Histological features of intracranial thrombi in stroke patients with cancer.

The histological features of thrombus in stroke patients with cancer are not well known. Using immunohistochemical staining of thrombi retrieved during mechanical thrombectomy in stroke patients, thrombus compositions were compared between 16 patients with active cancer, 16 patients with inactive cancer, and 16 patients without any history of cancer. The active cancer group showed higher platelet and lower erythrocyte fractions than the inactive cancer or the control group. Four patients with vegetation showed very high platelet and low erythrocyte fractions. Patients with cryptogenic etiology in the active cancer group showed a similar pattern to those with vegetation. These findings may aid the determination of treatment strategies in cancer-associated stroke. ANN NEUROL 2019.

Two Alternative Splicing Variants of AtERF73/HRE1, HRE1α and HRE1ß, Have Differential Transactivation Activities in Arabidopsis.

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1ß. In this study, we examined the differences between the molecular functions of HRE1α and HRE1ß. We found that HRE1α and HRE1ß are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1ß, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1ß also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1ß was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1ß might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1ß differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis.

Arabidopsis non-TZF gene AtC3H17 functions as a positive regulator in salt stress response.

Functional studies of CCCH-type zinc finger proteins in abiotic stress responses have largely focused on tandem CCCH-type zinc finger (TZF) genes, whereas the study of functional roles of non-TZF genes in abiotic stress responses has largely been neglected. Here, we investigated the functional roles of AtC3H17, a non-TZF gene of Arabidopsis, in salt stress responses. AtC3H17 expression significantly increased under NaCl, mannitol, and ABA treatments. AtC3H17-overexpressing transgenic plants (OXs) were more tolerant under NaCl and MV treatment conditions than the wild type (WT). atc3h17 mutants were more sensitive under NaCl and MV treatment conditions compared with the WT. The transcription of the salt stress-responsive genes in ABA-dependent pathway, such as RAB18, COR15A, and RD22, was significantly higher in AtC3H17 OXs than in WT both under NaCl-free condition and after NaCl treatment. Our results demonstrate that AtC3H17 functions as a positive regulator in salt stress response, via the up-regulation of ABA-dependent salt stress-response pathway.

Rh-catalyzed oxidative C-C bond formation and C-N bond cleavage: direct access to C2-olefinated free (NH)-indoles and pyrroles.

The rhodium-catalyzed oxidative C2-olefination of indoles and pyrroles containing N-arylcarboxamide directing groups with a range of alkenes and subsequent cleavage of directing groups is described. This method provides direct and efficient access to C2-functionalized free (NH)-heterocycles.

Low-moderate dose whole-brain γ-ray irradiation modulates the expressions of glial fibrillary acidic protein and intercellular adhesion molecule-1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mouse model.

The anti-inflammatory efficacy of radiation therapy (RT) with single fractions below 1.0 Gy has been demonstrated in Alzheimer's disease mouse models. As neuroinflammation is also a major pathological feature of Parkinson's disease (PD), RT may also be effective in PD treatment. Therefore, this study aimed to investigate the anti-inflammatory effect of low-moderate dose RT (LMDRT, 0.6 Gy/single dose, for 5 days) exposure in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg, intraperitoneally, for 5 consecutive days)-induced PD mouse model. Importantly, LMDRT reduced the levels of glial fibrillary acidic protein and intercellular adhesion molecule-1 (CD54) in the striatum region, which increased following MPTP administration. LMDRT also modulated inflammatory gene expression patterns in the substantia nigra region of the MPTP-treated mice. However, LMDRT had no direct effects on the severe loss of dopaminergic neurons and impaired motor behavior in the rotarod test. These results indicate that LMDRT has anti-inflammatory effects by modulating neuroinflammatory factors, including glial fibrillary acidic protein and intercellular adhesion molecule-1, but showed no behavioral improvements or neuroprotection in the MPTP-induced mouse model of PD.

AZD7648, a DNA-PKcs inhibitor, overcomes radioresistance in Hep3B xenografts and cells under tumor hypoxia.

