[The expression of cortactin in colorectal cancer and its relationship with clinicopathological and prognostic parameters].

Objective: To investigate the expression of cortactin in colorectal cancer and its correlation with clinicopathological parameters and prognosis. Methods: The expressions of cortactin in normal colorectal mucosal tissue and colorectal cancer tissue in paraffin-embedded tissue microarray from 319 patients who were diagnosed as colorectal cancer and treated in Cancer Hospital of Chinese Academy of Medical Sciences from 2006 to 2009 was detected by immunohistochemistry. Kaplan-Meier method and Log rank test were used for survival analysis, and Cox proportional risk regression model was used for multivariate analysis. Results: The positive expression rates of cortactin in colorectal cancer tissue and normal colorectal mucosal tissue were 61.1% (195/319) and 5.6% (18/319, P<0.001), respectively. T-stage, N-stage, American Joint Committee on Cancer (AJCC) stage, degree of tumor differentiation, neural invasion and preoperative carcinoembryonic antigen (CEA) levels were associated with the expression of cortactin (P<0.05). The positive expression of cortactin was associated with poorer disease-free survival (P=0.036) and overall survival (P=0.043), and the effect was more significant in patients with stage Ⅱ to Ⅲ. For patients with stage Ⅱ-Ⅲ colorectal cancer, postoperative adjuvant therapy was associated with disease-free survival (P=0.007) and overall survival (P=0.015). The vascular tumor embolus, pathological type, preoperative CEA level and cortactin expression were independent influencing factors for disease-free survival (P<0.05). The age, AJCC stage, preoperative CEA level and cortactin expression were independent influencing factors for overall survival (P<0.05). Preoperative CEA level and cortactin expression were independent influencing factors for disease-free survival and overall survival (P<0.05). Conclusion: Cortactin is expressed in colorectal cancer and in stage Ⅱ-Ⅲ patients, it is a potential predictor of colorectal cancer prognosis.

[One-year follow-up results of atrial fibrillation patients who undergoing transcatheter aortic valve implantation].

Objective: To investigate whether atrial fibrillation (AF) before transcatheter aortic valve implantation (TAVI) will affect the prognosis of patients post TAVI. Methods: This is a single center retrospective study. A total of 115 patients with severe aortic stenosis (AS) who were admitted to General Hospital of Northern Theater Command from May 2016 to November 2020 and successfully received TAVI treatment were included. According to absence or accompaniment of AF pre-TAVI, they were divided into AF group (21 cases) and non-AF group (94 cases). The patients were followed up for postoperative antithrombotic treatment and the occurrence of the net adverse clinical and cerebrovascular events (NACCE) at 12 months post TAVI, including cardiogenic death, readmission to hospital for heart failure, nonfatal myocardial infarction, ischemic stroke and severe bleeding (BARC levels 3-5). Univariate logistic regression was used to analyze the related factors of NACCE. Results: Among the 115 selected patients, age was (73.8±6.9) years, there were 63 males. And 21 cases (18.2%) were diagnosed as AFbefore TAVI. In terms of postoperative antithrombotic therapy, 48.9% (46/94) of the patients in the non-AF group received monotherapy and 47.9% (45/94) received dual antiplatelet therapy. In the AF group, 47.6% (10/21) received anticoagulants and 33.3% (7/21) received dual antiplatelet therapy. The proportion of patients in the AF group taking non-vitamin K antagonist oral anticoagulants (NOAC) was higher than that in the non-AF group (38.1% (8/21) vs. 2.1% (2/94), P<0.001). Patients in both groups were followed up to 12 months after TAVI. During the 12 months follow-up, the incidence of NACCE after TAVI was 14.3% (3/21) in the AF group, which was numerically higher than that in the non-AF group (6.4% (6/94)), but the difference was not statistically significant (P=0.441). The incidence of severe bleeding was significantly higher in the AF group than in the non-AF group (9.5% (2/21) vs. 0, P=0.032). Univariate logistic regression analysis showed that hypertension was associated with the risk of NACCE (OR=8.308, P=0.050), while AF was not associated with the risk of NACCE (P=0.235). Conclusion: The incidence of severe bleeding after TAVI is higher in patients with AF than in patients without AF prior TAVI, and there is a trend of increased risk of NACCE post TAVI in AF patients.

