Discovery of novel selective phosphodiesterase­1 inhibitors for the treatment of acute myelogenous leukemia.

Acute myelogenous leukemia (AML) is the most common form of acute leukemia in adults. PDE1 (Phosphodiesterase 1) is a subfamily of the PDE super-enzyme families that can hydrolyze the second messengers cAMP and cGMP simultaneously. Previous research has shown that suppressing the gene expression of PDE1 can trigger apoptosis of human leukemia cells. However, no selective PDE1 inhibitors have been used to explore whether PDE1 is a potential target for treating AML. Based on our previously reported PDE9/PDE1 dual inhibitor 11a, a series of novel pyrazolopyrimidinone derivatives were designed in this study. The lead compound 6c showed an IC50 of 7.5 nM against PDE1, excellent selectivity over other PDEs and good metabolic stability. In AML cells, compound 6c significantly inhibited the proliferation and induced apoptosis. Further experiments indicated that the apoptosis induced by 6c was through a mitochondria-dependent pathway by decreasing the ratio of Bcl-2/Bax and increasing the cleavage of caspase-3, 7, 9, and PARP. All these results suggested that PDE1 might be a novel target for AML.

[Screening and promoting effect of grow-promoting fungi in rhizosphere of Angelica dahurica var. formosana].

Excessive application of chemical fertilizer has caused many problems in Angelica dahurica var. formosana planting, such as yield decline and quality degradation. In order to promote the green cultivation mode of A. dahurica var. formosana and explore rhizosphere fungus resources, the rhizosphere fungi with nitrogen fixation, phosphorus solubilization, potassium solubilization, iron-producing carrier, and IAA-producing properties were isolated and screened in the rhizosphere of A. dahurica var. formosana from the genuine and non-genuine areas, respectively. The strains were identified comprehensively in light of the morphological characteristics and ITS rDNA sequences, and the growth-promoting effect of the screened strains was verified by pot experiment. The results showed that 37 strains of growth-promoting fungi were isolated and screened from the rhizosphere of A. dahurica var. formosana, mostly belonging to Fusarium. The cultured rhizosphere growth-promoting fungi of A. dahurica var. formosana were more abundant and diverse in the genuine producing areas than in the non-genuine producing areas. Among all strains, Aspergillus niger ZJ-17 had the strongest growth promotion potential. Under the condition of no fertilization outdoors, ZJ-17 inoculation significantly promoted the growth, yield, and accumulation of effective components of A. dahurica var. formosana planted in the soil of genuine and non-genuine producing areas, with yield increases of 73.59% and 37.84%, respectively. To a certain extent, it alleviated the restriction without additional fertilization on the growth of A. dahurica var. formosana. Therefore, A. niger ZJ-17 has great application prospects in increasing yield and quality of A. dahurica var. formosana and reducing fertilizer application and can be actually applied in promoting the growth of A. dahurica var. formosana and producing biofertilizer.

Identification of Novel Quinolin-2(1H)-ones as Phosphodiesterase 1 Inhibitors for the Treatment of Inflammatory Bowel Disease.

Phosphodiesterase 1 (PDE1) is a subfamily of PDE super enzyme families that can hydrolyze cyclic adenosine monophosphate and cyclic guanosine monophosphate simultaneously. Currently, the number of PDE1 inhibitors is relatively few, significantly limiting their application. Herein, a novel series of quinolin-2(1H)-ones were designed rationally, leading to compound 10c with an IC50 of 15 nM against PDE1C, high selectivity across other PDEs, and remarkable safety properties. Furthermore, we used the lead compound 10c as a chemical tool to explore whether PDE1 could work as a novel potential target for the treatment of inflammatory bowel disease (IBD), a disease which is a chronic, relapsing disorder of the gastrointestinal tract inflammation lacking effective treatment. Our results showed that administration of 10c exerted significant anti-IBD effects in the dextran sodium sulfate-induced mice model and alleviated the inflammatory response, indicating that PDE1 could work as a potent target for IBD.

[Bioinformatics and expression analysis on MYB-related family in Angelica dahurica var. formosana].

