Discovery of Antimicrobial Lysins from the "Dark Matter" of Uncharacterized Phages Using Artificial Intelligence.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

Safety evaluation of ceftazidime/avibactam based on FAERS database.

OBJECTIVE: To explore adverse event (AE) signals of Ceftazidime/avibactam (CZA) based on the FDA Adverse Event Reporting System (FAERS) database. METHODS: AE reports primarily associated with CZA were retrieved from the FAERS database from the second quarter of 2015 to the second quarter of 2023. Signal detection was conducted using the reporting odds ratio (ROR), proportional reporting ratio (PRR), Bayesian Confidence Propagation Neural Network (BCPNN), and Multi-item Gamma Poisson Shrinker (MGPS) methods. RESULTS: A total of 750 AEs reports with CZA as the preferred suspected drug were obtained, identifying 66 preferred terms (PTs) involving 24 system organ classes (SOCs). Besides, the AEs already mentioned in the drug label, this study also revealed some new, clinically valuable potential AEsignals, such as Cholestasis (n = 14, ROR 29.39, PRR 29.15, IC 3.34, EBGM 29.11), Drug-induced liver injury (n = 8, ROR 9.05, PRR 9.01, IC 2.25, EBGM 9.01), Hepatocellular injury (n = 7, ROR 13.90, PRR 13.84, IC 2.41, EBGM 13.63), Haemolytic anaemia (n = 5, ROR 24.29, PRR 24.22, IC 2.42, EBGM 40.53), etc. Additionally, AE signals with higher intensity were identified, such as Hypernatraemia (n = 5, ROR 40.73, PRR 40.61, IC 2.31, EBGM 24.19), Toxic epidermal necrolysis (n = 4, ROR 11.58, PRR 11.55, IC 1.89, EBGM 11.54). Therefore, special vigilance for these potential AEs is warranted when using CZA clinically. CONCLUSION: This study highlights the potential AEs and risks associated with the clinical use of CZA, particularly the risks related to Cholestasis, Drug-induced liver injury, Haemolytic anaemia, Hypernatraemia, and Toxic epidermal necrolysis.

Characterization of NMCR-3, NMCR-4 and NMCR-5, three novel non-mobile colistin resistance determinants: Implications for MCR-3, MCR-7, and MCR-5 progenitors, respectively.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

Unraveling the contributing factors of stale odor in Longjing tea through a sensomics approach.

Longjing tea is renowned for its fresh aroma and high value. However, during storage, the emergence of an off-flavor known as "stale odor" can significantly impact the flavor quality and economic benefits of Longjing tea. Yet, the specific volatiles responsible for this stale odor in Longjing tea remain unknown. In this study, Longjing tea samples with varying degrees of stale odor intensity were analyzed using simultaneous distillation extraction coupled with gas chromatography-mass spectrometry (SDE-GC-MS). Through odor activity value (OAV) and fractional omission testing, hexanoic acid and trans-2-nonenal were identified as the primary contributors to the stale odor. Moreover, the concentration of hexanoic acid was found to be valuable in predicting the intensity of the stale odor in Longjing tea. The oxidative degradation of linoleic acid was proved as the generation pathway of stale odor in Longjing tea. These findings provide essential theoretical principles for Longjing tea production and preservation.

Effects of cognitive behavioral therapy in patients with chronic obstructive pulmonary disease: A systematic review and meta-analysis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) causes airflow blockage and breathing-related issues. This chronic disease impacts people worldwide. Substantial evidence supports the use of cognitive behavioral therapy (CBT) to help patients with chronic illnesses cope with worrisome and painful symptoms. However, the impact of CBT on COPD outcomes is less understood. OBJECTIVE: In this study, we systematically summarized the effects of CBT on lung function, anxiety and depressive symptoms, and quality of life of patients with COPD. METHODS: Six English-language and four Chinese-language databases were systematically searched for relevant randomized controlled trials published through April 15, 2023. Studies in which CBT was the only difference in treatment administered to experimental and control groups were included in the review. The studies' risk of bias was evaluated using the Cochrane Criteria. RESULTS: Sixteen studies (1887 participants) were included. The meta-analysis showed that CBT improved the percent-predicted forced expiratory volume in 1 second (FEV1%), forced vital capacity (FVC), FEV1/FVC ratio, maximal voluntary ventilation, peak expiratory flow, treatment compliance, and World Health Organization abbreviated quality of life, Self-rating Anxiety and Depression Scale, and St George's Respiratory Questionnaire scores compared with the control (all p < .05). CONCLUSION: This review demonstrated that CBT improves the lung function, anxiety and depressive symptoms, treatment compliance, and quality of life of patients with COPD and can be used widely in the clinical treatment of this disease.

