docking and CoMFA of Di- phenyl Ether, Isoxazole Phenyl, and Pyrazole Phenyl Ether

Protoporphyrinogen oxidase （PPO, EC 1.3.3.4） is one of the most significant targets for a large family of in- hibitors that may be used as herbicide, bactericide, fungicide, or photosensitizing activator to treat cancer through photodynamic therapy （PDT）. Molecular docking and CoMFA were combined in a multistep framework with the ultimate goal of identifying important factor contributing to the activity of PPO inhibitors. As a continuation of the previous research work on the development of new PPO inhibitors, the bioassay results indicated that good PPO in- hibitors were discovered in all of the three chemical series with ICs0 values ranging from 0.010 to 0.061 pmol·L ^-1. Using the crystal structure of tobacco mitochondrial PPO （mtPPO） as template, all the compounds were docked into the enzyme active site. The docking pose of each compound was subsequently used in a receptor-based alignment, leading to the development of a significant CoMFA model with r^2 value of 0.98 and q^2 （cross validation r^2） value of 0.63. This novel multistep framework gives insight into the and it can be extended to other classes of PPO inhibitors. In be particularly applicable in virtual screening procedures. structural characteristics for the binding of inhibitors, addition, the simplicity of the proposed approach may

DISCOVERY OF TRIKETONE-QUINOXALINE HYBRIDS AS POTENT HPPD INHIBITORS USING STRUCTUREBASED DRUG DESIGN

p-Hydroxyphenylpyruvate dioxygenase(EC 1.13.11.27,HPPD)belongs to the family of Fe(II)-dependent non-heme oxygenases that occur in the majority of aerobic organisms.HPPD has proved to be a promising target in herbicide research and development.A battery of novel triketone-quinoxaline compounds has been designed using a structure-based drug design strategy and then prepared.Enzyme inhibition assays show that these synthesized derivatives possess favorable inhibition capability against Arabidopsis thaliana HPPD with IC50 values ranging from 0.317 to 0.891 mmol$L–1.Subsequently,the molecular docking results indicate that two adjacent carbonyls of the triketone moiety of the representative compound 2-(2,3-dimethyl-8-(o-tolyl)quinoxaline-6-carbonyl)-3-hydroxycyclohex-2-en-1-one(7d)engage in chelation with the ferrous ion of A.thaliana HPPD in a bidentate pose,and its quinoxaline scaffold forms two sets of parallelπ-stacking interaction between two phenylalanine residues(Phe424 and Phe381).In addition,the extended phenyl group also interacts with Phe392 in aπ-πstacking way.This study indicates that triketone-quinoxaline is a promising scaffold for discovering HPPD inhibitors with substantially increased potency,providing insight into the molecular design of new herbicides.

CROP PROTECTION OPENS UP NEW ERA OF CONSERVATION AND UTILIZATION OF GREEN OPTIONS

China is the largest agricultural producer in the world.Reducing yield losses caused by pests is an important issue and major challenge for China,especially when confronting global climate change,biological invasions and declining agricultural biodiversity of recent decades.Wang et al.(this issue)summarized the impacts of changing climate on two staple crops in China,wheat and rice(https://doi.org/FASE-2021432).