Optimization of the formulation of a porcine fibrin patch / 中国药房

OBJECTIVE To optimize the formulation of a porcine fibrin patch （abbreviated as “DBT”）. METHODS Based on single-factor tests， with the contents of fibrinogen， thrombin and collagen before freeze-drying as the factors， with the overall desirability （OD） value of adhesion strength， holding viscosity and water absorption as response value， the formulation of DBT was optimized by Box-Behnken-response surface methodology， and the verification tests were conducted. RESULTS According to the results of the single factor tests and Box-Behnken-response surface methodology， combined with the actual production， the optimal formulation of DBT was 6.5 mg/cm2 of fibrinogen， 8.0 IU/cm2 of thrombin and 5.6 mg/mL of collagen. The average OD value of 3 validation tests was 0.726 6 （RSD＝0.58%， n＝3）， and the relative error of which with the predicted value （0.733 0） was －0.87%. CONCLUSIONS The optimal formulation of DBT is stable and feasible.

Expression of Toll like receptor in pancreatic cancer cells / 中华肝胆外科杂志

Objective:To compare the expression difference of Toll like receptor (TLR) and inflammatory factors between pancreatic cancer and normal pancreatic epithelial cells, and explore the correlation between TLR and inflammatory microenvironment.Methods:Normal pancreatic duct epithelium cells (HPNE) and pancreatic cancer cells (Panc-1 and Mia-PACA-2) were cultured and proteins were obtained. The expression of TLR family protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and myeloid differentiation factor 88 (MyD88) were examined by western blot in HPNE, Panc-1 and Mia-PACA-2. The correlations between TLR and inflammation cytokines of pancreatic cancer were analyzed by Pearson correlation analysis.Results:Compared with HPNE, the TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 were highly expressed in Panc-1 and Mia-PACA-2 (all P<0.05). Compared with Panc-1, the expression of TLR2 and TLR4 in Mia-PACA-2 were increased obviously, while the TLR9 expression was mildly decreased (all P<0.05). The expression of IL-6 in HPNE was found less than that in Panc-1 (0.52±0.03 vs. 0.76±0.04) and Mia-PACA-2 (0.52±0.03 vs. 1.12±0.09) with statistical differences ( P<0.05). Similarly, the expression of TNF-α was found significantly less than that of Panc-1 cells (0.63±0.04 vs. 0.87±0.06) and Mia-PACA-2 cells (0.63±0.04 vs. 0.95±0.10) with statistical differences (all P<0.05). The expression of IL-6 was found positively correlated with expressions of TLR2 ( r=0.964), TLR4 ( r=0.968), TLR7 ( r=0.844), TLR8 ( r=0.668) (all P<0.05), and the expression of TNF-α was found positively correlated with expressions of TLR2 ( r=0.805), TLR4 ( r=0.893), TLR7 ( r=0.847), TLR8 ( r=0.780) (all P<0.05). In contrast with HPNE, the expression of MyD88 was found highly expressed in Panc-1 (0.91±0.10 vs. 0.33±0.03) and Mia-PACA-2 (1.14±0.10 vs. 0.33±0.03) (all P<0.001). Compared with Panc-1, the expression of MyD88 in Mia-PACA-2 was obviously increased (1.14±0.10 vs. 0.91±0.10) with statistical difference ( P=0.048). Conclusion:The TLR family may play a critical role in development of pancreatic cancer by regulating the immune microenvironment, and its mechanism may be through upregulating MyD88 which functions as key signal transduction.

Application of "blended learning + flipped classroom" in medical public elective courses / 中华医学教育探索杂志

Objective:To explore the application effect of "blended learning + flipped classroom" in public elective courses for medical students, and provide reference for the blended construction of related courses.Methods:Totally 229 medical students were selected as research participants, who were taught by "blended learning + flipped classroom". After the courses, the curriculum evaluation questionnaires and focus group interview were used to collect students' evaluation of this course. SPSS 22.0 statistical software was used to make descriptive statistics on the questionnaire data.Results:Totally 98.5% students preferred "blended learning + flipped classroom" teaching mode, and more than 93% students thought this teaching method could improve their learning interest, empathy, understanding and application of knowledge. The results of the qualitative survey extracted two themes, namely "improving learning autonomy" and "expanding comprehensive quality".Conclusion:Perfecting the "blended learning + flipped classroom" teaching mode of the medical public elective courses plays a positive role in improving the learning autonomy and comprehensive quality of medical students and optimizing the learning effect.

