Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis / 生物工程学报

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

Reliability and Validity of Extended Barthel Index for Stroke Patients / 中国康复理论与实践

Objective:To study the reliability and validity of extended Barthel Index (EBI) in the assessment of activities of daily living (ADL) for stroke patients. Methods:From January, 2018 to October 2019, 136 stroke inpatients from the First Affiliated Hospital of Soochow University were conveniently sampled. They were assessed with EBI by rater A twice within 48 hours after admission, with interval above twelve hours; and by rater B within 24 hours. They were also assessed with modified Barthel Index (MBI) and Function Independence Measure (FIM) within 48 hours after admission by rater C. The intraclass correlation coefficient (ICC) of retest and inter-rater of EBI were calculated, as well as the Cronbach's α coefficient, Spearman correlation coefficient between scores of items and total, and Spearman correlation coefficient of EBI to MBI or FIM. The construct validity of EBI was tested with factor analysis. Results:The retest ICC of items was 0.766 to 0.953; the retest ICC of motor and cognitive items was 0.938 and 0.845, respectively; the retest ICC of total was 0.891. The inter-rater ICC of items was 0.728 to 0.976, the inter-rater ICC of motor and cognitive items was 0.948 and 0.717, respectively; the inter-rater ICC of total was 0.866. The Cronbach's α coefficient of EBI was 0.885. The correlation coefficients were above 0.4 in all the items (P < 0.001) to the total, except the item of vision (r = 0.215, P < 0.05). The correlation of items of EBI to MBI was significant (r = 0.648 to 0.958, P < 0.01), as well as those of EBI to FIM (r = 0.722 to 0.976, P < 0.01). Four components were extracted with principal component analysis, accumulated to 72.19% of the variable; after the vision item was removed, three principal components were extracted, accumulated to 69.09% of the variable. The component 1 was mainly about sphincter control and some advanced brain functions (communication and social cognition), component 2 mainly about ADL related to lower extremities, and component 3 mainly about ADL related to upper extremities. Conclusion:EBI is reliable and valid in the assessment of ADL for stroke patients.

Effect of aerobic exercise on blood lipid and ICAM-1,LFA-1 in hyperlipidemia rats / 中国医师杂志

Objective To observe aerobic exercise on blood lipid and intercellular adhesion molecule-1 (ICAM-1),lymphocyte function-associated antigen-1 (LFA-1) in hyperlipidemia rats.Methods 120 male Sprague Dawley (SD) rats were randomized into the 4 groups (n =30):normal diet group (control group),high fat group (HF group),HF + SBC-115076 group [SBC-115076,8 mg/kg · w)] and HF + aerobic exercise group.Rats in HF group and HF + SBC-115076 group did not exercise during feeding and lived in normal cage.Rats in HF + SBC-115076 group recieved PCSK9 inhibitor SBC-115076 (8 mg/kg) injection once a week for 8 weeks.HF + aerobic exercise group underwent load-free swimming training 6 days a week for 8 weeks.The rats were sacrificed 8 weeks later.The serum levels of triglyceride (TG),total cholesterol (TC),low density lipoprotein (LDL),high density lipoprotein (HDL),ICAM-1 and LFA-1 were measured by apical blood sampling.Hematoxylin-eosin (HE) staining of thoracic aorta to observe pathological changes of aorta.Results After modeling,there was no significant difference in the food intake levels of the hyperlipidemia rats (P ＞ 0.05).The body weight of HF rats and HF + SBC-115076 rats increased significantly than HF + aerobic exercise rats (P ＜ 0.01).The levels of serum lipid profile,ICAM-1,and LFA-1 were significantly different between HF rats and control rats.Compared with HF rats,serum levels of TG,TC,LDL,ICAM-1 and LFA-1 in HF + SBC-115076 group and HF + aerobic exercise group were significantly lower and HDL levels were significantly higher.HE staining showed that the intimal thickening was significantly improved in HF + aerobic exercise group with less endothelial damage.Conclusions Aerobic exercise can reduce the levels of serum TG,TC,LDL and increase HDL level in rats with hyperlipidemia.It can improve the intimal thickening and endothelial damage caused by high-fat diet by reducing the expression levels of serum ICAM-1 and LFA-1,and has anti-atherosclerosis effect.

