The Value of VR-PVEP in Objective Evaluation of Monocular Refractive Visual Impairment.

OBJECTIVES: To study the virtual reality-pattern visual evoked potential (VR-PVEP) P100 waveform characteristics of monocular visual impairment with different impaired degrees under simultaneous binocular perception and monocular stimulations. METHODS: A total of 55 young volunteers with normal vision (using decimal recording method, far vision ≥0.8 and near vision ≥0.5) were selected to simulate three groups of monocular refractive visual impairment by interpolation method. The sum of near and far vision ≤0.2 was Group A, the severe visual impairment group; the sum of near and far vision <0.8 was Group B, the moderate visual impairment group; and the sum of near and far vision ≥0.8 was Group C, the mild visual impairment group. The volunteers' binocular normal visions were set as the control group. The VR-PVEP P100 peak times measured by simultaneous binocular perception and monocular stimulation were compared at four spatial frequencies 16×16, 24×24, 32×32 and 64×64. RESULTS: In Group A, the differences between P100 peak times of simulant visual impairment eyes and simultaneous binocular perception at 24×24, 32×32 and 64×64 spatial frequencies were statistically significant (P<0.05); and the P100 peak time of normal vision eyes at 64×64 spatial frequency was significantly different from the simulant visual impairment eyes (P<0.05). In Group B, the differences between P100 peak times of simulant visual impairment eyes and simultaneous binocular perception at 16×16, 24×24 and 64×64 spatial frequencies were statistically significant (P<0.05); and the P100 peak time of normal vision eyes at 64×64 spatial frequency was significantly different from the simulant visual impairment eyes (P<0.05). In Group C, there was no significant difference between P100 peak times of simulant visual impairment eyes and simultaneous binocular perception at all spatial frequencies (P>0.05). There was no significant difference in the P100 peak times measured at all spatial frequencies between simulant visual impairment eyes and simultaneous binocular perception in the control group (P>0.05). CONCLUSIONS: VR-PVEP can be used for visual acuity evaluation of patients with severe and moderate monocular visual impairment, which can reflect the visual impairment degree caused by ametropia. VR-PVEP has application value in the objective evaluation of visual function and forensic clinical identification.

[Distribution of Formic Acid after Methanol Intoxication in Rats].

OBJECTIVE: To investigate concentration and distribution in blood and tissues of formic acid after methanol intoxication in rats. METHODS: The Sprague-Dawley rats were divided into groups for control group and 3-day and 7-day intoxication treatment groups. The experimental groups were administered methanol by gavage with the initial dose of 8 mL/kg and followed with 4 mL/kg supplemental dose 24 h later. After 3 days and 7 days later, rats were killed by decapitation. Then samples of cardiac blood, liver, kidney, brain, heart and stomach of each group were collected. Formic acid concentrations were detected by high performance liquid chromatography. RESULTS: Formic acid concentrations in tissues were higher than in blood. Compared with 3-day intoxication group, there was an increase formic acid of concentration in brain and stomach in 7-day intoxication group, while a decrease in liver and kidney (P < 0.05). CONCLUSION: High performance liquid chromatography could be used to accurately detect formic acid. As the metabolite of methanol, formic acid accumulates in rat blood and tissues after intoxication and the concentrations in organs and tissues are obviously higher than in blood.

[Correlation between contrast vision and sweep visual evoked potential acuity].

OBJECTIVE: To compare the correlation between contrast vision (LV) and sweep visual evoked potential acuity (SVEP-A) among people with emmetropia, mild myopia, and moderate myopia. METHODS: The CV and SVEP-A were tested individually in 96 eyes from healthy young volunteers, including 37 eyes of emmetropia, 27 eyes of mild myopia, and 32 eyes of moderate myopia. The statistic analysis was done by ANOVA analysis and rank sum test. RESULTS: (1) With the decrease of contrast, CV and SVEP-A decreased in every group. (2) At 100% contrast, the difference of CV between emmetropia and mild myopia had statistical significance (P<0.05). At 100%, 25% and 10% contrast, the difference of CV between emmetropia and moderate myopia had statistical significance (P<0.05). (3) In the same group, the difference of 100% and 25% contrast had statistical significance (P < 0.05). So was between 100% and 10% contrast. (4) At 100% and 10% contrast, the difference of CV and SVEP-A had statistical significance (P < 0.05). CONCLUSION: The CV of myopia relates to many factors including ametropia and fundus lesions. The correction of ametropia is important to the values of CV and SVEP-A.