A retrospective study of ocular manifestations and aquaporin 4 antibody concentrations in 132 patients with neuromyelitis optica / 中华眼底病杂志

Objective To observe the ocular manifestations and the titer of aquaporin 4 antibody (AQP-4) in NMO patients,and to evaluate the BCVA prognosis in patients with different titers of AQP-4Ab.Methods A retrospective case study.From September 2009 to March 2014,132 NMO patients diagnosed in Department of Neurology and Ophthalmology in Huashan Hospital of Fudan University were included in the study.Among the patients,74 patients (56.06％) were involved in optic nerve for the first time,among which 63 patients (47.72％) were involved in optic nerve alone,and 11 patients (8.33％) were involved in optic nerve and spinal cord at the same time.The recurrence rate was 62.88％ (twice or more).All patients underwent BCVA,slit lamp microscope,fundus examination,thyroid function,sex hormones,and serum AQP-4Ab detection.BCVA was recorded at admission and before discharge from hospital,and worse BCVA was recorded in binocular patients.The BCVA of patients with different titers of AQP-4Ab were analyzed comparatively.Results Among the 74 patients with optic nerve involved in the first onset,50 patients with BCVA＜0.1 at the initial diagnosis (67.57％);AQP-4Ab positive was found in 56 patients,which including 13,9 and 34 patients of AQP-4Ab titer 5-60,61-100 and ＞ 100 RSRU/ml.After 2 weeks of treatment,BCVA improved in 40 patients (71.42％),including 11 (84.62％),6 (66.67％) and 23 (67.64％) ofAQP-4Ab titer 5-60,61-100 and ＞ 100 RSRU/ml.Among 132 patients,98 patients (74.24％) were AQP-4Ab positive.There were 73 patients (55.30％) with abnormal immune rheumatoid index.Conclusions The optic nerve is involved in 56.06％ patients with NMO for the first time,and 67.57％ of the patients had poor vision with BCVA＜0.1.BCVA prognosis is better in patients with serum AQP-4Ab titer of 5-60 RSRU/ml.

Clinical observation on macular edema treated with treating different diseases with the same acupuncture comprehensive therapy / 中国针灸

<p><b>OBJECTIVE</b>To compare the clinical efficacy of treating different diseases with the same acupuncture comprehensive therapy and intramuscular injection of ranibizumab in the treatment of macular edema, and to explore an effective treatment.</p><p><b>METHODS</b>A retrospective study was conducted, ①Acupuncture combined with EA at Xinming one (Extra), Sizhukong (TE 23), Tongziliao (GB 1), once every other day; ②acupoint injection, alternation with compound anisodine and mecobalamine injection at Qiuhou (EX-HN 7), Taiyang (EX-HN 5), once every other day; ③auricular acupressure at yan (LO), gan (CO), shen (CO) and other points; ④plum-blossom needle at Zhengguang 1 (Extra), Zhengguang 2 (Extra), once every other day were given in the acupuncture group (20 cases, 24 affected eyes). Intramuscular injection of 0.5 mg ranibizumab was given in the ranibizumab group (22 cases, 23 affected eyes). The macular foveal thickness, early treatment diabetic retinopathy study of (ETDRS) visual acuity chart, self-evaluation scores of visual function impairment ophthalmopathy patient's quality of life scale were observed before treatment, after 3, 6, 9 and 12 months of treatment, and the clinical efficacy was evaluated.</p><p><b>RESULTS</b>①At all the observation time points of the treatment, the macular thickness was lower than that before treatment in the two groups (all <0.05), and there was no significant difference between the acupuncture group and the ranibizumab group (all >0.05). ②Visual acuity was higher than that before treatment at all the time points in the two groups (all <0.05). After 3-months treatment, there was no statistical significance between the two groups (>0.05). After 6, 9, and 12 months treatment, the visual acuity in the acupuncture group was better than that in the ranibizumab group (<0.05, <0.01). ③At all the time points, the quality of life scores were lower than those before treatment in the two groups (all <0.05). There was no statistical significance in the ranibizumab group compared with those before treatment (all >0.05). In 3, 6, 9 and 12 months of treatment, the quality of life scores in the acupuncture group was better than those in the ranibizumab group (<0.05, <0.01). ④The total effective rate of the acupuncture group was 79.2% (19/24), which was better than 30.4% (7/23) in the ranibizumab group (<0.05). ⑤The improvement of visual acuity before and after treatment was negatively correlated with the course of disease (<0.05), ie, the longer the disease course of the eyes, the worse the visual acuity and the worse the effect.</p><p><b>CONCLUSION</b>Acupuncture comprehensive treatment can effectively treat macular edema, significantly improve the patient's vision, improve the subjective experience and the quality of life, and the shorter the course of the disease the more significant effect. Acupuncture comprehensive treatment is better than intramuscular injection of ranibizumab.</p>