Radiation therapy is one of the most commonly used cancer treatments. However, it has important concerns such as damage to normal tissues around cancers and radioresistance. To overcome these problems, combination therapy using radiosensitizer and radiotherapy will be a good alternative. The present study investigated the effects of AZD7648 on overcoming radioresistance as well as radiosensitizing in Hep3B xenografts and cells. The results showed that AZD7648 enhanced ionizing radiation (IR)-induced tumor growth not only in radiosensitive but also radioresistant tumors. In particular, the combination of AZD7648 with radiation reduced the expression of hypoxia induce factor-1α (HIF-1α) in radioresistant tumors. In vitro studies, AZD7648 plus IR increased IR-induced G2/M arrest and regulated cell cycle checkpoints such as cyclinB1, p-cdc2 in normoxia but not in hypoxia. AZD7648 induced more radiation-mediated ROS than radiation only under normoxia, but these ROS were not altered by AZD7648 under hypoxia. Interestingly, AZD7648 downregulated HIF-1α expression level under CoCl2-treated hypoxic condition but not in normoxic condition. In conclusion, AZD7648 synergistically increased radiosensitivity through accumulating IR-induced G2/M arrest and further improved radioresistance via regulation of HIF-1α. The present data suggest that AZD7648 may be a strong radiosensitizer in radioresistant as well as radiosensitive cancers.

BR101801 enhances the radiosensitivity of p53-deficient colorectal cancer cells by inducing G2/M arrest, apoptosis, and senescence in a p53-independent manner.

Inhibition of DNA-dependent protein kinase (DNA-PK) in the non-homologous end-joining repair pathway reportedly increases the radiation sensitivity of cancer cells. We have recently reported that BR101801, a novel triple inhibitor of PI3K-gamma (γ), delta (δ), and DNA-PK, functions as an efficient sensitizer of radiation-induced DNA damage in various human solid cancer cells and a xenograft mouse model. Given that the p53 tumor suppressor gene plays an important role in radiotherapeutic efficacy, in the current study, we focused on the impact of the p53 status on BR101801-induced radiosensitization using isogenic HCT116 p53+/+ and HCT116 p53-/- human colorectal cancer cell lines. In vitro, HCT116 p53+/+ and HCT116 p53-/- human colorectal cancer cells were pretreated with 1 µM BR101801 for 24 h before exposure to ionizing radiation (IR), followed by assays to analyze colony formation, DNA damage, cell cycle changes, senescence, autophagy, apoptosis, and DNA damage response-related proteins. Xenograft mouse models were constructed to examine the potential synergistic effects of BR101801 (50 mg/kg, orally administered once daily) and fractionated IR (2 Gy × 3 days) on tumor growth inhibition in vivo. BR101801 inhibited cell proliferation and prolonged DNA damage in both HCT116 p53+/+ and HCT116 p53-/- human colorectal cancer cells. Combined treatment with BR101801 and IR robustly induced G2/M phase cell cycle arrest, apoptosis, and cellular senescence in HCT116 p53-/- cells when compared with treatment with IR alone. Furthermore, BR101801 synergistically inhibited tumor growth in the HCT116 p53-/- xenograft mouse model. BR101801 enhanced the radiosensitivity of HCT116 human colorectal cancer cells regardless of their p53 status. Moreover, BR101801 exerted robust synergistic effects on IR-induced cell cycle arrest, apoptosis, and tumor growth inhibition, even in radioresistant HCT116 p53-/- cells. Overall, these findings provide a scientific rationale for combining BR101801 with IR as a new therapeutic strategy to overcome radioresistance induced by p53 deficiency.

Neutrophil Recruitment in Arterial Thrombus and Characteristics of Stroke Patients with Neutrophil-Rich Thrombus.