In recent years, the MYB-related gene family has been found pivotal in plant growth and development. MYB-related gene family in Angelica dahurica var. formosana was systematically investigated based on "Chuanzhi No. 2" through transcriptome database search and bioinformatics and the temporal and spatial expression patterns were analyzed through real-time fluorescence-based quantitative polymerase chain reaction(PCR). The results showed that 122 MYB-related proteins family were identified, mainly including the unstable hydrophilic proteins with good thermal stability. Most of the proteins were located in nuclei. The majority of the proteins had the structures of random coil and α-helix. Five MYB-related proteins family of A. dahurica var. formosana had membrane-binding domains. The conserved domain analysis of MYB-related proteins family of A. dahurica var. formosana showed that the MYB domains of genes in five subgroups, similar to 2 R-, 3 R-, and 4 R-MYB proteins, contained three evenly distributed Trp(W) residues in the MYB repeat sequence. The phylogenetic analysis of MYB-related proteins family in A. dahurica var. formosana and Arabidopsis thaliana showed that the MYB-related members were unevenly distributed in five subgroups, and A. thaliana and A. dahurica var. formosana had almost the same number of genes in the CCA1-like subgroup. There were differences in the number, type, and distribution of motifs contained in 122 encoded proteins. Transcription factors with similar branches had similar domains and motifs. The expression pattern analysis showed that the transcription factors AdMYB53, AdMYB83, and AdMYB89 responded to hormones to varying degrees, and they were highly expressed in leaves and responded quickly in roots. This study lays a foundation for further investigating the function of MYB-related transcription factors of A. dahurica var. formosana and solving the corresponding biological problems such as bolting early.

PDE1 inhibitors: a review of the recent patent literature (2008-present).

INTRODUCTION: PDE1 has been demonstrated to be a potential drug target for a variety of diseases, such as Alzheimer's disease and cardiovascular disease. In the past decades, numerous PDE1 inhibitors with structural diversities have been developed and patented by pharmaceutical companies, providing drug candidates for exploring novel disease indications of PDE1. AREA COVERED: This review aims to provide an overview of PDE1 inhibitors reported in patents from 2008 to present. EXPERT OPINION: Among current PDE1 inhibitors, only a few of them showed high selectivity over other PDEs, which might cause severe side effects in the clinic. The development of highly selective PDE1 inhibitors is still the 'top priority' in the following research. The selective recognition mechanism of PDE1 with inhibitors should be further elucidated by X-ray crystallography in order to provide evidence for the rational design of selective PDE1 inhibitors. In addition, PDE1 inhibitors should be applied to different clinical indications beyond CNS diseases.

Discovery of Potent Phosphodiesterase-9 Inhibitors for the Treatment of Hepatic Fibrosis.

Hepatic fibrosis commonly exists in chronic liver disease and would eventually develop to cirrhosis and liver cancer with high fatality. Phosphodiesterase-9 (PDE9) has attracted profound attention as a drug target because of its highest binding affinity among phosphodiesterases (PDEs) with cyclic guanosine monophosphate. However, no published study has reported PDE9 inhibitors as potential agents against hepatic fibrosis yet. Herein, structural modification from a starting hit LL01 led to lead 4a, which exhibited an IC50 value of 7.3 nM against PDE9, excellent selectivity against other PDE subfamilies, and remarkable microsomal stability. The cocrystal structure of PDE9 with 4a revealed an important residue, Phe441, capable of improving the selectivity of PDE9 inhibitors. Administration of 4a exerted a significant antifibrotic effect in bile duct-ligation-induced rats with hepatic fibrosis and transforming growth factor-ß-induced fibrogenesis. This therapeutic effect was indeed achieved by selectively inhibiting PDE9 rather than other PDE isoforms, identifying PDE9 inhibitors as potential agents against hepatic fibrosis.

[Bioinformatics analysis and expression pattern of NAC transcription factor family of Angelica dahurica var. formosana from Sichuan province].

NAC(NAM/ATAF/CUC) protein plays an important role in plant growth and development, secondary cell wall formation and stress response. In this study, based on the sequencing data of Angelica dahurica, the NAC family was systematically analyzed using bioinformatics methods and its expression pattern was analyzed. Studies showed that 75 candidate genes had been selected from the NAC transcription factor family of A. dahurica, with the protein size of 148-641, all of which were unstable hydrophilic proteins. Most NAC proteins were localized in the nucleus, and had complete NAC domain. Phylogenetic analysis of NAC family proteins of A.dahurica and Arabidopsis thaliana showed that among the 17 subfamilies, NAC members were unevenly distributed in each subfamily, indicating that the evolution of species is developing in multiple directions. Among them, ANAC063 subfamily contained no NAC sequence of A. dahurica, which might be due to the functional evolution of the species. Analysis of protein transmembrane structure and signal peptide showed that NAC transcription factor could carry out transmembrane transportation, but its signal peptide function had not been found. Expression analysis showed that most transcription factors responded to abiotic stress and hormones to varying degrees, and the effects of hormones were obvious, especially ABA and IAA. In different organs of A. dahurica, most members of the NAC family had higher expression in root phloem, followed by root xylem. This study lays a foundation for further research on the function of A. dahurica NAC transcription factor and for solving the biological problems of A. dahurica.