[Changes of oscillatory potentials of electroretinogram after methanol intoxication in rats].

OBJECTIVE: To study the characters of oscillatory potentials (OPs) of electroretinogram (ERG) after methanol intoxication in rats. METHOD: The SD rat models of methanol intoxication were established and divided into control group, 3-day intoxication group, 7-day intoxication group. The changes of OPs of ERG were recorded in a dark room. RESULTS: The total amplitudes of 3-day and 7-day intoxication groups decreased approximately 50% compared with that of the control group, while the schedule delayed approximately 16% and 61%, respectively. CONCLUSION: The characters of methanol intoxication in rats included delay in schedule and decline in the total amplitude of OPs.

[Expression and mechanism of alphaB-crystallin in retina and extraocular tissues and organs].

alphaB-crystallin is the structural protein of vertebrate lens, which is widely expressed in non-lens tissue. As one of the heat shock protein family members, alphaB-crystallin possesses biological properties of molecular chaperones and anti-apoptotic effects. Multi-factor injuries, such as retinopathy, inflammation and nervous system diseases, have a closely relationship with alphaB-crystallin. This paper reviews the research progress of the expression and mechanism of alphaB-crystallin in retina and extraocular tissues and organs.

[Comparison of anterior chamber angle examination by UBM, SL-OCT and gonioscopy].

OBJECTIVE: To compare the agreement of anterior chamber angle examination by ultrasound biomicroscope (UBM), slit lamp optical coherence tomography (SL-OCT), and gonioscopy in angle recession and angle closure. METHODS: The anterior chamber angle was measured with UBM, SL-OCT and gonioscopy in turns for temporal, nasal, superior and inferior quadrant in the same dark room. The results were compared with the agreement of the three methods in angle recession and angle closure by χ2 test and Kappa test. RESULTS: There were no statistically significant differences of the three methods in testing angle closure and angle recession (P>0.05). The consistency of UBM and gonioscopy was better (Kappa value of 0.882) than that of SL-OCT and gonioscopy (Kappa value of 0.624). CONCLUSION: When testing angle recession, UBM is better than SL-OCT with gonioscopy as the standard. When testing angle closure, UBM, SL-OCT and gonioscopy have good agreement.

Fast growth of single-crystal covalent organic frameworks for laboratory x-ray diffraction.

The imine-exchange strategy makes single-crystal growth of covalent organic frameworks (COFs) with large size (>15 microns) possible but is a time-consuming process (15 to 80 days) that has had limited success (six examples) and restricts structural characterization to synchrotron-radiation sources for x-ray diffraction studies. We developed a CF3COOH/CF3CH2NH2 protocol to harvest single-crystal COFs within 1 to 2 days with crystal sizes of up to 150 microns. The generality was exemplified by the feasible growth of 16 high-quality single-crystal COFs that were structurally determined by laboratory single-crystal x-ray diffraction with resolutions of up to 0.79 angstroms. The structures obtained included uncommon interpenetration of networks, and the details of the structural evolution of conformational isomers and host-guest interaction could be determined at the atomic level.

An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells.

A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells.

BACKGROUND: Gene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo) has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects. METHODS: The Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by checking the expression of CD34 and hexon proteins, respectively. RESULTS: dl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and the loss of p14(ARF) expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner. In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and oncolytic effects. CONCLUSIONS: Ad-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520 alone. These findings indicate that the combination of Ad-mediated antiangiogenic gene therapy and oncolytic Ad therapeutics could be one of promising comprehensive treatment strategies for GC.

Electroretinogram and histopathologic changes of the retina after methanol intoxication.

In order to study the functional and structural alterations of the retina in SD rat model after methanol intoxication, 35 rats were divided randomly into five groups administrated with saline, 3-day high dose, 7-day high dose, 3-day low dose and 7-day low dose methanol separately. The retinal function of each group was assessed by flash electroretinogram (F-ERG) 3 and 7 days after methanol poisoning. The microstructure and ultrastructure of the retina were observed at the same time. The high-dose methanol intoxication induced irreversible retinal functional and structural damages 3 days after poisoning, which included prolonged latency and reduced amplitude of the Max-reaction of F-ERG. These injuries were aggravated 7 days after poisoning. Meanwhile, the latency and amplitude of the Cone-reaction of F-ERG were also affected 3 days after poisoning, but there were no further worsening tendency 7 days after poisoning. The retinal histological analysis showed cellular edema, heteromorphy and disarrangement, tissular loosen of the inner nuclear layer and photoreceptors layer. The mitochondrial damage began at the photoreceptors layer and developed further into the inner nuclear layer. The low-dose methanol intoxication only caused transient damage of the retina. Our results showed that the function and structure of the photoreceptor and inner nuclear layer were the primary target of methanol intoxication and that the rod cells were more sensitive to methanol intoxication than the cone cells. The mitochondrial damage developed from outer layer to inner layer of the retina.