Stroke literacy in Singapore: data from a survey of public housing estate residents

<p><b>INTRODUCTION</b>Knowledge of stroke symptoms is associated with seeking medical attention early, and knowledge of risk factors is an essential factor in stroke prevention. In this study, we evaluated the level of stroke literacy in Singapore.</p><p><b>MATERIALS AND METHODS</b>A cross-sectional study of Singapore citizens and permanent residents aged 21 years and above was conducted in a public housing estate. Participants were randomly sampled using multi-stage stratified sampling. Assessment of awareness of stroke symptoms and risk factors was performed using open-ended questions. In total, 687 respondents were recruited, with a response rate of 69.7%.</p><p><b>RESULTS</b>Overall, 52.4% of respondents identified the brain as the source of pathology, and 47.6% could cite at least 1 of the 3 FAST symptoms (facial droop, arm weakness and speech difficulty), while 40% could name 2 or more of 7 established risk factors for stroke (high blood pressure, high cholesterol, cigarette smoking, diabetes mellitus, older age, previous heart attacks and stroke). Respondents at higher risk of stroke (older individuals and those with stroke risk factors) did not have greater awareness of stroke symptoms and risk factors. The majority of respondents reported they would seek immediate medical care if they experienced stroke symptoms. Only 59.4% knew the emergency ambulance service telephone number.</p><p><b>CONCLUSION</b>In a sample of Singaporean adults residing in a public housing estate, we found evidence of poor stroke literacy, highlighting the need for comprehensive population-based education efforts. There is a role for opportunistic education among those at higher risk of stroke.</p>

Rash in Psychiatric and Nonpsychiatric Adolescent Patients Receiving Lamotrigine in Korea: A Retrospective Cohort Study

OBJECTIVE: Lamotrigine is a widely used medication for psychiatric disorders and epilepsy, but the adverse effects of this drug in adolescent Korean patients have not yet been investigated. In the present study, we sought to compare the incidence and impact of lamotrigine-induced skin rashes and different pattern of adverse events in psychiatric and nonpsychiatric adolescent patients. METHODS: Using a retrospective cohort design, all of the charts were reviewed for adolescents (13 to 20 years old), treated with lamotrigine during the previous 2 years in the Child and Adolescent Psychiatric Clinic and Pediatric Neurologic Clinic of the Ulsan University Hospital in South Korea. RESULTS: Of the 102 subjects, 23 patients developed a skin rash. All of these rashes were observed within 7 weeks of the initiation of the lamotrigine therapy. Only one subject developed a serious rash, which was diagnosed as Stevens-Johnson syndrome. Although the psychiatric subjects were administered statistically lower doses of lamotrigine during weeks 1 through 5 and at week 12, the likelihood of developing a rash was not significantly different between the psychiatric and nonpsychiatric patients. CONCLUSION: Careful dose escalation and close observation of side effects for the first 7 weeks of treatment is important. The present study reveals the tolerability of lamotrigine in an adolescent population, although a double-blind, controlled trial is needed to confirm these findings.

Mechanism of arginine deiminase activity by site-directed mutagenesis / 生物工程学报

Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

Switching from Methylphenidate-Immediate Release (MPH-IR) to Methylphenidate-OROS (OROS-MPH): A Multi-center, Open-label Study in Korea

OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of methylphenidate HCL OROS extended-release (OROS-MPH) among children with attention deficit hyperactivity disorder (ADHD) who had been previously treated with methylphenidate HCL immediate-release (MPH-IR). METHODS: The sample included 102 children aged 6-12 (9.4+/-2.6) years who had been diagnosed with ADHD according the criteria of the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-IV; American Psychiatric Association, 1994) and who were attending seven centers in Korea. All participants had been medicated with a stable dose of MPH (10-60 mg/day) for at least 3 weeks before entry into the study. Doses of OROS-MPH were comparable to daily doses of MPH. Efficacy was assessed at baseline (day 0) and at day 28 with the Inattentive-Overactive with Aggression (IOWA) Conners Rating Scale, which was completed by parents/caregivers and teachers, the Peer Interaction Rating Items, which were completed by teachers, and the Clinical Global Impression (CGI) scale, which was completed by child psychiatrists. Paired t-tests were used, and P-values were set at the 0.05 level. RESULTS: Of the subjects, 92.2% were boys and 79.4% were students in the first to fourth grades of elementary school. 72% were diagnosed with the combined type of ADHD, 23% were diagnosed with the inattentive type, and 5% were diagnosed with the hyperactive-impulsive type. The results of the parents' responses to the Inattention/Hyperactivity (I/H) and Oppositional/Defiant (O/D) subscales of the IOWA Conners scale indicated statistically significant improvement in childrens behavior after 4 weeks of treatment with OROS-MPH (t=6.28, p<.001, t=4.12, p<.001). However, the teachers' responses to the Conners I/H and O/D subscales indicated no significant improvement at 4 weeks. The teachers also reported no significant improvements under the OROS-MPH compared with the MPH-IR condition with respect to peer interactions. Scores on the CGI scale showed that 46.1% of children with ADHD were rated by psychiatrists as "minimally improved", 27.5% as "much improved," 1.0% as "very much improved," 3.9% as "minimally worse," and 16.7% as showing "no change". Children exhibited significantly fewer tics with OROS-MPH treatment than with MPH-IR treatment (19.6% vs. 27.7%). We found no differences between in sleep and appetite problems according to medication. CONCLUSION: The results of this study indicated that an MPH-IR regimen can be successfully changed to a once-daily OROS-MPH regimen without any serious adverse effects. The changes in parent/caregiver IOWA Conners ratings suggested that OROS-MPH improved the control of symptoms after school, a finding that is consistent with the 12-h duration of action of this medication. Because the therapeutic effect of OROS-MPH is sufficiently longer than that of a b.i.d. dose of MPH-IR, OROS-MPH had significant positive effects on oppositional/defiant behavior in addition to its effects on the core symptoms of ADHD.