PURPOSE: Neutrophils contribute to thrombosis. However, there is limited information on the temporal course of neutrophil recruitment in thrombosis, the contribution of neutrophils to thrombus growth, and the characteristics of stroke patients with neutrophil-rich thrombi. MATERIALS AND METHODS: After inducing carotid artery thrombosis in Institute of Cancer Research mice using ferric chloride, aged thrombi were produced by ligating the distal portion of the carotid artery in mice for 0.5, 1, 2, 3, 6, or 24 h. For thrombus analysis in stroke patients, we used registry data and thrombi that were obtained during intra-arterial thrombectomy. Immunohistochemistry was performed to determine thrombus composition. RESULTS: In the thrombi of 70 mice, Ly6G positive cell counts (neutrophils) and histone H3-positive cell counts increased in a time-dependent manner (both p<0.001). Ly6G-positive cell count was strongly correlated with histone H3-positive cell counts (r=0.910, p<0.001), but not with thrombus size (p=0.320). In 75 stroke patients, atrial fibrillation and cardioembolism were more frequent in the higher neutrophil group (32/37, 86.5%) than in the lower neutrophil group (19/38, 50%) (p=0.002). The median erythrocyte fraction was higher [52.0 (interquartile range 39.9-57.8)] in the higher neutrophil group than in the lower neutrophil group [40.3 (interquartile range 23.5-53.2)]. The fraction of neutrophils was positively correlated with that of erythrocytes (R=0.35, p=0.002). CONCLUSION: Neutrophils were recruited and increased in arterial thrombosis in a time-dependent manner; however, they were not associated with the growth of formed thrombi. Neutrophil fractions in the thrombi of stroke patients appeared to be associated with atrial fibrillation and erythrocyte fraction.

Synergistic radiosensitizing effect of BR101801, a specific DNA-dependent protein kinase inhibitor, in various human solid cancer cells and xenografts.

DNA-dependent protein kinase (DNA-PK), an essential component of the non-homologous end-joining (NHEJ) repair pathway, plays an important role in DNA damage repair (DDR). Therefore, DNA-PK inhibition is a promising approach for overcoming radiotherapy or chemotherapy resistance in cancers. In this study, we demonstrated that BR101801, a potent DNA-PK inhibitor, acted as an effective radiosensitizer in various human solid cancer cells and an in vivo xenograft model. Overall, BR101801 strongly elevated ionizing radiation (IR)-induced genomic instability via induction of cell cycle G2/M arrest, autophagic cell death, and impairment of DDR pathway in human solid cancer cells. Interestingly, BR101801 inhibited not only phosphorylation of DNA-PK catalytic subunit in NHEJ factors but also BRCA2 protein level in homologous recombination (HR) factors. In addition, combination BR101801 and IR suppressed tumor growth compared with IR alone by reducing phosphorylation of DNA-PK in human solid cancer xenografts. Our findings suggested that BR101801 is a selective DNA-PK inhibitor with a synergistic radiosensitizing effect in human solid cancers, providing evidence for clinical applications.

Collateral augmentation treatment with a combination of acetazolamide and head-down tilt in a rat ischemic stroke model.

Cerebral collaterals is crucially important in the pathophysiology of acute ischemic stroke and associated with outcome after reperfusion therapy. We explored the effectiveness of collateral augmentation treatment with a combination of acetazolamide (ACZ) and head-down tilt (HDT) in the transient middle cerebral artery occlusion (MCAO) rat model. Transient MCAO was induced in all animals for 1.5 h, followed by reperfusion for 22.5 h. Seventy-two male Wistar rats were divided into four treatment groups: control, ACZ, HDT, and combination. Twenty sham rats, which underwent surgery, were randomly allocated to these groups. Twenty-four hours after MCAO or sham surgery, we measured the infarction volume, brain edema (aquaporin-4 [AQP4], and brain water content), and neurological deficits (Garcia and Longa tests). Collateral augmentation treatments were associated with reduced infarction volume, less brain edema, and better neurological outcomes compared with untreated animals. More specifically, ACZ and HDT treatments resulted in small infarction volumes, and HDT was associated with a low AQP4 expression and improved neurological score, while the combination of ACZ and HDT improved neurological scores and reduced brain water content. This study shows that collateral augmentation treatments are associated with a better stroke prognosis compared with untreated animals after transient MCAO. The combination of ACZ and HDT seems to have some synergistic effect, but was not proven to be superior to HDT treatment alone.

Predicting Stroke Outcomes Using Ankle-Brachial Index and Inter-Ankle Blood Pressure Difference.