Therapeutic Observation of Comprehensive Acupuncture Treatment for Primary Open-angle Glaucoma / 上海针灸杂志

Objective To observethe clinical efficacy of comprehensive acupuncture treatment in treating primary open-angle glaucoma, and to objectively evaluate the therapeutic efficacy.Method Twenty-eight patients (53 eyes) who received acupuncture treatment were recruited. By adopting a self-control design, the changes of intraocular tension, mean defect (MD) of vision field, mean sensitivity (MS), vision, and score of Quality of Life Scale for Patients with Visual Impairment (QLSPVI) were observed after 3-month acupuncture treatment.ResultThe intraocular tension of the 28 patients declined obviously after the treatment (P0.05); the QLSPVI score dropped significantly after the treatment (P0.05).Conclusion Acupuncture treatment can effectively reduce the intraocular tension, control the deterioration of MD and MS, maintain the level of vision, and enhance the quality of life of patients with primary open-angle glaucoma; with the same disease duration, the longer the treatment, the better the therapeutic efficacy.

Preparation and Characterization of Scutellarin Nanosuspension / 中国药师

Objective: To prepare scutellarin nanosuspension ( SCU-NS) and study the main influencing factors in the prepara-tion. Methods:The technology parameters were determined, and then the influencing factors of SCU-NS were studied. The optimal formula was confirmed by orthogonal design with zeta potential as the evaluation index. Results: The optimal formula process was as follows:drug amount was 0. 5 g, Pluronic? F68 amount was 0. 1 g, phospholipid amount was 0. 2 g, SDS amount was 0. 05 g and HPMC E5 amount was 0. 05 g. The average particle size and the zeta potential of SCU-NS was (122 ± 4) nm and ( -25. 5 ± 0. 6) mV, respectively. The result of transmission electron microscope showed that SCU-NS was spherical and uniform, and the dissolution of SCU-NS in 30 min was more than 90%. Conclusion:Nanosuspension can significantly enhance the dissolution of SCU.

Evaluation of the effect of fasudil hydrochloride on outflow facility in enucleated eyes of different animals / 眼科新进展

