Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

Delayed Administration of Nafamostat Mesylate Inhibits Thrombin-Mediated Blood-Spinal Cord Barrier Breakdown During Acute Spinal Cord Injury in Rats

Background: Nafamostat mesylate (NM), an FDA-approved serine protease inhibitor, exerts anti-neuroinflammation and neuroprotective effect on rat spinal cord injury (SCI). However, the time window for NM administration after SCI as well as its underlying mechanism remains unclear. Methods: A series of different first administration time points of NM was tested on rat contusive SCI model. The optimal time window of NM was screened by evaluating hindlimb locomotion and electrophysiology. We performed western blot and immunofluorescence to evaluate the drug target thrombin as well as its downstream Protease activated receptor 1 (PAR-1), and matrix metalloproteinase-9 (MMP9). Enzyme activity assay was used to test thrombin activity. The permeability of blood-spinal cord barrier (BSCB) was assessed by Evans Blue leakage. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.Results: The optimal administration time window of NM was 2-12 h. The thrombin specific inhibitor, Argatroban, had similar pattern. The temporal expression pattern of thrombin peaked at 12 hours and returned to normal level at 7 days post SCI. PAR-1, the thrombin receptor, was observed a significant upregulation after SCI. MMP9, downstream of PAR-1, was also increased along with thrombin and PAR1. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). NM significantly downregulated the thrombin and MMP9 expression as well as thrombin activity in the spinal cord, especially in ECs. NM administration at 2-12 h after SCI could inhibit the leakage of Evans blue in the epicenter and upregulate tight junction proteins (TJPs) expression. 8 h administration of NM effectively inhibited the infiltration of peripheral macrophages in the acute SCI. Conclusions: Our study provided preclinical data of NM administration time window in SCI model, which is clinically relevant in the acute SCI. We elucidated the protective mechanism of NM through BSCB protection and anti-neuroinflammation via thrombin intervention.

Bradykinin Is a Proximal Event in Experimental Cerebral Malaria

Human malaria is a complex disease and a leading cause of mortality in children under 5 years of age. Plasmodium falciparum (Pf) is the agent responsible for cerebral malaria. Parasite infected erythrocytes are sequestered in the brain vasculature, disrupting the blood-brain-barrier, and with systemic inflammation leading to progressive brain edema. The precise pathophysiologic mechanism(s) underlying brain swelling in CM is not known. Recent work from our laboratories indicates that there is a role for bradykinin (BK) in fluid transport in human brain microvascular endothelial cells (Front Med 6:75, 2019). We examined the role of bradykinin (BK) in pediatric CM. Initial studies showed recombinant falcipain-2, a cysteine protease contained in the parasite digestive vacuole, was inhibited by high molecular weight kininogen (HK), with an IC 50=36 nM. Further, falcipain-2, but not the related protease falcipain 3, hydrolyzed the chromogenic substrate S2302 (Pro-Phe-Arg-pNA) at pH 7.4 with an 88 nM K m. These results suggest that falcipain-2 has plasma kallikrein-like activity. HK is both an inhibitor and substrate of falcipain-2. Molar excess HK to falcipain-2 (ratio 8:1 to 2:1) blocked the proteolytic activity of the cysteine protease at pH 7.4. Equal molar falcipain-2 to HK (1:1) resulted in kallikrein-like cleavage of HK with stable BK liberation over 1 h. Molar excess falcipain-2 to HK (1:2 and greater) led to progressive HK cleavage into smaller proteins and peptides. The falcipain-2 major cleavages observed by N-terminal sequencing were in Domain 3 of the heavy chain of HK, the cysteine protease inhibitory region (I 292ASFSQNCDIYPGKDF 303, D 320IPTNSPELEETLT 334, and E 412KKIYPTVNCQPLG 425). P. falciparum trophozoite lysates completely hydrolyzed purified and plasma HK into a ~64 kDa heavy chain and ~46 kDa light chain in buffer containing EDTA, pepstatin, and PMSF. The cysteine proteinase inhibitor E64 blocked this cleavage, suggesting that the relevant activity was that of a cysteine protease. Plasma from Kenyan children presenting with CM (fever, parasitemia, coma) had evidence of circulating cHK, indicative of BK released from HK. Forty percent (8 of 20) of CM patients had no intact 120 kDa HK at hospital entry. In contrast, only 16% (3 of 8) of children with uncomplicated malaria had detectable cHK. In CM patients, the HK level before antimalarial treatment (58 ± 3.9 µg/ml) was significantly lower than the value after clinical recovery (69 ± 3.6 µg/ml; p<0.04) as measured by competitive ELISA. We also examined the roles of BK and HK in experimental cerebral malaria. 10 6 infected red blood cells with P. berghei ANKA were injected intraperitoneally into wild-type (C57BL/6) and total kininogen deficient (kgn1 -/-) C57BL/6 mice. The level of parasitemia on day 5 post-infection was ≥ 8% for both groups of mice (Figure 1). The kgn1 -/- mice had protected neuronal function measured by SHIRPA score relative to wild-type mice. Cerebral edema detected in wild- type mice by Evans Blue dye extravasation test was nearly completely attenuated in kgn1 -/- mice. Corroborative studies were performed in BK B2 receptor deleted (bdkrb2 -/-) mice. In mice with 15% parasitemia for both genotypes, there was significantly less neurologic function deterioration and a 30% reduction in cerebral Evans blue extravasation into brain parenchyma in the bdkrb2 -/- mice. These data strongly suggest that falcipain-2 liberates BK from HK by acting like plasma kallikrein and in high concentrations destroys HK's cysteine protease inhibitory region. Some children with CM have in vivo evidence of prior HK proteolysis. Total kininogen deficiency protects mice from lethal experimental CM. Taken together, these data suggest that bradykinin is a proximal mediator of cerebral malaria. Figure 1 Figure 1. Disclosures McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