Production of Double-Stranded RNA Using the Prokaryotic Promoter-Mediated Bidirectional Transcription.

Large-scale and cost-less production of double-stranded RNA (dsRNA) is the basis for the widespread application of dsRNA in agriculture. Bidirectional transcription of target sequence in RNase III-deficient Escherichia coli strain HT115 (DE3) is an efficient way to produce large amounts of dsRNA. Here, we present a detailed method for the production of dsRNA by bidirectional transcription in E. coli from vector construction, induction of expression by isopropylthio-ß-galactoside (IPTG), and purification of dsRNA from E. coli.

Environmental fate and risk evolution of calcium channel blockers from chlorine-based disinfection to sunlit surface waters.

Organic micropollutants present in disinfected wastewater and discharged to sunlit surface waters may be transformed by multiple processes, such as chlorination due to the presence of chlorine residuals, solar irradiation as well as solar-irradiated chlorine residues. This study reports, for the first time, the multi-scenario degradation kinetics, transformation products, and risk evolution of calcium channel blockers (CCBs), a class of emerging pharmaceutical contaminants with worldwide prevalence in natural waters and wastewater. It was found that the chlorination of the studied CCBs (amlodipine (AML) and verapamil (VER)) was dominated by the reaction of HOCl with their neutral species, with second-order rate constants of 6.15×104 M-1 s-1 (AML) and 7.93×103 M-1 s-1 (VER) at pH 5.0-11.0. Bromination is much faster than chlorination, with the measured kapp,HOBr values of 2.94×105 M-1 s-1 and 6.58×103 M-1 s-1 for AML and VER, respectively, at pH 7.0. Furthermore, both CCBs would undergo photolytic attenuations with hydroxyl and carbonate radicals as the dominant reactive species in water. Notably, free chlorine mainly contributed to their abatement during the solar/chlorine treatment. Additionally, the halogen addition on the aromatic ring was observed during chlorination and bromination of the two CCBs. Cyclization was observed under solar irradiation only, while the aromatic ring was opened in the solar/chlorine system. Some products generated by the three transformation processes exhibited non-negligible risks of high biodegradation recalcitrance and toxicity, potentially threatening the aquatic environment and public health. Overall, this study elucidated the environmental fate of typical CCBs under different transformation processes to better understand the resulting ecological risks in these environmental scenarios.

Rice volatile compound (E)-ß-caryophyllene induced by rice dwarf virus (RDV) attracts the natural enemy Cyrtorhinus lividipennis to prey on RDV insect vectors.

BACKGROUND: Rice dwarf virus (RDV)-induced rice plant volatiles (E)-ß-caryophyllene and 2-heptanol modulate the olfactory behavior of RDV insect vectors that promote viral acquisition and transmission. However, it remains elusive whether these two volatiles could influence the behaviors of the natural enemies of RDV insect vectors. Herein, we determined the effects of these two volatiles on the olfactory and predatory behaviors of Cyrtorhinus lividipennis (Hemiptera: Miridae), an important predator of RDV insect vectors in rice paddies. RESULTS: The results showed that C. lividipennis preferred RDV-infected rice plant odors over RDV-free rice plant odors. C. lividipennis was attracted by (E)-ß-caryophyllene, but showed no behavioral responses to 2-heptanol. The attraction of (E)-ß-caryophyllene towards C. lividipennis was further confirmed using oscas1 rice plants, which do not release (E)-ß-caryophyllene in response to RDV infection, through a series of complementary assays. The oviposition preference of the RDV vector insect Nephotettix cincticeps (Hemiptera: Cicadellidae) showed no significant difference between RDV-infected and RDV-free wild-type plants, nor between oscas1-RDV and oscas1 plants. However, the predation rate of C. lividipennis for N. cincticeps eggs on RDV-infected plants was higher than that on RDV-free plants, whereas there was no significant difference between oscas1-RDV and oscas1 plants. CONCLUSION: (E)-ß-caryophyllene induced by RDV attracted more C. lividipennis to prey on N. cincticeps eggs and played a crucial role in plant-virus-vector-enemy interactions. These novel findings will promote the design of new strategies for disease control by controlling the populations of insect vectors, for example recruiting more natural enemies by virus-induced plant volatiles. © 2023 Society of Chemical Industry.