[Application and prospect of cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy in the treatment of peritoneal metastasis from colorectal cancer].

Colorectal cancer is one of the common malignant tumors in China, and its incidence is increasing with years. As the second most common metastatic site of colorectal cancer, peritoneum is difficult to diagnose early and with a poor prognosis. Systemic intravenous chemotherapy was used as the main treatment strategy for peritoneal metastasis in the past, but its systemic toxic and side effects were obvious, and it could not effectively control tumor progression. In recent years, the continuous development of surgical techniques, concepts, and equipment, as well as the introduction of new chemotherapy drugs and targeted drugs have significantly improved the quality of life and prognosis of patients with peritoneal metastasis of colorectal cancer. Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) can effectively eradicated the intraperitoneal free cancer cells and subclinical lesions, while reducing systemic side effects of chemotherapy drugs, and achieve the radical cure of the tumor at the macro and micro levels to the greatest extent. It has been used as the first-line treatment program for peritoneal metastasis of colorectal cancer at home and abroad. This article focuses on the analysis and summary of the survival efficacy, prognostic factor analysis, and chemotherapy safety of CRS+ HIPEC in the treatment of colorectal cancer peritoneal metastasis. The existing problems and controversies of HIPEC therapy are discussed simultaneously.

Physical activity and sedentary behaviour among children and adolescents with intellectual disabilities during the COVID-19 lockdown in China.

BACKGROUND: In the wake of the COVID-19 pandemic, preliminary research has reported a significant decline in physical activity (PA) and an increase in sedentary behaviour (SB) among typically developed children and adolescents. Limited research has looked at the current situation of PA and SB during this pandemic among children and adolescents with intellectual disabilities (ID). This study investigated the situations about PA and SB among school-aged children and adolescents with ID on China's mainland during the COVID-19 outbreak. METHODS: In total, 837 parents of children and adolescents (ages 6-18 years) with ID from 15 special education schools of Shandong Province in China were recruited through convenience sampling in the study. Parents reported PA and SB among children and adolescents with ID through the Children's Leisure Activities Study Survey-Chinese version (CLASS-C) online questionnaires. RESULTS: From parents' reports, Chinese children and adolescents with ID during the COVID-19 pandemic participated in approximately 10 min of moderate-to-vigorous physical activity, and engaged in approximately 530 min of SB every day. Meanwhile, only 17.4% of children and adolescents with ID were able to achieve the recommendation of 60 min of daily moderate-to-vigorous physical activity and 76.1% of children and adolescents with ID spent more than 2 h on SB per day. Additionally, the problems of decrease PA and excessive SB were more prominent in older adolescents with ID compared with younger children with ID. CONCLUSION: In China, the low level of PA and high level of SB is particularly evident in children and adolescents with ID during the outbreak of COVID-19. The great majority of children and adolescents with ID did not meet the recommended amount of PA while undergoing excessive SB under the long-term home quarantine environment. Therefore, immediate attention and great effort should be made to deal with this severe situation among this vulnerable population in the mainland of China.

Efficacy of liposomal bupivacaine vs. traditional anaesthetic infiltration for pain management in total hip arthroplasty: a systematic review and meta-analysis.

In this review, we assess the effectiveness of liposomal bupivacaine against the traditional bupivacaine infiltration in the postoperative management of total hip arthroplasty (THA). Various databases including PubMed Central, Medline, Scopus, Embase, Google Scholar, Cochrane library and ScienceDirect (inception date till August 2020) were searched. The quality of published trials was assessed using Cochrane risk of bias tool, and a random-effects model was used for meta-analysis. We report pooled Risk ratios (RR) or pooled Standardized Mean difference (SMD) with 95% confidence intervals (CIs). We analyzed a total of 13 studies with 62,582 participants. The majority of the studies were retrospective with lower bias risks. Liposomal bupivacaine was significantly associated with the reduction in opioid requirement at 48 hours (SMD = -0.25; 95% CI: -0.40 to -0.09; p=0.002) and length of hospital stay (SMD = -0.25; 95% CI: -0.43 to -0.07, p=0.006) following THA compared with the control group. However, there was no statistically significant difference between the effect of liposomal bupivacaine and other agents for pain score (24 and 48 hours), opioid requirement at 24 hours and incidence of nausea. Liposomal bupivacaine has selective benefits in terms of opioid consumption and length of hospital stay against the traditional bupivacaine among the patients undergoing THA.