BACKGROUND: This study investigated the association of high ankle-brachial index difference (ABID) and systolic inter-ankle blood pressure difference (IAND) with short- and long-term outcomes in acute ischemic stroke patients without peripheral artery disease (PAD). METHODS: Consecutive patients with acute ischemic stroke who underwent ankle-brachial index (ABI) measurement were enrolled. ABID was calculated as |right ABI-left ABI|. IAND and systolic inter-arm blood pressure difference (IAD) were calculated as |right systolic blood pressure - left systolic blood pressure|. Poor functional outcome was defined as modified Rankin Scale score ≥3 at 3 months. Major adverse cardiovascular events (MACEs) were defined as stroke recurrence, myocardial infarction, or death. RESULTS: A total of 2901 patients were enrolled and followed up for a median of 3.1 (interquartile range, 1.6-4.7) years. Among them, 2643 (84.9%) patients did not have PAD. In the logistic regression analysis, ABID ≥ 0.15 and IAND ≥ 15 mmHg were independently associated with poor functional outcome (odds ratio (OR), 1.970, 95% confidence interval (CI), 1.175‒3.302; OR, 1.665, 95% CI, 1.188‒2.334, respectively). In Cox regression analysis, ABID ≥0.15 and IAND ≥ 15 mmHg were independently associated with MACEs (hazard ratio (HR), 1.514, 95% CI, 1.058‒2.166; HR, 1.343, 95% CI, 1.051‒1.716, respectively) and all-cause mortality (HR, 1.524, 95% CI, 1.039‒2.235; HR, 1.516, 95% CI, 1.164‒1.973, respectively) in patients without PAD. CONCLUSION: High ABID and IAND are associated with poor short-term outcomes, long-term MACE occurrence, and all-cause mortality in acute ischemic stroke without PAD.

Brachial-ankle pulse wave velocity for predicting functional outcomes in patients with cryptogenic stroke.

Even after extensive standard evaluation, the probable cause of stroke in some patients remains unclear; this condition is defined as cryptogenic stroke (CS). The prognosis of patients with CS is largely undetermined. We investigated whether higher brachial-ankle pulse wave velocities (baPWVs) can predict poor functional outcomes at 3 months after stroke onset in these patients. We investigated patients with CS with first-ever acute cerebral infarction who underwent baPWV measurements. The stroke subtypes were classified using the Trial of ORG 10172 in Acute Stroke Treatment classification. Poor functional outcomes were defined as modified Rankin Scale scores of >2 at 3 months after stroke onset. In total, 595 patients with CS were included; among them, 360 were men (60.5%). Their mean age was 65.0 ±â€¯12.4 years. One-hundred-eleven patients (18.7%) had poor functional outcomes. In the multivariable logistic regression analysis, the cutoff baPWV value based on the receiver-operating characteristic curve was >1968 cm/s, which was determined as a strong independent predictor (OR 3.159, 95% CI 1.487-6.715, p = 0.003). The OR of the cutoff value was higher in the patients with CS with initial National Institutes of Health Stroke Scale (NIHSS) scores of ≥5 (OR 4.252, 95% CI 1.596-11.324, p = 0.004); that in the patients with initial NIHSS scores of <5 was not significant (OR 1.671, 95% CI 0.620-4.505, p = 0.310). baPWV measurement during the acute stroke phase might be useful in identifying patients with CS at high risks of having a poor neurological prognosis.

Prediction of functional outcome using the novel asymmetric middle cerebral artery index in cryptogenic stroke patients.