The Side Effects and Correlates of OROS-Methylphenidate in the Treatment of Children and Adolescents with ADHD

OBJECTIVES: The aim of this study was to investigate the effect of the clinical and demographic variables such as body weight, dosage, family history of attention-deficit hyperactivity disorder (ADHD), and psychiatric co-morbidity on the side-effects of OROS-Methylphenidate (OROS-MPH), and to evaluate the relationship between drug response and side effect severity. METHODS: A total of 144 children (ages 6-18) with diagnosed ADHD were treated with OROS-MPH. Children were examined at baseline and after 1, 3, 6, 9, and 12 weeks of each treatment condition. The stimulant drug side effect rating scale (SERS), pulse rate, systolic blood pressure, diastolic blood pressure, and electrocardiogram (ECG) were evaluated to assess side effect profiles. Changes in these parameters from baseline were examined and analyzed. RESULTS: Anorexia (30.95%) and insomnia (13.10%) were the most commonly reported side effects during this study. Insomnia and loss of appetite score increased at one week follow-up, but was sustained or decreased as treatment progressed. Small but significant increases in pulse rate and diastolic blood pressure were observed during treatment; however, no clinically meaningful changes in ECG parameters were noted during the study. Low body weight, high dosage of OROS-MPH, and family history of ADHD were associated with cardiovascular side effect. In contrast, there was no significant relationship between OROS-MPH treatment response and the severity of side effect and no difference resulted between the responder and non-responder groups with respect to OROS-MPH dosage in the 12 weeks of follow-up. CONCLUSION: To the best of our knowledge, this study is the first Korean study to investigate comprehensive side effect profiles and their correlates in OROS-MPH treatment for ADHD children. OROS-MPH was well tolerated with no clinically significant side effects during the treatment period. In conclusion, low body weight, high dosage of OROS-MPH, and family history of ADHD could be used as predictive factors in increasing pulse rate and blood pressure.

Recent trend of nonaqueous enzymology and biocatalysis in nonaqueous media / 生物工程学报

With the rapid development of industrial biotechnology, breakthrough in enzymology and biocatalysis has been made in recent years, especially in areas of stability and activity of enzyme in nonaqueous media, screening, construction and modification of solvent-tolerant biocatalysts, as well as the development of green solvent with excellent biological and environmental compatibility. Recent trend and future focus include: in silico virtual screening and construction of solvent-tolerant biocatalysts based on bioinformatic technology, modification and construction of native solvent-tolerant biocatalysts, the development of environmental friendly green solvent such as ionic liquids.

Breeding of monofluoroacetate-resistant strains of Actinobacillus succinogenes and the mechanism based on metabolic flux analysis / 生物工程学报

Succinic acid has received a great deal of attention as an important green chemical stock for the manufacture of synthetic resins, biodegradable polymers and chemical intermediates. In this paper, the breeding mechanism of Actinobacillus succinogenes based on metabolic flux analysis was demonstrated to improve the yield of succinic acid by fermentation. After the NTG treatment, mutants from A. succinogenes CGMCC 1593 which were able to grow in medium containing concentrations of about 50-100 mmol/L of sodium monofluoroacetate were obtained. Among them, a mutant SF-9 was selected for producing more succinic acid and less acetic acid. When fermentations were conducted in a 5 L bioreactors, the final succinic acid concentration of SF-9 (34.8 g/L) increased 23.4%, and the mass ratio of succinic acid/acetic acid increased from 3.3 to 9 compared with those of the parent strain. Based on the metabolic flux analysis of A. succinogenes, PEP was found to be a key node which has an important effect on the production of succinic acid, and the flux ratio of by-productions (acetic, formic, lactic acid) was influenced by PYR node. Compared with the parent strain, the flux to succinic acid of mutant (A. succinogenes SF-9) was significantly increased, while the flux to by-productions had an obvious decline. Therefore, PEP and PYR are not rigid nodes in the metabolic regulation of A. succinogenes.