[Analysis of risk factors of severe postoperative complications in elderly patients with colorectal cancer aged over 80 years].

Objective: To investigate the risk factors of severe postoperative complications in elderly patients with colorectal cancer aged over 80 years old. Methods: A retrospective case-control study was conducted to collect and analyze the clinicopathological data of patients (≥80 years old) who underwent radical colorectal cancer surgery at department of Colorectal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from January 2010 to December 2018. A total of 269 patients were included in the study, including 160 males and 109 females. The average age was 83 (80-94) years. Among them, the pathological TNM stage was 16 in stage I, 76 in stage II, 167 in stage III, and 10 in stage IV. According to Clavien-Dindo classification, the postoperative complications of grade III and above were defined as serious complications. To analyze the relationship between the patient's clinical data, such as general information, the surgeon's experience (whether to complete more than 500 radical colorectal cancer surgery), intraoperative conditions and the occurrence of severe complications. Univariate analysis was conducted with the chi-squared test. Multivariate logistic regression analysis was used for statistically significant variables in univariate analysis. Results: Of the 269 patients, 34 (12.6%) had severe complications after surgery. The incidence of postoperative complications ranged from high to low, respectively, for pulmonary infection (8/269,3.0%), intestinal obstruction (8/269, 3.0%) and anastomotic leakage (7/269, 2.6%). One patient died of pulmonary embolism and one patient died of multiple organ failure, with a perioperative mortality rate of 0.7% (2/269). On univariate analysis, the occurrence of severe postoperative complications was associated with age (χ(2)=8.181, P=0.004), American society of anesthesiologists grade (χ(2)=7.945, P=0.005), preoperative albumin level (χ(2)=9.088, P=0.003), operation experience (χ(2)=9.395, P=0.002). Multivariable logistic regression analysis showed that age ≥85 years old (OR=4.415, 95% CI: 1.702-11.453, P=0.080), preoperative albumin <35 g/L (OR=2.544, 95%CI: 1.083-5.974, P=0.032), and less-experieced group (OR=2.475, 95% CI:1.082-5.661, P=0.032) was independent risk factor for severe postoperative complications. The incidence of serious postoperative complications was similar in patients undergoing laparoscopy and laparotomy [10.1% (17/169) vs. 17.0% (17/100), χ(2)=2.741, P=0.098]. Conclusion: Adequate preoperative evaluation, appropriate perioperative nutritional support and experienced specialists are the key factors to ensure the successful perioperative period of elderly patients with colorectal cancer aged over 80 years old. In addition, more attention should be paid to the elderly patients aged ≥85 years.

[Risk factor analysis for perineal incision complications after abdominoperineal resection in elderly patients with rectal cancer].

Objective: To evaluate the risk factors of perineal incision complications after abdominal abdominoperineal resection (APR) in elderly patients with rectal cancer. Methods: From January 2007 to September 2018, the clinical data of 72 elderly rectal cancer patients (age≥80 years) underwent abdominoperineal resection at Department of Colorectal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College were collected and retrospectively analyzed. Univariate and multivariate analyses were performed to determine the risk factors of perineal incision complications in elderly patients with rectal cancer after APR. Results: Of the 76 patients, 47 were male and 25 were female, with an average age of (81.8±1.8) years. The incidence of postoperative perineal incision complications was 23.6% (17/72), including 5 cases of wound infection, 4 cases of incision fat liquefaction, and 8 cases of delayed wound healing. All of the patients were well recovered and discharged without death. The result of univariate analysis showed that, the occurrence of perineal incision complications was associated with serum albumin level < 35g/L (χ(2)=4.860, P=0.027), intraperitoneal chemotherapy with fluorouracil sustained release/lobaplatin rinse (χ(2)=8.827, P=0.003), pelvic restoration (χ(2)=9.062, P=0.003), diabetes (χ(2)=6.387, P=0.011) and coronary heart disease (χ(2)=7.688, P=0.006). Multivariable logistic regression analysis showed that the intraoperative pelvic restoration (OR=0.17, 95% CI: 0.04~0.82, P=0.027) and diabetes (OR=4.32, 95% CI: 1.05~17.81, P=0.043) were independent risk factors for perineal incision complications. Conclusions: Elderly patients with rectal cancer who undergo APR should preserve and restore the pelvic peritoneum as much as possible. Moreover, perioperative blood glucose monitoring is a powerful guarantee for preventing complications of perineal incision.

