Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

Direct Laser Writing Photonic Crystal Hydrogels with a Supramolecular Sacrificial Scaffold.

Photonic crystal hydrogels (PCHs), with smart stimulus-responsive abilities, have been widely exploited as colorimetric sensors for years. However, the current fabrication technologies are mostly applicable to produce PCHs with simple geometries at the sub-millimeter scale, limiting the introduction of structural design into PCH sensors as well as the accompanied advanced applications. This paper reports the microfabrication of three-dimensional (3D) PCHs with the help of supramolecular agarose PCH as a sacrificial scaffold by two-photon lithography (TPL). The supramolecular PCHs, formulated with SiO2 colloidal nanoparticles and agarose aqueous solutions, show bright structural color and are degradable upon short-time dimethyl sulfoxide treatment. Leveraging the supramolecular PCH as a sacrificial scaffold, PCHs with precise 3D geometries can be fabricated in an economical and efficient way. This work demonstrates the application of such a strategy in the creation of structural-designed PCH mechanical microsensors that have not been explored before.

MRI-Based Kinetic Heterogeneity Evaluation in the Accurate Access of Axillary Lymph Node Status in Breast Cancer Using a Hybrid CNN-RNN Model.

BACKGROUND: Accurate evaluation of the axillary lymph node (ALN) status is needed for determining the treatment protocol for breast cancer (BC). The value of magnetic resonance imaging (MRI)-based tumor heterogeneity in assessing ALN metastasis in BC is unclear. PURPOSE: To assess the value of deep learning (DL)-derived kinetic heterogeneity parameters based on BC dynamic contrast-enhanced (DCE)-MRI to infer the ALN status. STUDY TYPE: Retrospective. SUBJECTS: 1256/539/153/115 patients in the training cohort, internal validation cohort, and external validation cohorts I and II, respectively. FIELD STRENGTH/SEQUENCE: 1.5 T/3.0 T, non-contrast T1-weighted spin-echo sequence imaging (T1WI), DCE-T1WI, and diffusion-weighted imaging. ASSESSMENT: Clinical pathological and MRI semantic features were obtained by reviewing histopathology and MRI reports. The segmentation of the tumor lesion on the first phase of T1WI DCE-MRI images was applied to other phases after registration. A DL architecture termed convolutional recurrent neural network (ConvRNN) was developed to generate the KHimage (kinetic heterogeneity of DCE-MRI image) score that indicated the ALN status in patients with BC. The model was trained and optimized on training and internal validation cohorts, tested on two external validation cohorts. We compared ConvRNN model with other 10 models and the subgroup analyses of tumor size, magnetic field strength, and molecular subtype were also evaluated. STATISTICAL TESTS: Chi-squared, Fisher's exact, Student's t, Mann-Whitney U tests, and receiver operating characteristics (ROC) analysis were performed. P < 0.05 was considered significant. RESULTS: The ConvRNN model achieved area under the curve (AUC) of 0.802 in the internal validation cohort and 0.785-0.806 in the external validation cohorts. The ConvRNN model could well evaluate the ALN status of the four molecular subtypes (AUC = 0.685-0.868). The patients with larger tumor sizes (>5 cm) were more susceptible to ALN metastasis with KHimage scores of 0.527-0.827. DATA CONCLUSION: A ConvRNN model outperformed traditional models for determining the ALN status in patients with BC. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

Perioperative tislelizumab plus chemotherapy for locally advanced resectable thoracic esophageal squamous cell carcinoma trial: a prospective single-arm, phase II study (PILOT trial).