Considerations in engineering viral vectors for genome editing in plants.

Plant viruses have been engineered to express proteins and induce gene silencing for decades. Recently, plant viruses have also been used to deliver components into plant cells for genome editing, a technique called virus-induced genome editing (VIGE). Although more than a dozen plant viruses have been engineered into VIGE vectors and VIGE has been successfully accomplished in some plant species, application of VIGE to crops that are difficult to tissue culture and/or have low regeneration efficiency is still tough. This paper discusses factors to consider for an ideal VIGE vector, including insertion capacity for foreign DNA, vertical transmission ability, expression level of the target gene, stability of foreign DNA insertion, and biosafety. We also proposed a step-by-step schedule for excavating the suitable viral vector for VIGE.

How Does Aging Affect Whole-brain Functional Network Connectivity? Evidence from An ICA Method.

Many studies have shown that changes in the functional connectivity are diverse along with aging. However, few studies have addressed how aging affects connectivity among large-scale brain networks, and it is challenging to examine gradual aging trajectories from middle adulthood to old age. In this work, based on large-sample fMRI data from 6300 subjects aged between 49 to 73 years, we apply an independent component analysis-based method called NeuroMark to extract brain functional networks and their connectivity (i.e., functional network connectivity (FNC)), and then propose a two-level statistical analysis method to explore robust aging-related changes in functional network connectivity. We found that the enhanced FNCs mainly occur between different functional domains, involving the default mode and cognitive control networks, while the reduced FNCs come from not only between different domains but also within the same domain, primarily relating to the visual network, cognitive control network and cerebellum. Our results emphasize the diversity of brain aging and provide new evidence for non-pathological aging of the whole brain.Clinical Relevance-This provides new evidence for non-pathological aging of functional network connectivity in the whole brain.

Decoding the Chemical Signatures and Sensory Profiles of Enshi Yulu: Insights from Diverse Tea Cultivars.

Enshi Yulu, a renowned Chinese steamed green tea, is highly valued for its unique sensory attributes. To enhance our comprehensive understanding of the metabolic variation induced by steaming fixation, we investigated the overall chemical profiles and organoleptic quality of Enshi Yulu from different tea cultivars (Longjing 43, Xiapu Chunbolv, and Zhongcha 108). The relationships between sensory traits and non-volatiles/volatiles were evaluated. A total of 58 volatiles and 18 non-volatiles were identified as characteristic compounds for discriminating among the three tea cultivars, and the majority were correlated with sensory attributes. The "mellow" taste was associated with L-aspartic acid, L-asparagine, L-tyrosine, L-valine, EGC, EC, and ECG, while gallic acid and theobromine contributed to the "astringent" taste. "Kokumi" contributors were identified as L-methionine, L-lysine, and GCG. Enshi Yulu displayed a "pure" and "clean and refreshing" aroma associated with similar volatiles like benzyl alcohol, δ-cadinene, and muurolol. The composition of volatile compounds related to the "chestnut" flavor was complex, including aromatic heterocycles, acids, ketones, terpenes, and terpene derivatives. The key contributors to the "fresh" flavor were identified as linalool oxides. This study provides valuable insights into the sensory-related chemical profiles of Enshi Yulu, offering essential information for flavor and quality identification of Enshi Yulu.

Risk factors for delirium among hospitalized adults with COVID-19: A systematic review and meta-analysis of cohort studies.