BACKGROUND: Etiology is unknown in approximately one-quarter of stroke patients after evaluation, which is termed cryptogenic stroke (CS). The prognosis of CS patients is largely undetermined. We created a novel index from transcranial Doppler parameters including mean flow velocity (MV) and pulsatility index (PI) and investigated whether the calculation of asymmetry in the novel parameter can predict functional outcomes in CS patients. METHODS: We made the middle cerebral artery (MCA) index (%) as a novel parameter, which was calculated as 100 X (MCA MV + MCA PI X 10) / (MCA MV-MCA PI X 10). The MCA asymmetry index (%) was also calculated as 100 X (|Rt MCA index-Lt MCA index|) / (Rt MCA index + Lt MCA index) / 2. Poor functional outcomes were defined as modified Rankin Scale score (mRS) ≥3 at 3 months after stroke onset. RESULTS: A total of 377 CS patients were included. Among them, 52 (13.8%) patients had a poor outcome. The overall MCA asymmetry index was two-fold higher in CS patients with a poor outcome (10.26%) compared to those with a good outcome (5.41%, p = 0.002). In multivariable analysis, the overall MCA asymmetry index (OR, 1.054, 95% CI, 1.013-1.096, p = 0.009) and the cutoff value of the overall MCA asymmetry index >9 were associated with poor outcomes at 3 months (OR, 3.737, 95% CI, 1.530-9.128, p = 0.004). CONCLUSION: We demonstrated that the novel asymmetric MCA index can predict short-term functional outcomes in CS patients.

Impact of white matter hyperintensities on the prognosis of cryptogenic stroke patients.

BACKGROUND: To our knowledge, little is known regarding whether white matter hyperintensities (WMH) affect the prognosis of cryptogenic stroke (CS) patients. Understanding this association may be helpful with expecting the prognosis of CS patients. METHODS: This retrospective observational study enrolled consecutive CS patients who underwent brain MRI and comprehensive cardiac evaluation. Severe WMH was defined as Fazekas' score ≥3. We defined poor functional outcome as modified Rankin Scale score ≥3 at 3 months. Long-term mortality and causes of death were identified using national death certificates and assessed by Kaplan-Meier method and regression analysis model. RESULTS: Among 2732 patients with first-ever ischemic stroke, 599 (21.9%) patients were classified as having CS. After exclusions, 235 patients were enrolled and followed up for a median of 7.7 years (IQR, 6.7-9.0). Severe WMH were found in 81 (34.5%) patients. After adjustments, severe WMH were an independent predictor for poor functional outcomes at 3 months (OR 5.25, 95% CI, 2.07-13.31). Subgroup analysis showed that severe WMH were an independent predictor for long-term mortality only in younger patients (age < 65) (HR 3.11, 95% CI, 1.29-7.50), but not in older patients (HR 1.19, 95% CI, 0.63-2.23). CONCLUSIONS: Severe WMH were independently associated with short-term functional outcomes in CS patients and independently associated with long-term mortality in younger CS patients. Grading WMH is of value in predicting prognosis of CS patients with young age.

Dabigatran reduces endothelial permeability through inhibition of thrombin-induced cytoskeleton reorganization.

Dabigatran etexilate (DE), a new oral anti-coagulant, is a direct thrombin inhibitor. Clinical trials showed the favorable benefit-to-risk profile of DE compared to warfarin for the prevention of ischemic stroke in patients with atrial fibrillation. Remarkably, patients treated with dabigatran showed reduced rates of intracerebral hemorrhage compared to warfarin. As the breakdown of endothelial barrier integrity is associated with hemorrhagic events and as thrombin increases endothelial permeability, we hypothesized that dabigatran preserves the endothelial barrier by inhibiting thrombin-induced permeability. We assessed leakage of fluorescein isothiocyanate (FITC)-dextran through the endothelial monolayer and measured trans-endothelial electrical resistance of the endothelial monolayer after treatment of thrombin or thrombin pre-incubated with dabigatran. Thrombin increased the permeability of endothelial cells. Dabigatran effectively blocked the ability of thrombin to increase permeability. Dabigatran inhibited the formation of actin stress fibers induced by thrombin and inhibited consequent destabilization of junctional protein complexes and intercellular gap formation. The interaction of thrombin with protease activated receptor-1 activates the Rho A guanosine triphosphate (GTP)ase-myosin light chain (MLC) phosphorylation signaling axis, leading to actin cytoskeleton changes. This signaling pathway was effectively inhibited by dabigatran in endothelial cells. Consistently, the number of phosphorylated MLC-positive cells was significantly decreased in ischemic tissue of rat brains. These results indicate dabigatran blocks the ability of thrombin to induce vascular permeability and the resulting underlying signaling cascade in endothelial cells. Our findings provide evidence that dabigatran may confer a lower risk of intracerebral hemorrhage by preserving endothelial barrier integrity.