Objective To evaluate the influence of a selective Rho-associated protein kinase inhibitor (fasudil hydrochloride) on outflow facility in enucleated porcine,rabbit and bovine eyes.Methods At the constant perfusion pressure of 15 mm-Hg (1 kPa =7.5 mmHg),the baseline coefficient of outflow facility (C0) of the isolated porcine,rabbit and bovine eyes was recorded respectively.The enucleated porcine eyes were divided into two groups randomly (n =6),and they were control group and experimental group.The same grouping method was also used-C0 the ribbit and bovine eyes.The control group was subjected to GPBS perfusion,while the experimental group was treated with 100 μmol · L-1 fasudil solution,followed by recording the experimental coefficient of outflow facility (C1),as well as calculating ΔC (ΔC =C1-C0) and ΔC％ (ΔC％ =ΔC/C0).Finally,the paired t test and one-way analysis of variance were performed using SPSS 17.0.Results As for porcine eyes,the ΔC％ of the control group was (17.83 ± 3.84) ％ while the experimental group was (44.00 ± 6.44) ％;as for rabbit eyes,the ΔC％ of the control group was (15.50 ± 2.93) ％,while the experimental group was (31.67 ±6.54)％;as for bovine eyes,the ΔC％ of the control group was (11.67 ± 1.17)％,while the experimental group was (37.17 ± 4.48)％.The ΔC％ in the experimental group was significantly increased when compared with the control group in three animals,with significant difference (all P ＜ 0.05).There was no statistical difference in ΔC％ of three experimental groups among different kinds of animals (all P ＜ 0.05).Conclusion Fasudil can improve outflow facility in enucleated eyes of animals,and it can redistribute aqueous humor drainage to a wider area through directly regulating the cytoskeleton of cells and matrix,resulting in increased coefficient of outflow facility.

Advances in stress response of DNA binding with one finger transcription factor family genes in graminaceous plants / 生物工程学报

Transcription factor is a key trans-acting factor to mediate stress response by regulating gene expression. Plants have developed a series of mechanisms to modulate development, stress response, signaling and disease resistance at transcription level. DNA binding with one finger (DOF), containing one C₂-C₂ zinc finger domain, is a special plant transcription factor. Specifically, the conserved domain at N-terminus of DOF has multiple functions, including interacting with DNA and protein, which could be involved in plant development and stress response. Although many DOF family genes are characterized in plant stress response, it is not clear if DOF genes have functions in cereal plants. In the present paper, the role of DOF family genes on cereal plants were discussed based on a comprehensive phylogenetic relationship analysis, expression profiles in different tissues and various environmental conditions. The results obtained here will provide an important reference for further understanding the mechanism of gramineous crops in stress resistance.

The current status of the patient safety culture in intensive care unit nurses and influencing factors / 中国实用护理杂志

Objective To investigate the current status of cognitive level of patient safety culture in intensive care unit (ICU) nurses and analyze the influencing factors.Methods Totally 373 ICU nurses were investigated with the general information questionnaire,the Hospital Survey on Patient Safety Culture instrument (HSOPSC),the Report Barriers Questionnaire and the Condition of Work Effectiveness Questionnaire (CWEQ-Ⅱ).Results The mean rate of positive response on safety culture dimensions was 68.08％ in ICU nurses,Organizational learning-continuous improvement was the safety culture dimension with the highest positive response(89.95％),the lowest positive response was staffing(35.53％).Multiple regression analysis showed that the meaning of report,punitive culture,resources,opportunity,formal empowerment and ICU department were influencing factors of patient safety culture (P ＜ 0.05).Conclusions The level of patient safety culture in ICU nurses was medium,it remains a further improvement.Nursing managers should take targeted measures according to the influencing factors to enhance the cognitive level of patient safety culture in ICU nurses,strengthen the consciousness of safety culture,and improve the quality of intensive care.

Clinical and pathological features of IgA nephropathy with macrohematuria in history / 中华肾脏病杂志