Discovery of effective phosphodiesterase 2 inhibitors with antioxidant activities for the treatment of Alzheimer's disease.

The multi-target-directed-ligand (MTDL) strategy has been widely applied in the discovery of novel drugs for the treatment of Alzheimer's disease (AD) because of the multifactorial pathological mechanisms of AD. Phosphodiesterase-2 (PDE2) has been identified to be a novel and promising target for AD. However, MTDL combining with the inhibitory activity against PDE2A and other anti-AD factors such as antioxidants has not been developed yet. Herein, a novel series of PDE2 inhibitors with antioxidant capacities were designed, synthesized, and evaluated. Most compounds showed remarkable inhibitory activities against PDE2A as well as antioxidant activities. Compound 6d was selected, which showed good IC50 of 6.1 nM against PDE2A, good antioxidant activity (ORAC (Trolox) = 8.4 eq.) and no cytotoxicity to SH-SY5Y cells. Molecular docking and dynamics simulations were applied for the rational design and explanation of structure-activity relationship (SAR) of lead compounds.

Screening and identification of endometrial proteins as novel potential biomarkers for repeated implantation failure.

Inadequate endometrial receptivity may be responsible for the low implantation rate of transferred embryos in in vitro fertilization (IVF) treatments. Patients with repeated implantation failure (RIF) impact the clinical pregnancy rate for IVF. We collected endometrial tissue during the implantation window of hysteroscopy biopsies from September 2016 to December 2019 and clinical data were collected simultaneously. Patients were divided into RIF and pregnant controls group according to pregnancy outcomes. A total of 82 differentially expressed endometrial proteins were identified, including 55 up-regulated proteins (>1.50-fold, P < 0.05) and 27 down-regulated proteins (<0.67-fold, P < 0.05) by iTRAQ labeling coupled with the 2D LC MS/MS technique in the RIF group. String analysis found interactions between these proteins which assembled in two bunches: ribosomal proteins and blood homeostasis proteins. The most significant enriched Gene Ontology terms were negative regulation of hydrolase activity, blood microparticle, and enzyme inhibitor activity. Our results emphasized the corticosteroid-binding globulin and fetuin-A as the specific proteins of endometrial receptivity by Western-blot. Our study provided experimental data to establish the objective indicator of endometrial receptivity, and also provided new insight into the pathogenesis of RIF.

Therapeutic targets and potential agents for the treatment of COVID-19.

The outbreak of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global crisis. As of November 9, COVID-19 has already spread to more than 190 countries with 50,000,000 infections and 1,250,000 deaths. Effective therapeutics and drugs are in high demand. The structure of SARS-CoV-2 is highly conserved with those of SARS-CoV and Middle East respiratory syndrome-CoV. Enzymes, including RdRp, Mpro /3CLpro , and PLpro , which play important roles in viral transcription and replication, have been regarded as key targets for therapies against coronaviruses, including SARS-CoV-2. The identification of readily available drugs for repositioning in COVID-19 therapy is a relatively rapid approach for clinical treatment, and a series of approved or candidate drugs have been proven to be efficient against COVID-19 in preclinical or clinical studies. This review summarizes recent progress in the development of drugs against SARS-CoV-2 and the targets involved.