Diagnosis of solitary pulmonary lesions with intravoxel incoherent motion diffusion-weighted MRI and semi-quantitative dynamic contrast-enhanced MRI.

AIM: To evaluate the diagnostic value of intravoxel incoherent motion (IVIM) diffusion-weighted (DWI) magnetic resonance imaging (MRI) and semi-quantitative dynamic contrast-enhanced MRI (DCE-MRI) to help diagnose indeterminate solitary pulmonary lesions (SPLs) and the subgroups of lung cancer (LC), and to explore the relationship between IVIM and DCE-MRI. MATERIALS AND METHODS: Sixty-four consecutive patients (44 male, 20 female; age, 52.77±10.46 years) from February 2014 to September 2016 with SPLs, were involved in this prospective study. Total apparent diffusion coefficient (ADCtotal), tissue diffusivity (D), pseudo-diffusion coefficient (D*), perfusion fraction (F), maximum enhancement ratio (MER), Tmax, slope, and washout were compared between the lung cancer (LC) and benign group and among the subtypes of LC. Time-intensity curves (TICs) were drawn. Receiver operating characteristic (ROC) curves were constructed to estimate the diagnostic performance. The correlation of both tools was assessed. RESULTS: ADCtotal, D, and Tmax were significantly higher for benignity than for LC (p=0.005, p=0.002 and p<0.001 respectively). D* and slope were significantly higher in LC than benignity (p=0.005 and p=0.011, respectively). D and Tmax had the highest sensitivity and accuracy, respectively. A combination of D and Tmax improved the sensitivity to 90.5%, the specificity to 86.4%, and the accuracy to 89.1%. Poor correlations were found between parameters derived from IVIM and DCE-MRI. ADCtotal values of SCC and SCLC were found to be significantly lower compared with that in adenocarcinoma. CONCLUSION: Both IVIM-DWI and DCE-MRI were useful for discriminating benignity from LC. ADCtotal was helpful for distinguishing adenocarcinoma and non-adenocarcinoma. A combination of DCE-MRI and IVIM could provide a robust method to determine the microstructural characteristics of SPLs.

Eomesodermin, HAND1, and CSH1 proteins are induced by cellular stress in a stress-activated protein kinase-dependent manner.

Eomesodermin (Eomes) is a transcription factor essential for trophoblast development. Stress stimuli activate stress-activated protein kinase (MAPK8/9) and modulate transcription factors in trophoblast stem cells (TSC). In this study, we test the hypothesis that stress-induced Eomes upregulation and downstream trophoblast development are MAPK8/9-dependent. Immunocytochemical and immunoblot assays suggest that Eomes is induced by hyperosmolar stress in a dose- and time-dependent manner. Two MAPK8/9 inhibitors that work by different mechanisms, LJNKl1 and SP600125, block induction of Eomes protein by stress. During normal TSC differentiation, the transcription factor heart and neural crest derivatives expressed 1 (HAND1) is dependent on Eomes, and chorionic somatomammotropin hormone 1 (CSH1) expression is dependent on HAND1. Similar to Eomes, HAND1 and CSH1 induction by stress are MAPK8/9-dependent, and CSH1 is induced in nearly all stressed TSC. CSH1 induction normally requires downregulation of the transcription factor inhibitor of differentiation 2 (ID2) as well as HAND1 upregulation. It was shown previously that hyperosmolar stress induces AMP-activated protein kinase (PRKAA1/2)-dependent ID2 loss in a MAPK8/9-independent manner. Inhibition of PRKAA1/2 with compound C and LJNKl1, more than MAPK8/9 inhibitors alone, inhibits the induction of CSH1 by stress. Taken together these data suggest that stress-induced MAPK8/9 and PRKAA1/2 regulate transcription factors Eomes/HAND1 and ID2, respectively. Together this network mediates induction of CSH1 by stress. Therefore, stress triggers a proportional increase in a normal early TSC differentiation event that could be adaptive in inducing CSH1. But the flexibility of TSC to undergo stress-induced differentiation could lead to pathophysiological consequences if stress endured and TSC differentiation became unbalanced.