Objective To investigate the clinical and pathological characteristics of IgA nephropathy (IgAN) with macrohematuria (MH).Method 1512 consecutive patients with biopsyproven IgAN diagnosed from January 2006 to December 2011 were enrolled,and divided into MH group and control group respectively,according to whether there existed episodes of MH before renal biopsy.The clinical and pathological characteristics were compared between two groups.Patients in MH group were then divided into three groups according to the interval from the last episode of MH to renal biopsy to clarify the concomitant clinicopathological changes associated with occurrence of MH.Results The rate of MH in history was 22.1％.MH group patients had significantly lower serum creatinine,slighter proteinuria,lower prevalence of hypertension and heavier microhematuria than control group (all P ＜ 0.001).The prebiopsy durations were similar in two groups (P=0.627).In MH group,chronic pathological indicators,including global/segmental sclerosis,tubule atrophy/interstitial fibrosis were all slighter (all P＜ 0.001),whereas activity indicators,including necrosis lesions,crescents and mesangial proliferation were all more severe compared with control group (all P ＜ 0.05).Those who underwent renal biopsy within 30 days of the last episode of MH had more severe proteinuria and microhematuria,higher prevalence of necrosis lesions,more severe crescents formation,and endothelial proliferation (all P ＜ 0.05).Conclusions IgAN patients with MH in history have relatively milder clinical and chronic pathological manifestations,however more active pathological changes especially in those who suffer episode of MH recently.

Meta analysis on risk factors of chronic obstructive pulmonary disease among Chinese people in mainland / 重庆医学

Objective To explore the main risk factorrelated to the incidence of chroniobstructive pulmonary disease (COPD) among Chinese people in mainland so ato provide the basifothe decision making on COPD prevention .MethodEighpublished literatureof case-control studieon the risk factoof COPD were collected and analyzed quantitatively and synthetically by the metanalysi;the RevMan 5 .2 software waadopted to perform the consistency tesand calculate the pooled oddradio (OR) value and 95% CI .The risk factorwith the Ovalue＞1 were performed the calculation of population attributable risk pro-portion(PARP) .ResultThe pooled oddradio value,95% CI and PARP were smoking OR= 2 .12(1 .58 -2 .86) ,PARP=28 .16% ;occupational exposure OR=1 .82(1 .04-3 .18) ,PARP=11 .60% ;family history of respiratory disease OR=1 .82(1 .36-2 .44) ,PARP=14 .25% ;coal and biomasfuel focooking and heating OR=3 .29(1 .01-10 .67) ,PARP=41 .29% ;low body masindex OR=2 .58(1 .78-3 .74) ,PARP=5 .71% ;low educational degree OR=1 .24(1 .02 -1 .50) ,PARP=12 .93% ;history of re-currenrespiratory tracinfection during childhood OR= 2 .10 (0 .99 -4 .47 ) ,PARP= 13 .39% ;passitive smoking OR= 1 .00 (0 .89-1 .11) .Conclusion Smoking ,occupational exposure ,family history of respiratory disease ,coal and biomasfuel focooking and heating ,low body masindex ,low educational degree and history of recurrenrespiratory tracinfection during childhood are the risk factorinfluencing the incidence of COPD among Chinese people in mainland .

Therapeutic Observation of Comprehensive Acupuncture for Wet Age-related Macular Degeneration / 上海针灸杂志

ObjectiveTo systematically and objectively evaluate the therapeutic efficacy of comprehensive acupuncture treatment in managing wet age-related macular degeneration (ARMD), and to seek a standard acupuncture protocol for this disease, for promoting the application in clinic.MethodTotally 37 patients (67 eyes) were observed to compare the vision, Amsler grid and rating scale of quality of life for visual impairment before and after the 3-month treatment. The clinical efficacy was then evaluated. ResultThe vision and score of quality of life were significantly changed after intervention (P<0.05); according to Amsler grid, the total improved rate was 79.2% and the total effective rate was 77.6%; age and disease duration were negatively correlated with the therapeutic efficacy (P<0.05).ConclusionComprehensive acupuncture treatment can significantly improve the vision and quality of life in patients with wet ARMD, and the younger the patients and the shorter the disease duration, the higher the therapeutic efficacy.