OBJECTIVES: This study aims to systematically assess the risk factors, the overall strength of association, and evidence quality related to delirium among adults hospitalized with COVID-19. METHODS: A comprehensive search was conducted in thirteen databases from inception to February 10, 2023. The included databases were thoroughly searched, including PubMed, Web of Science, Proquest, Ovid MEDLINE, CINAHL, Scopus, the Cochrane Library, FMRS, Wanfang Database, Chinese Biomedical Database (CBM), China Knowledge Resource Integrated Database (CNKI), Weipu Database (VIP), and Embase. The search was limited to articles published in English and Chinese. The selected studies were screened, data were extracted, and the quality was evaluated using the Newcastle-Ottawa Scale. Meta-analysis was performed using RevMan 5.4 software. The certainty of the evidence was assessed using the GRADE criteria. RESULTS: A total of 22 cohort studies with a sample size of 11,957 individuals were included in the analysis. Among these studies, 20 were of high quality, while the remaining 2 were of moderate quality. The risk factors that showed the strongest association with delirium were prior cognitive impairment (including dementia), mechanical ventilation, and ICU admission. Age, frailty (Clinical Frailty Scale score > 5), antipsychotic use, benzodiazepine use, neutrophil-to-lymphocyte ratio, and vasopressor use were identified as moderate risk factors for delirium. According to the GRADE evaluation, ICU admission, benzodiazepine use, neutrophil-to-lymphocyte ratio, and vasopressor use had a high-quality body of evidence, while antipsychotic usage had an intermediate-quality body of evidence. All other risk factors had a low-quality body of evidence. CONCLUSIONS: This systematic review and meta-analysis identified several medium- to high-intensity risk factors for delirium in hospitalized adults with COVID-19. ICU admission, benzodiazepine usage, neutrophil-to-lymphocyte ratio, antipsychotic use, and vasopressor use were associated with delirium and were supported by medium- to high-quality evidence. These findings provide healthcare professionals with an evidence-based basis for managing and treating delirium in hospitalized adults with COVID-19.

Bempedoic Acid Unveils Therapeutic Potential in Non-Alcoholic Fatty Liver Disease: Suppression of the Hepatic PXR-SLC13A5/ACLY Signaling Axis.

The hepatic SLC13A5/SLC25A1-ATP-dependent citrate lyase (ACLY) signaling pathway, responsible for maintaining the citrate homeostasis, plays a crucial role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Bempedoic acid (BA), an ACLY inhibitor commonly used for managing hypercholesterolemia, has shown promising results in addressing hepatic steatosis. This study aimed to elucidate the intricate relationships in processes of hepatic lipogenesis among SLC13A5, SLC25A1, and ACLY and to examine the therapeutic potential of BA in NAFLD, providing insights into its underlying mechanism. In murine primary hepatocytes and HepG2 cells, the silencing or pharmacological inhibition of SLC25A1/ACLY resulted in significant upregulation of SLC13A5 transcription and activity. This increase in SLC13A5 activity subsequently led to enhanced lipogenesis, indicating a compensatory role of SLC13A5 when the SLC25A1/ACLY pathway was inhibited. However, BA effectively counteracted this upregulation, reduced lipid accumulation, and ameliorated various biomarkers of NAFLD. The disease-modifying effects of BA were further confirmed in NAFLD mice. Mechanistic investigations revealed that BA could reverse the elevated transcription levels of SLC13A5 and ACLY, and the subsequent lipogenesis induced by PXR activation in vitro and in vivo. Importantly, this effect was diminished when PXR was knocked down, suggesting the involvement of the hepatic PXR-SLC13A5/ACLY signaling axis in the mechanism of BA action. In conclusion, SLC13A5-mediated extracellular citrate influx emerges as an alternative pathway to SLC25A1/ACLY in the regulation of lipogenesis in hepatocytes, BA exhibits therapeutic potential in NAFLD by suppressing the hepatic PXR-SLC13A5/ACLY signaling axis, while PXR, a key regulator in drug metabolism may be involved in the pathogenesis of NAFLD. SIGNIFICANCE STATEMENT: This work describes that bempedoic acid, an ATP-dependent citrate lyase (ACLY) inhibitor, ameliorates hepatic lipid accumulation and various hallmarks of non-alcoholic fatty liver disease. Suppression of hepatic SLC25A1-ACLY pathway upregulates SLC13A5 transcription, which in turn activates extracellular citrate influx and the subsequent DNL. Whereas in hepatocytes or the liver tissue challenged with high energy intake, bempedoic acid reverses compensatory activation of SLC13A5 via modulating the hepatic PXR-SLC13A5/ACLY axis, thereby simultaneously downregulating SLC13A5 and ACLY.