[The inhibitory effect of amygdaloid stimulation on the "on-off" response of medial geniculate body neurons in rabbits].

Experiments were performed on 23 New Zealand rabbits immobilized with Flaxedil. The characteristics of on-off responses of medial geniculate body (MGB) neurons to tone bursts of varying frequency, intensity and duration were affected by lateral amygdaloid nucleus (LAm) stimulation, which was in form of altering the pattern of discharge or complete suppression. The effect also varied with the changes of the interval between amygdaloid stimulation and the tone bursts. The results of the present work show that LAm could modulate temporal processing of auditory information.

[GABA mediated inhibitory effect of amygdala on the activity of medial geniculate body neurons].

On 10 New Zealand white rabbits immobilized with Flaxedil, the inhibitory effect of amygdaloid stimulation on the responses of medial geniculate body (MGB) neurons to tone bursts and the involved neurotransmitter mechanism were investigated with microiontophoresis technique. The results showed that application of GABA could cause a suppression of spontaneous activity of MGB neurons while GABAA antagonist bicuculline had an opposite effect. Iontophoretic injection of GABA gave an inhibitory effect on MGB neurons similar to that caused by stimulating the amygdala or the auditory cortex behind the rhinal sulcus (ACBRS), and in particular, the GABA induced suppression could be completely antagonized by application of bicuculline. Taken together, these data suggested that GABA mediated the amygdaloid inhibitory effect. It seemed unlikely that glycine was involved in the effect, since strychnine, a glycine antagonist, could not affect the descending inhibition from ACBRS area.

[Efficacy of prostaglandin E1 in treatment of cor pulmonale patients with heart failure].

OBJECTIVE: To investigate the effects of prostaglandin E1 (PGE1) by intravenous drip in cor pulmonale patients with heart failure. METHODS: Thirty caseas of cor pulmonale with heart failure were treated with PGE1 at a dose of 200 micrograms.d-1 for 5 days and in 30 patients with cor pulmonale heart failure (no PGE1) and in 20 healthy subjects as controls. The level of serum endogenous digoxin-like factor (EDF), serum lipoid superoxide (LPO), serum superoxide dismutase (SOD) and blood PaO2, PaCO2 were determined. Their symptoms and signs were observed before and after treatment. RESULTS: The level of serum EDF, LPO were significantly increased (P < 0.001) and serum SOD was significantly decreased (P < 0.001) in PGE1 group and controls group (no PGE1) as compared with healthy subjects. In PGE1 group the level of serum EDF, LPO were markedly decreased (P < 0.001), and serum SOD was markedly elevated (P < 0.001), and their symptoms, signs and blood gas parameters had noteworthyly improved (P < 0.01-0.001). But in the control group (no PGE1) the level of serum EDF, LPO, SOD, blood gas parameters and their symptoms and signs, showed no significant difference (P > 0.05). CONCLUSIONS: PGE1 is effective in therapy of cor pulmonale with heart failure. Treatment with PGE1 shows more significant benefits in cor pulmonale patients with heart failure.

[Effect of amygdaloid stimulation on receptive field of "on" LGB neurons in rabbit].

With the aim of study of the effect of stimulating amygdaloid body (basal) on the responsive characteristics of the receptive field (RF) of on-response LGB neurons, electrophysiological experiments were performed on 37 New Zealand rabbits anaethetized with urethane and immobilized by flaxedil. Of the 117 recorded neurons, 47 showed changes in inresponse to light stimulation (facilitation or inhibition) and/or changes in RF configuration, although stimulation of amygdaloid body alone resulted in no visible change of the neuronal activities. The above results suggested that amygdaloid may modulate ascending visual information proccessing at thalami level by changing various combinations of the response characteristics of the on-response LGB neurons.

[A study on the effect of stimulation of amygdaloid complex on the electrical response of auditory cortex in rabbits].