[Correlativity between projected visual chart vision and contrast vision].

OBJECTIVE: To study the correlativity between the projected visual chart vision ("projector vision" in this paper) and the measured values of contrast vision, and to explore the application of contrast vision in identifying visual impairment after eye injury in the clinical forensic medicine. METHODS: One hundred and twenty nine eyes of seventy-nine young volunteers were collected for examination both of projector vision and contrast vision. In the examination of contrast vision, the contrast of optotypes were set to 100%, 25% and 10%, respectively. The correlativity of both collected experimental data were statistically analyzed. RESULTS: Projector vision was consistent with 100% contrast vision. There was a close and positive correlation between the contrast of optotypes and measured vision values. Under the same contrast of optotypes, projector vision presented a linear correlation with the average value of contrast vision. CONCLUSION: There is a close correlation between projector vision and contrast vision. The contrast vision can be applied to judicial identification in the clinical forensic medicine.

[Multifocal visual electrophysiology in visual function evaluation].

Multifocal visual electrophysiology, consisting of multifocal electroretinography (mfERG) and multifocal visual evoked potential (mfVEP), can objectively evaluate retina function and retina-cortical conduction pathway status by stimulating many local retinal regions and obtaining each local response simultaneously. Having many advantages such as short testing time and high sensitivity, it has been widely used in clinical ophthalmology, especially in the diagnosis of retinal disease and glaucoma. It is a new objective technique in clinical forensic medicine involving visual function evaluation of ocular trauma in particular. This article summarizes the way of stimulation, the position of electrodes, the way of analysis, the visual function evaluation of mfERG and mfVEP, and discussed the value of multifocal visual electrophysiology in forensic medicine.

Predicting visual acuity with machine learning in treated ocular trauma patients.

AIM: To predict best-corrected visual acuity (BCVA) by machine learning in patients with ocular trauma who were treated for at least 6mo. METHODS: The internal dataset consisted of 850 patients with 1589 eyes and an average age of 44.29y. The initial visual acuity was 0.99 logMAR. The test dataset consisted of 60 patients with 100 eyes collected while the model was optimized. Four different machine-learning algorithms (Extreme Gradient Boosting, support vector regression, Bayesian ridge, and random forest regressor) were used to predict BCVA, and four algorithms (Extreme Gradient Boosting, support vector machine, logistic regression, and random forest classifier) were used to classify BCVA in patients with ocular trauma after treatment for 6mo or longer. Clinical features were obtained from outpatient records, and ocular parameters were extracted from optical coherence tomography images and fundus photographs. These features were put into different machine-learning models, and the obtained predicted values were compared with the actual BCVA values. The best-performing model and the best variable selected were further evaluated in the test dataset. RESULTS: There was a significant correlation between the predicted and actual values [all Pearson correlation coefficient (PCC)>0.6]. Considering only the data from the traumatic group (group A) into account, the lowest mean absolute error (MAE) and root mean square error (RMSE) were 0.30 and 0.40 logMAR, respectively. In the traumatic and healthy groups (group B), the lowest MAE and RMSE were 0.20 and 0.33 logMAR, respectively. The sensitivity was always higher than the specificity in group A, in contrast to the results in group B. The classification accuracy and precision were above 0.80 in both groups. The MAE, RMSE, and PCC of the test dataset were 0.20, 0.29, and 0.96, respectively. The sensitivity, precision, specificity, and accuracy of the test dataset were 0.83, 0.92, 0.95, and 0.90, respectively. CONCLUSION: Predicting BCVA using machine-learning models in patients with treated ocular trauma is accurate and helpful in the identification of visual dysfunction.

Integrin α6 Targeted Near Infrared Fluorescent Imaging and Photoacoustic Imaging of Hepatocellular Carcinoma in Mice.