Cigarette smoke extract promotes senescence by reducing expression of HDAC2 in C2C12 myoblasts / 中国病理生理杂志

AIM:To investigate whether the cigarette smoke extract (CSE) causes senescence of C2C12 myo-blasts and the relationship between senescence and histone deacetylase 2(HDAC2).METHODS: Murine C2C12 cells were induced to differentiate into myoblasts.The HDAC2 activator and inhibitor were used to investigate the effects of CSE in the myoblasts on cell senescence and the expression of HDAC2.The expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blot, respectively, and the positive cell rate of β-galactosidase staining for cell senescence was also detected.RESULTS:The optimal concentration of CSE was 60 mL/L and the intervention time was 24 h.After the intervention of CSE, the positive cell rate ofβ-galactosidase staining was increased, accompanied with the reduction of HDAC2 expression at mRNA and protein levels.The expression of HDAC2 at mRNA and protein levels was increased by 4, 5, 6, 7-tetrabromobenzotriazole (TBB), accompanied with the reduction of positive cell rate ofβ-galacto-sidase staining.Furthermore, when HDAC2 expression at mRNA and protein levels was reduced by HDAC2 inhibitor valp-roic acid, the positive cell rate of β-galactosidase staining was increased.CONCLUSION: CSE promotes the senescence by reducing the expression of HDAC2 in C2C12 myoblasts.

Biotin-streptavidin system-mediated folate receptor-targeted quantum dot in vitro imaging of ovarian cancer SKOV3 cells / 中国肿瘤临床

Objective:To develop a biotin-streptavidin system (BAS)-mediated folate receptor (FR)-targeted quantum dot (QD) fluorescent probe and preliminarily validate the targeting ability and signal amplification effect of the probe. Methods: Streptavidin (SA) was covalently coupled with QD through the active ester method;the physical characteristics of the prepared QD-SA were veri-fied. Biotinylated folate was synthesized through the carrier bovine serum albumin using the same method and then reacted with QD-SA to form the special probe. The probe was used to identify SKOV3 cells and FR-negative A549 cells to verify its targeting speci-ficity. QD-SA was used as the contrast. SKOV3 cells were imaged using the BAS-mediated FR-targeted QD probe with a biotinylated folate incubation time of 1 or 4 h. Various reaction times were also tested between the probe and the QD-FA that was formed without BAS mediation. Results:The BAS-mediated FR-targeted QD probe specifically recognized FR-positive SKOV3 cells. The probe ob-tained higher fluorescent intensity after 4 h than after 1 h of biotinylated folate incubation. The BAS-mediated FR-targeted QD probe al-so had a stronger fluorescent signal than the QD-FA probe. Conclusion:The proposed probe presents a great potential in the early diag-nosis of ovarian cancer because of its high specificity and sensitivity.

Development of a concentration method for detection of tobacco mosaic virus in irrigation water / 中国病毒学·英文版

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantiifcation of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was conifrmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 102 viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.

Audiological assessment value of click-evoked auditory brainstem response combined with single stimulation of the auditory steady-state evoked response on normal young people / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#Analysis of normal young people multiple stimuli ASSR (m-ASSR), single stimuli ASSR (s-ASSR), C-ABR, PTA threshold, exploratory investigation of relevance of hearing test combination thresholds (C-ABR combines with 0.5, 1.0 kHz s-ASSR) predict behavioral thresholds, using a within-subjects design.@*METHOD@#Forty-three normal hearing adults (86 ears) were treated with pure tone audiometry (PTA), m-ASSR, 0.5, 1.0 kHz s-ASSR, C-ABR. Data were analyzed by SPSS 18.0 for bivariate correlation analysis.@*RESULT@#There was significant correlation between thresholds of hearing test combination and PTA,and the correlation coefficient (r) were 0.803, 0.650, 0.649, 0.734 at 0.5, 1.0, 2.0 and 4.0 kHz; while the correlation coefficient (r) between thresholds of m-ASSR and PTA were 0.48, 0.64, 0.57, 0.64.@*CONCLUSION@#There is significant correlation between thresholds of hearing test combination and PTA, hearing test combination can be more accurate, more reliable assessment of adults with normal hearing threshold level.

Agrobacterium-mediated genetic transformation of secondary somatic embryos in alfalfa / 生物工程学报

We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and npt II gene as selectable marker. The infected primary embryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated npt II gene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.