Discovery of Novel Selective and Orally Bioavailable Phosphodiesterase-1 Inhibitors for the Efficient Treatment of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and devastating lung disease lacking effective therapy. To identify whether phosphodiesterase-1 (PDE1) inhibition could act as a novel target for the treatment of IPF, hit-to-lead structural optimizations were performed on the PDE9/PDE1 dual inhibitor (R)-C33, leading to compound 3m with an IC50 of 2.9 nM against PDE1C, excellent selectivity across PDE subfamilies, reasonable drug-like properties, and remarkable pharmacodynamic effects as an anti-IPF agent. Oral administration of compound 3m (10 mg/kg) exerted more significant anti-pulmonary fibrosis effects than pirfenidone (150 mg/kg) in a bleomycin-induced IPF rat model and prevented transforming growth factor-ß-induced fibroblast-to-myofibroblast conversion in vitro, indicating that PDE1 inhibition could serve as a novel target for the efficient treatment of IPF.

Design, synthesis and evaluation of pyrazolopyrimidinone derivatives as novel PDE9A inhibitors for treatment of Alzheimer's disease.

Phosphodiesterase-9 (PDE9) is a promising target for the treatment of Alzheimer's disease (AD). To discover efficient PDE9 inhibitors with good metabolic stability and solubility, a series of novel pyrazolopyrimidinone derivatives have been designed with the assistance of molecular docking and dynamics simulations. All the fourteen synthesized compounds gave excellent inhibition ratio against PDE9 at 10 nM. Compound 1k with the IC50 of 2.0 nM against PDE9, showed good metabolic stability (t1/2 of 57 min) in the RLM as well as good solubility (195 mg/L). The analysis on binding modes of targeted compounds may provide insight for further structural modification.

An Fe-V@NiO heterostructure electrocatalyst towards the oxygen evolution reaction.

The development of a nonprecious and Earth-abundant electrocatalyst with high electrocatalytic activity for the oxygen evolution reaction (OER) is an emerging hot issue and remains a grand challenge. In the present work, we proposed a facile strategy to construct ultrathin NiO nanosheets decorated with Fe-V nanoparticles on nickel foam (Fe-V@NiO/NF) for use as an OER electrocatalyst. Due to the 3D rational configuration, the Fe-V@NiO/NF with a heterostructure shows excellent electrocatalytic activity towards the OER. Interestingly, it is found that in situ oxidation by galvanostatic electrolysis in alkaline solution is beneficial to enhance the OER performance. After 10 h of electrolysis, a current density of 50 mA cm-2 is achieved at a low overpotential of 271.1 mV. This is because during the in situ oxidation process, iron and vanadium ions insert into the NiO lattice and lead to the generation of highly active α-FeOOH and an amorphous (oxy)-hydroxide layer. Additionally, the charge transfer resistance dramatically reduces with the prolonging of oxidation time.

[Photosynthetic physiological characteristics of Angelica dahurica var.formosana from Sichuan province in different growth years].

In this study,the leaves of autumn-sown Angelica dahurica var. formosana from Sichuan province in different growth years was used to explore the fitting model of photosynthetic response curve and the different photosynthetic physiological characteristics between annual and biennial A. dahurica var. formosana from Sichuan province. The results showed that the fitting model of the optimum light response curve of the leaves of A. dahurica var. formosana from Sichuan province with different growth years was all rectangular hyperbolic correction model. The light saturation points were 1 600,1 700 µmol·m-2·s-1,the light compensation points were17. 98,52. 23 µmol·m-2·s-1 in the leaves of annual and biennial plant,respectively. The diurnal variation curves of net photosynthetic rate,transpiration rate and stomatal conductance in the leaves all acted as a single peak value wave. The daily mean values of net photosynthetic rate and transpiration rate in the leaves of biennial plant were significantly higher than that of annual plant. There was no significant difference in daily mean stomatal conductance. The net photosynthetic rate was significantly positively correlated with stomatal conductance in both of the different growth years. The net photosynthetic rate of annual and biennial A. dahurica var. formosana from Sichuan province had extremely significant and significantly negative correlation with the intercellular CO2 respectively. The transpiration rate of annual plant was positively correlated with the effective photosynthetic radiation intensity and air temperature,but had significantly negative correlation with the intercellular CO2 concentration. The transpiration rate of biennial plant had extremely positive correlation with the effective photosynthetic radiation intensity,and negatively correlated with the intercellular CO2 concentration. In conclusion,the photosynthetic efficiency of the leaves in biennial plant of A. dahurica var. formosana from Sichuan province was higher than that in annual plant,but the ability to utilize weak light was lower than that of annual plant. It should be planted in the sunny field.

Down-Regulation of SSSII-2 Gene Expression Results in Novel Low-Amylose Rice with Soft, Transparent Grains.