The herbicide bensulfuron-methyl inhibits rice seedling development by blocking calcium ion flux in the OsCNGC12 channel.

Identification of translocator protein-related genes involved in bensulfuron-methyl (BSM) uptake and transport in rice could facilitate the development of herbicide-tolerant cultivars by inactivating them. This study found that the OsCNGC12 mutants not only reduced BSM uptake but also compromised the Ca2 ⁺ efflux caused by BSM in the roots, regulating dynamic equilibrium of Ca2 ⁺ inside the cell and conferring non-target-site tolerance to BSM.

P1 of turnip mosaic virus interacts with NOD19 for vigorous infection.

P1 protein, the most divergent protein of virus members in the genus Potyvirus of the family Potyviridae, is required for robust infection and host adaptation. However, how P1 affects viral proliferation is still largely elusive. In this work, a total number of eight potential P1-interacting Arabidopsis proteins were identified by the yeast-two-hybrid screening using the turnip mosaic virus (TuMV)-encoded P1 protein as the bait. Among which, the stress upregulated NODULIN 19 (NOD19) was selected for further characterization. The bimolecular fluorescent complementation assay confirmed the interaction between TuMV P1 and NOD19. Expression profile, structure, and subcellular localization analyses showed that NOD19 is a membrane-associated protein expressed mainly in plant aerial parts. Viral infectivity assay showed that the infection of turnip mosaic virus and soybean mosaic virus was attenuated in the null mutant of Arabidopsis NOD19 and NOD19-knockdown soybean seedlings, respectively. Together, these data indicate that NOD19 is a P1-interacting host factor required for robust infection.

Aging brain shows joint declines in brain within-network connectivity and between-network connectivity: a large-sample study (N > 6,000).

Introduction: Numerous studies have shown that aging has important effects on specific functional networks of the brain and leads to brain functional connectivity decline. However, no studies have addressed the effect of aging at the whole-brain level by studying both brain functional networks (i.e., within-network connectivity) and their interaction (i.e., between-network connectivity) as well as their joint changes. Methods: In this work, based on a large sample size of neuroimaging data including 6300 healthy adults aged between 49 and 73 years from the UK Biobank project, we first use our previously proposed priori-driven independent component analysis (ICA) method, called NeuroMark, to extract the whole-brain functional networks (FNs) and the functional network connectivity (FNC) matrix. Next, we perform a two-level statistical analysis method to identify robust aging-related changes in FNs and FNCs, respectively. Finally, we propose a combined approach to explore the synergistic and paradoxical changes between FNs and FNCs. Results: Results showed that the enhanced FNCs mainly occur between different functional domains, involving the default mode and cognitive control networks, while the reduced FNCs come from not only between different domains but also within the same domain, primarily relating to the visual network, cognitive control network, and cerebellum. Aging also greatly affects the connectivity within FNs, and the increased within-network connectivity along with aging are mainly within the sensorimotor network, while the decreased within-network connectivity significantly involves the default mode network. More importantly, many significant joint changes between FNs and FNCs involve default mode and sub-cortical networks. Furthermore, most synergistic changes are present between the FNCs with reduced amplitude and their linked FNs, and most paradoxical changes are present in the FNCs with enhanced amplitude and their linked FNs. Discussion: In summary, our study emphasizes the diversity of brain aging and provides new evidence via novel exploratory perspectives for non-pathological aging of the whole brain.

Virus-Induced Plant Volatiles Promote Virus Acquisition and Transmission by Insect Vectors.