Experiments were performed on 40 adult rabbits immobilized with Flaxedil. The effect of stimulation of amygdaloid complex on the click evoked potential of Woolsey's AI, AII and the auditory cortex behind the rhinal sulcus (ACBRS) was examined by single unit analysis. The results showed that stimulation of lateral nucleus and basal nucleus of amygdala could induce either a facilitory or an inhibitory effect on the evoked potential and the unit discharges. The latency of the inhibitory effect was about 10-25ms, and lasted for 20-115ms. A facilitory effect with a latency as short as 2ms was also observed in one animal. The experimental results indicate that the effect of amygdaloid complex stimulation as transmitted through polysynaptic circuit while the facilitatory effect was monosynaptic. The functional significance of the amygdaloid effect was discussed.

Five-year follow-up of immune response to hepatitis B vaccine in juveniles.

The persistence of anti-HBs was investigated in 95 juveniles who received plasma-derived HBV vaccine (vaccine group) and 63 counterparts who got anti-HBs from natural HBV infection (infection group) for a period of five years. The positive rates of anti-HBs from the first to fifth year in the vaccine group are 97.89%, 98.95%, 81.05%, 78.95% and 72.63% respectively with one recipient remaining anti-HBs negative after being immunized with three dosages of 10 micrograms plasma-derived HBV vaccine in 0, 1st, 2nd month and the mean S/N values (GMV) are 30.94, 22.18, 13.61, 12.02 and 9.18 respectively. There are 26 recipients whose anti-HBs turned from positive to negative at the end of the study with a negative rate of 27.37%. The S/N GMVs in the infection group are 36.37, 27.33, 24.08 at the first, third and fifth year of the study, respectively. Both the S/N GMV and negative rate are lower than that of the vaccine group (P < 0.01). No one was found to have positive HBsAg or elevated ALT in both groups. Questions such as immune dosage, immune program and booster immunization in juvenile population are discussed.

Study of descending inhibitory and facilitory effect of the orbital cortex on the electrical discharges of medial geniculate body (MGB) neurons in rabbits.

Experiments were performed on 16 rabbits immobilized with flaxedil. The recording of spontaneous discharges and the discharges of single MGB neurons caused by clicks showed that when the cerebral-orbital cortex was stimulated, of 92 MGB neurons responding to clicks, 25 units (27.2%) were inhibited, 14 units (15.2%) were facilitated, and 15 units with spontaneous discharges were completely inhibited. The topographical distribution of stimulated points with such inhibitory and facilitory effect is quite concentrated on the orbital cortex. Stimulation of two adjacent points of the orbital cortex could respectively result in different kinds of inhibitory and facilitory effect on discharges of a same MGB neuron. Due to the existence of this descending effect with a short latent period (2-3 ms), the authors have reason to believe that there might be an "orbital cortex-MGB" direct pathway. Moreover, application of 2% procaine to block the auditory cortex behind the rhinal sulcus (ACBRS) could abolish the facilitory effect, with a long latency, of the orbital cortex. Therefore, the influence of the orbital cortex on MGB might be also transmitted via the "orbital-ACBRS-MGB" circuit.

Study of inhibitory effect of amygdaloid stimulation on auditory response of medial geniculate body (MGB) and analysis of transmissive pathway of the said effect.

Experiments performed on 103 rabbits immobilized with Flaxedil, and the recording of the electrical discharges of single MGB neuron caused by clicks, burst or sustained tone showed marked inhibition of the reaction of 47 out of 75 (62.7%) MGB units responding to sound stimuli, when the amygdaloid complex was stimulated. The latent period of such an inhibitory effect was 5-12 msec and its duration about 2-13 msec. Evoked responses with the latency of 1-2 msec for the stimulation of medial, central and lateral nuclei of amygdala could be recorded from the temporal and parietal cortex. The largest response was recorded from Woolsey's AI area of the temporal cortex. Stimulation of the Woolsey's AI auditory area could also result in a distinct inhibition of the spontaneous as well as the evoked discharges of MGB neurons in response to sound stimulation. Moreover, application of 1% procaine to the surface of auditory cortex could block the inhibitory effect of the amygdala on the MGB neurons. Based on the facts mentioned above, the authors have reason to believe that the inhibitory effect of the amygdaloid complex on the activity of MGB units might be transmitted via the amygdala-auditory cortex-MGB circuit. The biological significance of such an suppressive effect has been discussed.