BACKGROUND: The promising therapeutic outcomes of neoadjuvant immunotherapy combined with chemotherapy in the treatment of locally advanced esophageal squamous cell carcinoma (ESCC) have been confirmed by several phase II clinical trials and have been widely demonstrated in clinical work. Theoretically, postoperative adjuvant immunotherapy may further improve the therapeutic effect, but there is still lack of evidence. The aim of this study was to analyse the safety and efficacy of perioperative immunotherapy (tislelizumab) in locally advanced resectable thoracic ESCC (PILOT trial). METHODS: Seventy-three eligible patients with pathologically confirmed thoracic ESCC of clinical T1b-3N1-3M0 or T3N0M0 stage were allocated to receive neoadjuvant immunotherapy (tislelizumab 200 mg d1, q3w × 2 cycles) plus chemotherapy (nad-paclitaxel 260 mg/m2 d1 + carboplatin AUC = 5 d1, q3w × 2 cycles) treatment. Patients with pathologic complete response (pCR) after esophagectomy received adjuvant tislelizumab (200 mg every 3 weeks for up to one year), and patients with non-pCR were assigned adjuvant tislelizumab plus chemotherapy for two cycles and then maintenance tislelizumab (200 mg every 3 weeks for up to 15 cycles). The primary endpoint of this study is 2-year disease-free survival (DFS) in non-pCR patients. The secondary endpoints include pCR rate, major pathological response rate, 2-year DFS in pCR patients, R0 resection rate, adverse events, and overall survival. DISCUSSION: This protocol was reviewed and approved by the Ethics Committee of Shanghai Chest Hospital (IS23059). This is the first prospective clinical trial to investigate the safety and efficacy of perioperative immunotherapy for locally advanced resectable thoracic ESCC. We hypothesize that perioperative immunotherapy could be a promising therapeutic strategy that can provide better 2-year DFS in non-pCR patients. TRIAL REGISTRATION: ClinicalTrial.gov: NCT0605633.

Association between TH/MAOB gene single nucleotide polymorphisms and excitability in Labrador Retrievers.

Excitability is a pivotal quality in guide dogs because moderately active dogs are more trainable. Excessive activity is associated with behavioral problems and pet surrender. Excitability is a highly heritable trait, yet the relevant genetic factors and markers associated with this condition are poorly characterized. In the present study, we selected six single nucleotide polymorphisms (SNPs) of two genes that are possibly related to excitability in dogs (TH c.264G > A, TH c.1208A > T, TH c.415C > G, TH c.168C > T, TH c.180C > T and MAOB c.199 T > C). We measured the excitability of dogs using seven variables from three behavioral tests: the play test (interest in play, grabbing in throw and tug-of-war), the chase test (following and forward grabbing) and the passive test (moving range and moving time). These behavioral tests are part of the Dog Mentality Assessment developed by Svartberg & Forkman. The activity scores in the guide dog group were higher than in the temperament withdrawal group, and significant differences were detected in the aggregate score (p = 0.02), passive activity score (p = 0.007) and moving range score (p = 0.04). Analysis using the Kruskal-Wallis test and non-parametric Steel-Dwass test to evaluate the relationship between these SNPs and behavioral variable scores revealed that TH c.264G > A was associated with aggregate scores of excitability-related behavioral variables (adj. p = 0.03), object-interaction activity scores (adj. p = 0.03), following scores (adj. p = 0.03) and forward grabbing scores (adj. p = 0.03) in Labrador dogs and MAOB c.199 T > C was associated with moving range scores in these dogs (adj. p = 0.004). However, these results had low power. To explain the behavioral traits, further genetic studies more reliable than candidate gene studies are needed.

Programming colloidal bonding using DNA strand-displacement circuitry.

As a strategy for regulating entropy, thermal annealing is a commonly adopted approach for controlling dynamic pathways in colloid assembly. By coupling DNA strand-displacement circuits with DNA-functionalized colloid assembly, we developed an enthalpy-mediated strategy for achieving the same goal while working at a constant temperature. Using this tractable approach allows colloidal bonding to be programmed for synchronization with colloid assembly, thereby realizing the optimal programmability of DNA-functionalized colloids. We applied this strategy to conditionally activate colloid assembly and dynamically switch colloid identities by reconfiguring DNA molecular architectures, thereby achieving orderly structural transformations; leveraging the advantage of room-temperature assembly, we used this method to prepare a lattice of temperature-sensitive proteins and gold nanoparticles. This approach bridges two subfields: dynamic DNA nanotechnology and DNA-functionalized colloid programming.

Publisher Correction: An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.

Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.

6-acrylic phenethyl ester-2-pyranone derivative induces apoptosis and G2/M arrest by targeting GRP94 in colorectal cancer.