EXTH-07. TEMPORAL ASSESSMENT OF ALTERED PERI-ABLATION BLOOD-BRAIN BARRIER PERMEABILITY FOLLOWING TUMOR CYTOREDUCTION BY THERMAL THERAPY IN A RAT ORTHOTOPIC GLIOBLASTOMA MODEL

Laser interstitial thermal therapy (LITT) is a minimally invasive tumor cytoreductive treatment for recurrent gliomas, brain tumors in eloquent regions and/or otherwise inaccessible. Following reports of persistent peri-ablation blood-brain barrier (BBB) opening in humans, we examined this phenomenon using a rat glioblastoma model. Athymic female rats were implanted with U251 tumor cells in one brain hemisphere. Tumor growth was monitored using magnetic resonance imaging (MRI) and dynamic contrast enhanced (DCE)-MRI. When tumors reached about 4 mm in diameter, they were ablated under supervision of diffusion-weighted MRI using Visualase®, a clinical LITT system. Four rats were used as controls. Longitudinal MRI data were obtained before LITT, and at post-LITT 2 (n=9), 3 (n=3) and 4 (n=9) weeks. After the terminal MRI at each time point, rats were injected intravenously with fluorescent isothiocyanate dextran (FITC-dextran; 2000 kDa) and Evans Blue (68 kDa after binding to plasma albumin) and the brains immersion fixed in 10% paraformaldehyde. The brains were cut into 100 μM thick slices in a vibratome and examined for the distribution of the two fluorophores. All rats survived the LITT procedure. The sham controls showed increased tumor burden by 2 weeks and were sacrificed. DCE-MRI data and fluorescent data showed elevated BBB permeability in peri-ablation regions, with leakage of a gadolinium contrast on DCE-MRI and of Evans Blue, but not of FITC-dextran. Histology showed little tumor tissue at 2 weeks, but evidence of recurrence at ablation margins at later times. These data demonstrate that LITT is adaptable to rat glioma models and can be performed under MRI monitoring. Peri-ablation regions showed selective increase in BBB permeability acutely due to sublethal heating, but later increases in permeability may be due to tumor recurrence. We suggest this model is useful for examining the temporal and spatial development of peri-ablation BBB opening following LITT.