Although soft rice, with low amylose content (AC), has high eating and cooking quality (ECQ), its appearance is poor due to the opaque endosperm. Here, a novel soft rice with low AC but a transparent appearance was generated by knocking-down the expression of SSSII-2, a gene encoding one isoform of soluble starch synthase (SSS). The physicochemical properties of the SSSII-2 RNAi rice are quite different from the control but more like the popular soft rice "Nanjing 46". The taste value assay further demonstrated that the ECQ of SSSII-2 RNAi rice was as high as "Nanjing 46", but only SSSII-2 RNAi rice retained the transparent endosperm under low moisture conditions. Further examination showed that the different morphologies and fine structures of the starch granules may contribute to the specific properties of SSSII-2 RNAi rice. Therefore, SSSII-2 has potential application in future high quality rice breeding programs.

The protective effects of electro-acupuncture in thoracic surgery on trauma stressed rats involve the rostral ventrolateral medulla and supraoptic nucleus.

The present study was designed to explore whether the rostral ventrolateral medulla (RVLM) and supraoptic nucleus (SON) were involved in the protective effects of electro-acupuncture (EA) in thoracic surgery on trauma-stressed rats. The rats were randomly divided into a non-stressed group (Control), surgical trauma-stressed group (Trauma), and Neiguan EA applied on the surgical trauma-stressed group (Trauma+EA-PC 6). RVLM neuron discharge was observed by using an in vivo electrophysiological method, and micro-dialysis combining high-performance liquid chromatography with fluorometric detection (HPLC-FD) was used to assess expression of amino acids in the RVLM. Immunohistochemical methods were used to assess c-Fos expression in SON neurons. The trauma of surgical stress was shown to dramatically increase the discharge frequency of RVLM neurons and promote the release of glutamate and taurine in the RVLM. The expression of c-Fos was also significantly increased in the SON of traumatized rats. EA application at Neiguan acupoints significantly suppressed trauma-induced increase of discharge frequency of the RVLM neurons, almost completely suppressed the trauma-induced increase of glutamate release but only very slightly reduced the trauma-enhanced taurine release, and inhibited the increase of c-Fos expression in these SON neurons of traumatized rats. These results indicate that Neiguan EA may improve cardiac function by modulating neurons in the RVLM and the SON in surgically traumatized rats. The taurine-mediated negative feedback may be involved in the protective effect of EA on cardiac function.

Repeated electroacupuncture attenuating of apelin expression and function in the rostral ventrolateral medulla in stress-induced hypertensive rats.

Studies have revealed that apelin is a novel multifunctional peptide implicated both in blood pressure (BP) regulation and cardiac function control. Evidence shows that apelin and its receptor (APJ) in the rostral ventrolateral medulla (RVLM) may play an important role in central BP regulation; however, its role is controversial and very few reports have shown the relationship between acupuncture and apelin. Our study aims to both investigate the apelinergic system role in stress-induced hypertension (SIH) and determine whether acupuncture therapy effects on hypertension involve the apelinergic system in the RVLM. We established the stress-induced hypertensive rat (SIHR) model using electric foot-shock stressors with noise interventions. The expression of both apelin and the APJ receptor in the RVLM neurons was examined by immunohistochemical staining and Western blots. The results showed apelin expression increased remarkably in SIHR while APJ receptor expression showed no significant difference between control and SIHR groups. Microinjection of apelin-13 into the RVLM of control rats or SIHR produced pressor and tachycardic effects. Furthermore, effects induced by apelin-13 in SIHR were significantly greater than those of control rats. In addition, repetitive electroacupuncture (EA) stimulation at the Zusanli (ST-36) acupoint attenuated hypertension and apelin expression in the RVLM in SIHR; it also attenuated the pressor effect elicited by exogenous apelin-13 microinjection in SIHR. The results suggest that augmented apelin in the RVLM was part of the manifestations of SIH; the antihypertensive effects of EA might be associated with the attenuation of apelin expression and function in the RVLM, which might be a novel role for EA in SIH setting.

Protective effects of electroacupuncture on cardiac function in rats subjected to thoracic surgery trauma.