Colorectal cancer (CRC) is ranked the third driving reason for cancer death in the world. Surgery and chemotherapy have long been the first choices for cancer patients. However, the prognosis of CRC has never been satisfying, necessitating new effective treatment strategies. In our previous study, we synthesized compound5othat showed high anticancer potential with a 6-acrylic phenethyl ester-2-pyranone backbone, but its mechanism of action (MOA) is not understood. To articulate the MOA of 5o against colon cancer, we evaluated the anti-cancer effect of compound5oon CRC cells by cell proliferation assays. The MOA of5owas explored through cell cycle assays and apoptosis assays. The target of 5o was identified by molecular dynamic assays, ATPase assays, and surface plasmon resonance (SPR) analysis. We discovered 5o, a compound capable of inhibiting CRC cell proliferation with 1/25 folds in IC50 values compared with NCM460 cells (normal human colonic epithelial cell line). 5o induces cell apoptosis in a dose-dependent manner through PI3K/Akt/FoxO1 and NF-κB signaling pathways. In addition, 5o arrests cell cycle at G2/M by regulating MAPKs (ERK1/2 and p38) pathway. We further confirmed that 5o inhibits ATPase activity of GRP94 (Glucose-regulated protein 94) with the IC50 1.45 ± 0.06 µM. Compound 5o inhibits GRP94 to trigger regulation of PI3K/Akt and MAPKs pathways. This study reveals that 5o is a promising therapeutic agent against CRC as a novel GRP94 inhibition.

Potential Alterations of Functional Connectivity Analysis in the Patients with Chronic Prostatitis/Chronic Pelvic Pain Syndrome.

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is one of the most common diseases in urology, but its pathogenesis remains unclear. As a kind of chronic pain which the patients suffered for more than 3 months, we investigated the influence on patients' brain functional connectivity in resting state. Methods: We recruited a cohort of 18 right-handed male patients with CP/CPPS and 21 healthy male right-handed age-matched controls. Their resting-state fMRI data and structural MRI data were preprocessed and processed by RESTPlus V1.22. To assess the integrity of the default mode network (DMN), we utilized the voxel-wised analysis that we set medial prefrontal cortex (mPFC) and posterior cingulate gyrus (PCC) as seed points to compare the global functional connectivity (FC) strength. Results: Compared with healthy control, the FC strength between left mPFC and posterior DMN decreased in the group of CP/CPPS (P < 0.05, GFR correction, voxel P < 0.01, cluster P < 0.05), and the FC strength between the left anterior cerebellar lobe and posterior DMN increased (P < 0.05, GFR correction, voxel P < 0.01, cluster P < 0.05). In the patient group, there was a positive correlation between the increased FC strength and the score of the Hospital Anxiety and Depression Scale (HADS) anxiety subscale (r = 0.5509, P = 0.0178) in the left anterior cerebellar lobe, a negative correlation between the decreased FC strength and the score of the National Institutes of Health Chronic Prostatitis Symptom Index (r = -0.6281, P = 0.0053) in the area of left mPFC, and a negative correlation between the decreased FC strength and the score of HADS anxiety subscale (r = -0.5252, P = 0.0252). Conclusion: Patients with CP/CPPS had alterations in brain function, which consisted of the default mode network's compromised integrity. These alterations might play a crucial role in the pathogenesis and development of CP/CPPS.