Background and Aims: Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death and ranks sixth in terms of incident cases worldwide. The purpose of this study was to develop an effective and sensitive method to distinguish liver cancer tissues from normal tissues in HCC patients. Integrin α6 is a promising cell surface target for molecular imaging of HCC, where it is overexpressed and is a prognostic biomarker. We previously identified an integrin α6-targeted peptide CRWYDENAC (RWY) that has been used for positron emission tomography (PET) imaging of HCC in mouse models. Methods: We labeled the integrin α6-targeted RWY peptide with cyanine 7 (Cy7) to form an optical probe (Cy7-RWY) for near infrared fluorescent (NIRF) and photoacoustic (PA) imaging in HCC. Mice transplanted with subcutaneous HCC-LM3 or orthotopic HCC-H22 cells that overexpressed integrin α6 were intravenously injected with Cy7-RWY and its corresponding Cy7-control. NIRF and PA images of mice were collected from 0 to 48 h after injection. Results: Both NIRF and PA signals started to accumulate in the tumor 2 h after injection of Cy7-RWY and peaked at 24 h. Conclusions: Cy7-RWY is a promising optical probe for NIRF and PA imaging of HCC in mice, and has potential clinical application for HCC detection.

The efficacy of TACE combined sorafenib in advanced stages hepatocellullar carcinoma.

BACKGROUND: The long-term survival in hepatocellullar carcinoma (HCC) patients after transarterial chemoembolization (TACE) remains dismal due to local and/or regional recurrence as well as distant metastasis. The efficacy of sorafenib in advanced HCC has been demonstrated and brought great hope. Recently, the use of sorafenib in combination with TACE for BCLC stage B and C HCC patients was recommended. However, data on this dual-modality treatment is little, and its advantage over TACE alone has not been addressed. The present study sought to understand the efficacy of the combination of TACE and sorafenib in the treatment of advanced HCC. METHODS: Between June 2008 and Feb 2011, 45 patients with advanced HCC were enrolled and treated with sorafenib in combination with TACE according to an institutional protocol of the Zhongshan hospital, Fudan University. The control group of 45 other HCC patients with similar characteristics treated with TACE alone in the same period of time in our institute were selected for retrospective comparison of the treatment outcomes especially overall survival time. Adverse reactions induced by sorafenib were observed and recorded. RESULTS: The median overall survival time of the combined treatment group was 27 (95% Confidence Interval: 21.9-32.1) months, and that of TACE alone group was 17 months (95% Confidence Interval: 8.9-25.0) months (P = 0.001). Patients required significantly less frequent TACE for their symptomatic treatment after the initiation of sorafenib therapy. The most common adverse events associated with sorafenib were hand-foot skin reaction, rash and diarrhea. Of CTCAE grade IV or V toxicity was observed. CONCLUSION: TACE combined sorafenib significantly prolonged median overall survival time of patients with advanced HCC.

[Application of contrast vision in identifying the malingering decreased vision].

OBJECTIVE: To study the application value of contrast vision in identifying the malingering decreased vision in the practice of clinical forensic medicine. METHODS: Thirty-one young and middle aged volunteers were selected and divided randomly into group 1 (16 persons with 32 eyes) and group 2 (15 persons with 30 eyes). The optotype contrast was 100%, 25%, 10% and 5%, respectively. The contrast vision of group 1 was tested. The contrast vision of group 2 was tested as follows: (1) the volunteers cooperated without inspector's interference; (2) the volunteers cooperated under inspector's interference; (3) the volunteers disguised decreased vision without inspector's interference; (4) the volunteers disguised decreased vision under inspector's interference. The data was then analyzed by statistics. RESULTS: There was a close correlation between contrast vision and contrast. As the contrast decreased, the vision acuity also decreased. The contrast vision curve of former two methods showed a good reproducibility while the contrast vision curve of latter two methods had a bad reproducibility. CONCLUSION: The repetition of contrast curve with or without inspector's interference can be used to discriminate malingering vision. The acquired contrast curves can be provided to the court as direct evidence and can help enhance the verification conclusion.

[Features of blood supply and results of transarterial infusion and embolization in spinal metastases].