Rice dwarf virus (RDV) is transmitted by insect vectors Nephotettix virescens and Nephotettix cincticeps (Hemiptera: Cicadellidae) that threatens rice yield and results in substantial economic losses. RDV induces two volatiles ((E)-ß-caryophyllene (EBC) and 2-heptanol) to emit from RDV-infected rice plants. However, the effects of the two volatiles on the olfactory behavior of both non-viruliferous and viruliferous N. virescens are unknown, and whether the two volatiles could facilitate the spread and dispersal of RDV remains elusive. Combining the methods of insect behavior, chemical ecology, and molecular biology, we found that EBC and 2-heptanol influenced the olfactory behavior of non-viruliferous and viruliferous N. virescens, independently. EBC attracted non-viruliferous N. virescens towards RDV-infected rice plants, promoting virus acquisition by non-viruliferous vectors. The effect was confirmed by using oscas1 mutant rice plants (repressed EBC synthesis), but EBC had no effects on viruliferous N. virescens. 2-heptanol did not attract or repel non-viruliferous N. virescens. However, spraying experiments showed that 2-heptanol repelled viruliferous N. virescens to prefer RDV-free rice plants, which would be conducive to the transmission of the virus. These novel results reveal that rice plant volatiles modify the behavior of N. virescens vectors to promote RDV acquisition and transmission. They will provide new insights into virus-vector-plant interactions, and promote the development of new prevention and control strategies for disease management.

Risk factors for delirium in advanced cancer patients: A systematic review and meta-analysis.

PURPOSE: To systematically collect published research in order to identify and quantify risk factors for delirium in advanced cancer patients. METHODS: The Cochrane Library, PubMed, Proquest, CINAHL, Web of Science, Ovid MEDLINE, Embase, Scopus, Chinese Wanfang Data, Chinese Periodical Full-text Database (VIP), Chinese Biomedical Literature Database (CBM), and Chinese National Knowledge Infrastructure (CNKI) were systematically searched for cohort or case-control studies reporting individual risk factors for delirium among advanced-stage cancer patients published prior to March 2022. The Newcastle-Ottawa Scale was used to assess the methodological quality of included studies. The pooled adjusted odds ratio (aOR) and its 95% confidence interval were calculated using the RevMan 5.4 software package. RESULTS: A total of 15 studies with data from 3106 advanced cancer patients were included in our analysis. Nine studies were high-quality and six were of moderate quality. Pooled analyses revealed that 11 risk factors were statistically significant. High-intensity risk factors included sleep disturbance, infection, cachexia and the Palliative Prognostic Index; medium-intensity risk factors included male sex, renal failure, dehydration and drowsiness; low-intensity risk factors included age, total bilirubin and opioid use. Antibiotic use was found to have been a protective factor. CONCLUSIONS: We identified 12 independent risk factors that were significantly associated with delirium in advanced cancer patients and provide an evidence-based foundation to implement appropriate preventive strategies.

The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain.

Cluster of differentiation 31 (CD31), phalloidin and alpha smooth muscle actin (α-SMA) have been widely applied to label the cerebral blood vessels in the past years. Although CD31 is mainly used as endothelial marker in determining the cerebral capillaries, it seems likely that its labeling efficiency is closely correlated with the antibodies from the polyclonal or monoclonal one, as well as the conditions of blood vessels. In order to test this phenomenon, we compared the labeling characteristics of goat polyclonal anti-CD31 (gP-CD31) and mouse monoclonal anti-CD31 (mM-CD31) with those of phalloidin and α-SMA on the rat brain in health and ischemia/reperfusion (I/R) with the middle cerebral artery occlusion. By multiple immunofluorescence staining, it was found that gP-CD31 labeling expressed extensively on the cerebral capillaries forming the vascular networks on the normal and ischemic regions, but mM-CD31 labeling mainly presented on the capillaries in the ischemic region. In contrast to the vascular labeling with gP-CD31, phalloidin and α-SMA were mainly expressed on the wall of cortical penetrating arteries, and less on that of capillaries. By three-dimensional reconstruction analysis, it was clearly shown that gP-CD31 labeling was mainly located on the lumen side of vascular wall and was surrounded by phalloidin labeling and α-SMA labeling. These results indicate that gP-CD31 is more sensitive than mM-CD31 for labeling the cerebral vasculature, and is highly compatible with phalloidin and α-SMA for evaluating the cerebral vascular networks under the physiological and pathological conditions.