Characterization and validation of long noncoding RNAs as new candidates in prostate cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been proved to be an important regulator in gene expression. In almost all kinds of cancers, lncRNAs participated in the process of pathogenesis, invasion, and metastasis. Meanwhile, compared with the large amounts of patients, there is rare knowledge about the role of lncRNAs in prostate cancer (PCa). MATERIAL/METHOD: In this study, lncRNA expression profiles of prostate cancer were detected by Agilent microarray chip, 5 pairs of case and control specimens were involved in. Differentially expressed lncRNAs were screened out by volcano plot for constructing lncRNA-miRNA-mRNA central network. Then, the top ten up-regulated and down-regulated lncRNAs were validated by qRT-PCR in another 5 tumor specimens and 7 para-cancerous/benign contrasts. Furthermore, we searched for the survival curve of the top 10 upregulated and downregulated lncRNAs. RESULTS: A total of 817 differentially expressed lncRNAs were filtered out by the criteria of fold change (FC) and t-test p < 0.05. Among them, 422 were upregulated, whereas 395 were downregulated in PCa tissues. Gene ontology and KEGG pathway analyses showed that many lncRNAs were implicated in carcinogenesis. lnc-MYL2-4:1 (FC = 0.00141, p = 0.01909) and NR_125857 (FC = 59.27658, p = 0.00128) had the highest magnitude of change. The subsequent qPCR confirmed the expression of NR_125857 was in accordance with the clinical samples. High expression of PCA3, PCAT14 and AP001610.9 led to high hazard ratio while low expression of RP11-279F6.2 led to high hazard ratio. CONCLUSIONS: Our study detected a relatively novel complicated map of lncRNAs in PCa, which may have the potential to investigate for diagnosis, treatment and follow-up in PCa. Our study revealed the expression of NR_125857 in human PCa tissues was most up-regulated. Further studies are needed to investigate to figure out the mechanisms in PCa.

A Scalable "Junction Substrate" to Engineer Robust DNA Circuits.

Versatile building blocks are essential for building complex and scaled-up DNA circuits. In this study, we propose a conceptually new scalable architecture called a "junction substrate" (J-substrate) that is linked by prepurified double-stranded DNA molecules. As a proof-of-concept, this novel type of substrate has been utilized to build multi-input DNA circuits, offering several advantages over the conventional substrate (referred to as a "linear substrate", L-substrate). First, the J-substrate does not require long DNA strands, thus avoiding significant synthetic errors and costs. Second, the traditional PAGE purification method is technically facilitated to obtain high-purity substrates, whereby the initial leakage is effectively eliminated. Third, the asymptotic leakage is eliminated by introducing the "junction". Finally, circuits with the optimized J-substrate architecture exhibit fast kinetics. We believe that the proposed architecture constitutes a sophisticated chassis for constructing complex circuits.

Phosphorylation of connexin 43 induced by traumatic brain injury promotes exosome release.

Traumatic brain injury (TBI) caused by the external force leads to the neuronal dysfunction and even death. TBI has been reported to significantly increase the phosphorylation of glial gap junction protein connexin 43 (Cx43), which in turn propagates damages into surrounding brain tissues. However, the neuroprotective and anti-apoptosis effects of glia-derived exosomes have also been implicated in recent studies. Therefore, we detected whether TBI-induced phosphorylation of Cx43 would promote exosome release in rat brain. To generate TBI model, adult male Sprague-Dawley rats were subjected to lateral fluid percussion injury. Phosphorylated Cx43 protein levels and exosome activities were quantified using Western blot analysis following TBI. Long-term potentiation (LTP) was also tested in rat hippocampal slices. TBI significantly increased the phosphorylated Cx43 and exosome markers expression in rat ipsilateral hippocampus, but not cortex. Blocking the activity of Cx43 or ERK, but not JNK, significantly suppressed TBI-induced exosome release in hippocampus. Furthermore, TBI significantly inhibited the induction of LTP in hippocampal slices, which could be partially but significantly restored by pretreatment with exosomes. The results imply that TBI-activated Cx43 could mediate a nociceptive effect by propagating the brain damages, as well as a neuroprotective effect by promoting exosome release. NEW & NOTEWORTHY We have demonstrated in rat traumatic brain injury (TBI) models that both phosphorylated connexin 43 (p-Cx43) expression and exosome release were elevated in the hippocampus following TBI. The promoted exosome release depends on the phosphorylation of Cx43 and requires ERK signaling activation. Exosome treatment could partially restore the attenuated long-term potentiation. Our results provide new insight for future therapeutic direction on the functional recovery of TBI by promoting p-Cx43-dependent exosome release but limiting the gap junction-mediated bystander effect.

Coatomer subunit beta 2 (COPB2), identified by label-free quantitative proteomics, regulates cell proliferation and apoptosis in human prostate carcinoma cells.

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA.