OBJECTIVE: To study the features of blood supply and results of transarterial infusion and embolization in spinal metastases. METHODS: Forty-one patients with spinal metastasis received transarterial infusion and embolization between March 2001 and June 2008. The inclusion criteria were: The metastatic lesion caused back pain; The metastatic lesion involved vertebra at or below T3 level. There were 29 males and 12 females with a mean age of 56.0 (33 - 71) years. Epirubicin was used as the chemotherapeutic agent. Lipoid Ultra-Fluid, Contour SE or gelfoam particles were used as embolitic material. RESULTS: The technical success of therapy was achieved in 52 vertebrae (100%) including 14 thoracic, 35 lumbar and 3 sacral vertebrae. 105 arteries were used for infusion and embolization (16 intercostal arteries, 78 lumbar arteries, 4 iliolumbar arteries, 4 branches of iliac arteries, and 3 median sacral arteries). Lipoid Ultra-Fluid (2 - 8 ml) was used in 15, Contour SE (300 approximately 500 microm, 20 - 100 mg) in 20, and gelfoam particles in 33 arteries. Three days after treatment, complete pain relief (CR) was achieved in 17 patients, partial pain relief (PR) in 20, and moderate pain relief (MR) in 4, with an effective rate of 90.2%. Two weeks after treatment, CR was achieved in 17 patients, PR in 21, and MR in 3, with an effective rate of 92.7%. No adverse nervous system effect occurred. 16 patients developed swelling and pain of normal tissues which were alleviated after symptomatic treatment. CONCLUSION: Transarterial infusion and embolization is an effective therapy in relieving pain resulting from spinal metastases.

[Advancement of methanol poisoning mechanism research].

The methanol poisoning by oral intake or skin contact occurs occasionally, which may have serious consequences including blindness and/or death. Methanol and its metabolites, formaldehyde and formic acid, are associated with metabolic acidosis, visual dysfunction and neurological symptoms. At present, the mechanism of methanol poisoning primarily focuses on the cell hypoxia, the alteration of structure and biological activity induced by free radical and lactic acid. Meanwhile, methanol poisoning causes changes in the balance between the production of free radicals and antioxidant capacity and in the proteases-protease inhibitors system, which lead to a series of disturbances.

[The relationship between eyeball structure and visual acuity in high myopia].

OBJECTIVE: To explore the relationship between eyeball structure and visual acuity in high myopia. METHODS: Totally, 152 people (283 eyeballs) with different levels of myopia were tested for visual acuity, axial length, and fundus. All cases were classified according to diopter, axial length, and fundus. The relationships between diopter, axial length, fundus and visual acuity were studied. The mathematical models were established for visual acuity and eyeball structure markers. RESULTS: The visual acuity showed a moderate correlation with fundus class, comus, axial length and diopter ([r] > 0.4, P < 0.000 1). The visual acuity in people with the axial length longer than 30.00 mm, diopter above -20.00 D and fundus in 4th class were mostly below 0.5. The mathematical models were established by visual acuity and eyeball structure markers. CONCLUSION: The visual acuity should decline with axial length extension, diopter deepening and pathological deterioration of fundus. To detect the structure changes by combining different kinds of objective methods can help to assess and to judge the vision in high myopia.

Significance of serum fibroblast growth factor-23 and miR-208b in pathogenesis of atrial fibrillation and their relationship with prognosis.

BACKGROUND: The incidence and prevalence of atrial fibrillation are increasing each year, and this condition is one of the most common clinical arrhythmias. AIM: To investigate the levels and significance of serum fibroblast growth factor 23 (FGF-23) and miR-208b in patients with atrial fibrillation and their relationship with prognosis. METHODS: From May 2018 to October 2019, 240 patients with atrial fibrillation were selected as an observation group, including 134 with paroxysmal atrial fibrillation and 106 with persistent atrial fibrillation; 150 patients with healthy sinus rhythm were selected as a control group. The serum levels of FGF-23 and miR-208b in the two groups were measured. In the observation group, cardiac parameters were determined by echocardiography. RESULTS: The serum levels of FGF-23 and miR-208b in the observation group were 210.20 ± 89.60 ng/mL and 5.30 ± 1.22 ng/mL, which were significantly higher than the corresponding values in the control group (P < 0.05). In the observation group, the serum levels of FGF-23 and miR-208b in patients with persistent atrial fibrillation were 234.22 ± 70.05 ng/mL and 5.83 ± 1.00 ng/mL, which were significantly higher than the corresponding values in patients with paroxysmal atrial fibrillation (P < 0.05). The left atrial dimension (LAD) of patients with persistent atrial fibrillation was 38.81 ± 5.11 mm, which was significantly higher than that of patients with paroxysmal atrial fibrillation (P > 0.05). The serum levels of FGF-23 and miR-208b were positively correlated with the LAD (r = 0.411 and 0.382, P < 0.05). In the observation group, the serum levels of FGF-23 and miR-208b in patients with a major cardiovascular event (MACE) were 243.30 ± 72.29 ng/mL and 6.12 ± 1.12 ng/mL, which were significantly higher than the corresponding values in patients without a MACE (P < 0.05). CONCLUSION: The serum levels of FGF-23 and miR-208b are increased in patients with atrial fibrillation and are related to the type of disease, cardiac parameters, and prognosis.