Đánh giá tác dụng chống viêm, giảm đau và cầm máu của “Viên trĩ HV” trên động vật thực nghiệm

Nghiên cứu được thực hiện nhằm đánh giá tác dụng chống viêm, giảm đau và cầm máu của “Viên trĩ HV” trên thực nghiệm. Nghiên cứu thực nghiệm, có đối chiếu với nhóm chứng. Khảo sát tác dụng chống viêm trực tràng trên mô hình chuột cống trắng gây trĩ, “Viên trĩ HV” được dùng ở liều 0,7 g/kg/ngày và 1,4 g/kg/ngày, mức độ viêm tại chỗ được đánh giá bởi hình ảnh đại thể, chỉ số trực tràng, mức độ thoát mạch vào mô trực tràng xác định bằng lượng xanh evans (evans blue) có trong mô trực tràng, và hình ảnh vi thể của trực tràng.Nghiên cứu tác dụng giảm đau trên mô hình gây đau quặn bụng bằng acid acetic, “Viên trĩ HV” được dùng ở liều 1,2 g/kg/ngày và 2,4 g/kg/ngày trên chuột nhắt (tương đương liều dùng trên lâm sàng và gấp 2 liều lâm sàng) để nghiên cứu tác dụng giảm đau trên thực nghiệm. Đánh giá tác dụng cầm máu trên mô hình gây chảy máu do cắt đuôi chuột cống trắng, “Viên trĩ HV” được dùng ở liều 0,7 g/kg/ngày và 1,4 g/kg/ngày, tác dụng cầm máu được đánh giá dựa trên thời gian chảy máu và lượng máu mất. Kết quả nghiên cứu cho thấy viên nang cứng “Viên trĩ HV” dùng liều 0,7 g/kg/ngày và 1,4 g/kg/ngày có tác dụng chống viêm trên mô hình gây trĩ ở chuột cống trắng bằng dung dịch croton oil và có tác dụng cầm máu, làm rút ngắn thời gian chảy máu, tác dụng này của chế phẩm tương đương với thuốc tham chiếu carbazochrom 12mg/kg/ngày. Viên nang cứng “Viên trĩ HV” dùng trên chuột nhắt trắng liều 1,2 g/kg/ ngày và 2,4 g/kg/ngày làm giảm đau rõ rệt trên mô hình gây đau quặn bằng acid acetic. Nghiên cứu có thể kết luận “Viên trĩ HV” có tác dụng chống viêm, giảm đau và cầm máu trên thực nghiệm.

Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue (EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley (SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12wk and the diabetic groups for 4, 8, and 12wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation. RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was (0.54±0.23)%, (0.65±0.11)%, and (0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%, (0.03±0.05)%, and (0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were (0.53±0.22)%, (0.69±0.16)%, and (0.52±0.11)%. The leakage areas of deep blood vessels were (0.54±0.50)%, (1.42±0.16)%, and (1.80±0.07)% at 4, 8, and 12wk, respectively. There was a statistically difference of the leakage area between the 8th week and the 4th week of diabetes group (P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4wk and 8wk (P<0.001). CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.

Nickel doped Nano Cobalt (II) Oxide used as Photocatalyst for Degradation of Evans Blue Dye

The photocatalytic degradation of Evans blue (EB) has been studied under visible light in the presence of nanocrystalline nickel doped cobalt (II) oxide as a photocatalyst. Nickel-cobalt (II) oxide was synthesized by using Sol-gel technique. The photocatalyst was characterized by Field Emission Scanning Electron Microscopy (FESEM), Energy-dispersive X-ray spectroscopy (EDX), X-ray Diffraction (XRD), Fourier-transform infrared (FTIR) and High-resolution transmission electron microscopy (HRTEM). Effect of various working parameters like pH, concentration, amount of nickel doped and undoped cobalt (II) oxide, dose of dopants, light intensity etc. on the rate of degradation of Evans blue was also investigated. On the basis of observations, a suitable mechanism for the photocatalytic degradation of Evans blue dye has been proposed.

Apolipoprotein M and Sphingosine-1-Phosphate Receptor 1 Promote the Transendothelial Transport of High-Density Lipoprotein

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P 1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P 1 ) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P 1 ) endothelium-specific knockin of S1P 1 . The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125 I-HDL by human aortic endothelial cells was increased by an S1P 1 agonist but decreased by an S1P 1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125 I-HDL transport by the S1P 1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P 1 , apoE-haploinsufficient mice with endothelium-specific knockin of S1P 1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P 1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.