Hydrophobic Inorganic Oxide Pigments via Polymethylhydrosiloxane Grafting: Dispersion in Aqueous Solution at Extraordinarily High Solids Concentrations.

Building on the recent demonstration of aqueous-dispersible hydrophobic pigments that retain their surface hydrophobicity even after drying, we demonstrate the synthesis of surface-modified Ti-Pure R-706 (denoted R706) titanium dioxide-based pigments, consisting of a thin (one to three monolayers) grafted polymethylhydrosiloxane (PMHS) coating, which (i) are hydrophobic in the dry state according to capillary rise and dynamic vapor sorption measurements and (ii) form stable aqueous dispersions at solid contents exceeding 75 wt % (43 vol %), without added dispersant, displaying similar rheology to R706 native oxide pigments at 70 wt % (37 vol %) consisting of an optimal amount of conventional polyanionic dispersant (0.3 wt % on pigment basis). The surface-modified pigments have been characterized via 29Si and 13C cross-polarization/magic angle spinning solid-state NMR spectroscopy; infrared spectroscopy; thermogravimetric and elemental analyses; and ζ potential measurements. On the basis of these data, the stability of the surface-modified PMHS-R706 aqueous dispersions is attributed to steric effects, as a result of grafted PMHS strands on the R706 surface, and depends on the chaotropic nature of the base used during PMHS condensation to the pigment/polysiloxane interface. The lack of water wettability of the surface-modified oxide particles in their dry state translates to improved water-barrier properties in coatings produced with these surface-modified pigment particles. The synthetic approach appears general as demonstrated by its application to various inorganic-oxide pigment particles.

Involvement of Connexin40 in the Protective Effects of Ginsenoside Rb1 Against Traumatic Brain Injury.

Ginsenosides are the major active components of ginseng, which have been proven to be effective in therapies for neurodegenerative diseases. Ginsenoside Rb1 (GS-Rb1) is the most abundant among all the identified ginsenosides and has been shown to exert neuroprotective effects, although the underlying molecular mechanisms remain unclear. Connexins are a family of transmembrane proteins that form gap junctions, which are important for diffusion of cytosolic factors such as ions and second messenger signaling molecules. Previous studies have shown that a subset of connexin proteins is involved in neuroprotection. We investigated the protective effects of GS-Rb1 against traumatic brain injury (TBI) and the potential mechanism using TBI mouse model. We discovered that TBI-induced brain injury and up-regulation of connexin40 (Cx40) protein expression as early as 6 h post-TBI, which was reversed by administration of GS-Rb1. In addition, we found that the protective effects of GS-Rb1 are dose and time dependent and are partially mediated through phosphorylation of ERK1/2 signaling pathway, as evidenced by the abolishment of GS-Rb1-mediated elevation of p-ERK1/2 expression and inhibition of Cx40 expressions when ERK inhibitor U0126 was used. Our study provides evidence that Cx40 is implicated in TBI-induced brain injuries, and GS-Rb1 exerts neuroprotective activity against TBI involving down-regulation of Cx40 expression.

Single Step Stone-Wales Transformation Linking Two Thermodynamically Stable Sc2O@C78 Isomers.

Among the very recently reported dimetallic oxide fullerenes Sc2O@C2n (n = 35-47), a representative Sc2O@C78 still lacks of further characterizations. Herein, a systematical investigation on Sc2O@C78 has been performed by density functional theory combined with statistical thermodynamic studies. Two isolated pentagon rule (IPR) satisfying isomers, Sc2O@D3h(24109)-C78 and Sc2O@C2v(24107)-C78, are disclosed to possess prominent thermodynamic stabilities at the temperature region of fullerene formation. Significantly, these two structures are related by a single Stone-Wales transformation. Moreover, bonding critical points, bond orders, and delocalization indices have been analyzed to uncover covalent interactions in both isomers. In addition, (13)C NMR spectra and UV-vis-NIR adsorptions of the two stable structures are introduced to assist experimental identification and characterization in the future.

Theoretical prediction of the host-guest interactions between novel photoresponsive nanorings and C60: a strategy for facile encapsulation and release of fullerene.