Screening for Attention Deficit/Hyperactivity Disorder for Children in Seoul / 신경정신의학

OBJECTIVES: The purpose of this study is to investigate a cut-off value for screening children with ADHD in community based sample using the Korean Child Behavior Checklist (K-CBCL) and the Korean ADHD Rating Scale (K-ARS). METHODS: Children aged 6 to 8 (n=661) participated in this study. The K-CBCL and the K-ARS were used as the screening instruments. Diagnoses were confirmed through the Diagnostic Interview Schedule for Children Version IV (DISC-IV). RESULTS: Of the 189 subjects who underwent the DISC-IV, 26 subjects were diagnosed as ADHD. In the K-CBCL, scores in the attention problems, externalizing problems and total behavioral problems were positively correlated with the total score of the K-ARS. The K-ARS-Parent version (K-ARS-P) and above three scales in the K-CBCL showed significant screening validity in the ROC curve analysis. The reasonable level of sensitivity, specificity, and negative predictive value were obtained in the total scores > or =90th percentile in the K-ARS-Parent and > or =60T scores in the K-CBCL attention problems. The optimal level of specificity, positive predictive value and negative predictive value were obtained when combined the K-CBCL (> or =60T scores in the attention problems and > or =63T scores in the total problems) and the total scores > or =90th percentile in the K-ARS-P. CONCLUSION: The combined use of the K-CBCL and the K-ARS-P may serve as useful screening methods for ADHD children.

Effect of Different Carbon Sources on Succinic Acid Production of Actinobacillus succinogenes and Metabolic Flux Analysis / 微生物学通报

In anaerobic bottles fermentation,glucose,fructose,xylose,lactose,maltose,sucrose and sugar alcohols could be used to produce succinic acid with Actinobacillus succinogenes. When sorbitol was utilized as the carbon source in the batch fermentation,more succinate and ethanol were produced compared with those using glucose,while producing less acetate and formate. The metabolic flux analysis results showed that the flux partitioning at PEP node was stable when glucose was replaced by sorbitol,but the flux partitioning at PYR and AcCoA nodes changed a lot because more reducing power(NADH) was generated to meet the more requirement the synthesis of succinate and ethanol.

Environmental factors affecting the succinic acid production by Actinobacillus succinogenes CGMCC 1593 / 生物工程学报

Actinobacillus succinogenes is a promising candidate for the production of bio-based succinic acid. Previously, we isolated a succinic acid-producing strain Actinobacillus succinogenes CGMCC 1593 from bovine rumen. In this paper, the influence of the environmental factors such as gas phase, pH, ORP, on succinic acid production by A. succinogenes CGMCC 1593 was studied. The results showed that CO2 was the optimum gas phase for anaerobic fermentation ofA. succinogenes CGMCC 1593 as well as one of the substrate for the succinic acid synthesis. Using MgCO3 as a pH regulator, the pH was maintained within 7.1-6.2 during the anaerobic fermentation for the cell growth and acid production of A. succinogenes CGMCC 1593. Our results showed that low initial ORP was disadvantageous for the growth of A. succinogenes CGMCC 1593 and an ORP of -270 mV was demonstrated to be beneficial to the succinic acid production. By adding Na2S.9H2O to decrease ORP to -270 mV at the end of exponential growth phase in batch culture of A. succinogenes CGMCC 1593, the succinic acid concentration reached 37 g/L and the yield of succinic acid was 129% at 48 h. This work might provide valuable information for further optimization of succinic acid fermentation by A. succinogenes CGMCC 1593.

Breeding Actinobacillus succinogenes with Acid-tolerance by Genome Shuffling / 微生物学通报

A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain.After UV-EMS and UV-DES treatments respectively,seven mutated strains with subtle improvements in acid tol-erance were obtained,and were subjected for recursive protoplast fusion.Through three rounds of genome shuffling,four shuffled strains with both higher yield and acid tolerance were obtained.The shuffled strain namely F3-21 could even survive at pH 5.2.The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here.After 48 h of shake-flask fermentation,the succinic acid concentration of F3-21 was 48% higher than that of the parent strain.When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0,the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L,which was increased by 45% compared with that of the parent strain(26.2 g/L).While pH was controlled at 6.5~7.0,the production of succinic acid in 32 h fermentation attained 40.7 g/L.When F3-21 was carried out in fed-batch fermentation,succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation.These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A.succinogenes CGMCC 1593.