The present study investigates the protective effects of electroacupuncture (EA) application on cardiac function, while simultaneously exploring the underlying neurobiological mechanisms, in rats that have experienced thoracic surgery-induced stress. Mean arterial and left intraventricular pressures were monitored as indicators of cardiac function. Meanwhile, the immunohistochemistry for c-Fos protein expression and electrophysiology in vitro in brain nuclei, known to regulate cardiac function, provide insights into the effects of EA on the central nervous system. The results show that cardiac function was dramatically suppressed with thoracic surgery trauma, the expression levels of c-Fos in the paraventricular nucleus (PVN) and the rostral ventrolateral medulla (RVLM) significantly increased, the rheobase intensity of the intracellular current injection needed to initiate the action potential decreased, membrane resistance in the PVN neurons significantly increased, and the inductivity of the postsynaptic potentials in the PVN neurons of the surgery-treated rats significantly decreased. EA application at the Neiguan acupoints (PC6) attenuated the decreases in almost all investigated functional parameters of the heart. EA significantly decreased the number of Fos-immunoreactive neurons in the PVN and RVLM, significantly decreased the Max L. slope of the PVN neurons, and increased the inductivity of the postsynaptic potentials in the PVN neurons of the surgery-treated rats. These data indicate the protective effects of EA application on cardiac function in rats that have experienced surgery-induced stress and show that EA application at the Neiguan acupoints may produce its protective effects through the neurons in the PVN and the RVLM.

Nitric oxide modulates cardiovascular function in the rat by activating adenosine A2A receptors and inhibiting acetylcholine release in the rostral ventrolateral medulla.

1. Nitric oxide (NO), a gas transmitter, modulates many physiological processes, including the central regulation of cardiovascular activity. However, the mechanisms underlying the regulation of cardiovascular activity remain relatively unexplored. In the present study, we hypothesized that central NO-dependent sympathetic inhibition is mediated by activation of adenosine A(2A) receptors (A(2A)R) and inhibition of acetylcholine (ACh) release in the rostral ventrolateral medulla (RVLM). 2. L-Arginine (L-Arg; an NO donor; 100 nmol/100 nL) was microinjected into the RVLM of male Sprague-Dawley rats and heart rate variability (HRV) was assessed as an index of cardiac sympathovagal balance. Following microinjection of L-Arg, decreases were seen in mean arterial pressure (MAP), heart rate (HR) and the ratio of the low- to high-frequency components (LF/HF) of HRV. Pretreatment of rats with SCH58261 (40 pmol/60 nL into the RVLM), a competitive antagonist of the A(2A) R, attenuated these effects. 3. Western blot analysis and ELISA revealed that adenosine and A(2A)R levels increased in the RVLM following L-Arg microinjection, whereas ACh and muscarinic M(1) receptor levels decreased significantly, in parallel with the cardiovascular responses to L-Arg microinjection. The decrease in ACh levels was abolished by SCH58261 pretreatment. 4. Microinjection of N(G)-nitro-L-arginine methyl ester (a non-selective inhibitor of NO synthase; 15 nmol/100 nL) into the RVLM significantly increased MAP, HR and sympathetic activity, as evidenced by HRV (LF, HF and the LF/HF ratio were all increased). 5. The results indicate that the central NO/NO synthase system in the RVLM may modulate cardiovascular activity by activating the A(2A)R, which subsequently inhibits activation of the muscarinic M(1) receptor.

[The application of transcranial Doppler in the evaluation of brain function in deeply comatose patients].

OBJECTIVE: To study the cerebral blood flow changes in irreversible comatose patients and investigate the correlation between the results of transcranial Doppler (TCD) and clinical evaluation of brain function. METHODS: TCD studies were performed in 92 patients who were excluded from the possibility of reversible coma and all of them had Glasgow Coma Scale of 3. Their brain stem reflections all disappeared. Detection of the blood flow in bilateral middle cerebral arteries was carried out through temporal window in all the patients and in 42 of the 92 cases the blood flow of the basilar artery was detected through occipital or peri-occipital window. Bilateral middle cerebral artery were taken as the main arteries for evaluation. The positive standard is the appearance of one of the following characteristic blood flow, i.e. brief systolic forward flow or systolic spikes and diastolic reverse flow, brief systolic forward flow or systolic spikes and no diastolic flow and no detectable flow signals. The examination was repeated after 12 h. RESULTS: 68 cases were positive in the initial examination (73.9%) and 74 cases were positive after 12 h (80.4%). CONCLUSION: TCD can be an effective and objective index in the evaluation of irreversible termination of brain function.