A series of photoresponsive-group-containing nanorings hosts with 12∼14 Šin diameter is designed by introducing different number of azo groups as the structural composition units. And the host-guest interactions between fullerene C60 and those nanoring hosts were investigated theoretically at M06-2X/6-31G(d)//M06-L/MIDI! and wB97X-D/6-31G(d) levels. Analysis on geometrical characteristics and host-guest binding energies revealed that the designed nanoring molecule (labeled as 7) which is composed by seven azo groups and seven phenyls is the most feasible host for encapsulation of C60 guest among all candidates. Moreover, inferring from the simulated UV-vis-NIR spectroscopy, the C60 guest could be facilely released from the cavity of the host 7 via configuration transformation between trans-form and cis-form of the host under the 563 nm photoirradiation. Additionally, the frontier orbital features, weak interaction regions, infrared, and NMR spectra of the C60@7 host-guest complex have also been investigated theoretically.

Theoretical Insight into the Ambiguous Endohedral Metallofullerene Er3C74: Covalent Interactions among Three Lanthanide Atoms.

All of C74-based endohedral metallofullerenes (EMFs) are found to be monometallofullerenes with the same D3h(14246)-C74 cage so far. An opening question is whether other C74 cages could survive during the production of some novel C74-EMFs. Theoretically, we studied the trimetallic endohedral fullerene Er3C74, the existence of which had been proven without any further characterizations. Two thermodynamically stable Er3C74 isomers were obtained, both of which could be expressed as Er3@C74, meaning that previously synthesized Er3C74 is indeed an endohedral trierbium fullerene. Besides the isomer with well-known D3h(14246)-C74 cage which obeys isolated pentagon rule (IPR), another one possesses the C1(13771)-C74 cage with two adjacent pentagons. Notably, it is the first time an endohedral metallofullerene containing the C1(13771)-C74 cage has been reported. Frontier orbitals analysis, bonding analysis in terms of quantum theory of atoms-in-molecule (QTAIM) and Mayer bond order, together with two-dimensional maps of electron localization function (ELF) and Laplacian of electron density of Er3@D3h(14246)-C74 and Er3@C1(13771)-C74 show obvious covalent interactions not only between metallic atoms and carbon cage but also among three erbium atoms. Finally, simulated IR spectra of Er3@D3h(14246)-C74 and Er3@C1(13771)-C74 were simulated, which should be useful to distinguish those two isomers.

Protective effect of ginsenoside Rb1 on integrity of blood-brain barrier following cerebral ischemia.

Ginsenosides, the major bioactive compounds in ginseng root, have been found to have antioxidant, immunomodulatory, and anti-inflammatory activities. In the present study, we sought to investigate whether and how ginsenoside Rb1 (GS-Rb1), the most abundant ginsenoside, can protect blood-brain barrier (BBB) integrity following cerebral ischemia in middle cerebral artery occlusion (MCAO) animal model. ICR mice underwent MCAO and received GS-Rb1 by intraperitoneal injection at 3 h after reperfusion. We evaluated infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression at 48 h after MCAO. We further examined whether GS-Rb1 protected BBB integrity by suppressing post-ischemic inflammation-induced activity of matrix metalloproteinase-9 (MMP-9) and nicotinamide adenine dinucleotide phosphate oxidase (NOX). First, GS-Rb1 decreased infarction and improved neurological deficits in MCAO animals. In addition, GS-Rb1 reduced EB extravasation and brain edema and preserved expression of tight junction proteins in the ischemic brain. Moreover, GS-Rb1 inhibited expression of pro-inflammatory factors including nitric oxide synthase and IL-1ß, but increased expression of anti-inflammatory markers arginase 1 and IL-10 in the ischemic brain. Consistently, GS-Rb1 attenuated ischemia-induced expression and activity of MMP9. Finally, GS-Rb1 reduced NOX-4 mRNA expression and NOX activity in ischemic brain. These results suggest that GS-Rb1 protects loss of BBB integrity in ischemic stroke by suppressing neuroinflammation induction of MMP-9 and NOX4-derived free radicals, and indicate its potential for treating brain injuries, such as ischemia and stroke.