A Case of Anterior uveitis Following Influenza Vaccination

PURPOSE: To report one case of anterior uveitis following influenza vaccination in immunecompromised patient. METHODS: A 63-year-old woman with anterior uveitis following influenza vaccination 9 days after ovarian cancer chemotherapy was examined systematically and treated with 1% prednisolone eyedrop and dexamethasone subconjunctival injection. RESULTS: At the first visit, the corrected vision and intraocular pressure were 0.3 and 23 mmHg in both eyes. And, corneal edema and inflammatory cells in anterior chamber and vitreous were found, but there was no abnormal finding in fundus photography, fluorescein angiography and systemic evaluation. Although the response to steroid treatment was slow, she got better ophthalmologically and no problem in follow checkup 6 months later. CONCLUSIONS: We report a case of anterior uveitis following influenza vaccination in an immunocompromised patient after chemotheraphy and recommend to be cautious in influenza vaccination in the suspicious case of immunecompromized state.

Cloning of the Phosphatidylserine Synthase Gene and Its Secretive Expression in Bacillus subtilis / 中国生物工程杂志

The gene encoding the phosphatidylserine synthase in Escherichia coli K12 Sgal-(ExPASy P23830) was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive shuttle vector pBES of Bacillus subtilis, which was constructed in the lab, and the recombinant plasmid pBES-pss was transformed into competent cells of the Bacillus subtilis strain DB104. The positive transformant DB104 (pBES-pss) was grown on Bacillus subtilis common fermentation medium, which contained 30?g/ml kanamycin. After 2 hours cultivation, sucrose was added and increased to the final concentration of 2% for induction and this phosphatidylserine synthase was secreted into the medium. The result of SDS-PAGE showed that the molecular weight of the protein was 52kDa and the result of enzyme coupling colorimetric method showed that the enzyme activity was 1.50U/ml. The recombinant Bacillus subtilis has increased the yield of phosphatidylserine synthase which will be used for industrial biosynthesis of phosphatidylserine.

Two Cases of Boston (Dohlman-Doane) Type I Keratoprosthesis

PURPOSE: This article reports two cases of Boston (Dohlman-Doane) Type I Keratoprosthesis procedures that were performed for the first time in Korea. METHODS: Using the Boston Type I keratoprosthesis, we implanted ultimately these to two cases of corneal blinded patients. RESULTS: Two Boston (Dohlman-Doane) Type I Keratoprosthesis procedures were performed successfully. The first case had poor visual prognosis due to preexisting optic neuropathy and vitreous hemorrhage, but was showed better for cosmetic effect. The second case had maintained an uncorrected visual acuity of 0.1 at postoperative 3 months. CONCLUSIONS: We hope to perform additional procedures and long-term follow-ups to further evaluate this new surgical technique for stability and efficiency.

Correlation between Retinal Nerve Fiber Layer Thickness and Visual Field in Normal Tension Glaucoma

PURPOSE: This study was performed to evaluate the validity of optical coherence tomography 3000 (OCT3) in the diagnosis of normal tension glaucoma (NTG). METHODS: One-hundred and eighty eyes with NTG or glaucomatous optic disc changes by direct ophthalmoscope and 51 normal eyes were included. Mean deviation (MD) and pattern standard deviation (PSD) were acquired by the Humphrey visual field analyzer. Mean retinal nerve fiber layer (RNFL) thickness was measured by OCT3. We analyzed the relationships between MD and mean RNFL thickness, PSD, and mean RNFL thickness. We also analyzed differences in the change of mean RNFL thickness according to the change of visual field defect progression. RESULTS: MD and PSD were proportional to the mean RNFL thickness, using the Pearson correlation coefficient: 0.418 (p=0.001) and -0.397 (p=0.002), respectively. There was a significant decrease in the mean RNFL thickness according to the visual field defect severity. Particular, the RNFL thickness of the superior and inferior quadrants were significantly decreased in the severe visual field defect groups. The difference between the mean RNFL thicknesses in pre-perimetric glaucoma between the normal eye and early glaucoma groups was not statistically significant. CONCLUSIONS: The mean RNFL thickness measured by OCT3 could be considered as valid parameter for the diagnosis of NTG. For early diagnosis of glaucoma, however, visual field test must be considered with OCT3.