Impact of near-infrared fluorescence imaging with indocyanine green on structural sequelae of anastomotic leakage after laparoscopic intersphincteric resection of malignant rectal tumors.

BACKGROUND: Recent studies have indicated the potential benefit of intraoperative near-infrared fluorescence imaging (NIR-FI) with indocyanine green in reducing early anastomotic leakage in colorectal surgery. Nonetheless, whether NIR-FI is effective in reducing structural sequelae of anastomotic leakage (SSAL) remains unclear. The aim of the present study was to investigate the impact of NIR-FI on SSAL after laparoscopic intersphincteric resection (ISR) of malignant rectal tumors. METHODS: This study was a retrospective single-center cohort study. A total of 293 consecutive patients who underwent elective laparoscopic ISR from May 2010 to August 2017 were included. Patients were divided into 2 groups; those who underwent elective laparoscopic ISR with lymphadenectomy for malignant rectal tumors using NIR-F (NIR-FI group) and those who underwent elective laparoscopic ISR with lymphadenectomy for malignant rectal tumors without using NIR-FI (control group). Thirty were excluded from the analyses (13 died, 7 had pelvic recurrence, and 10 were lost to follow-up). The primary endpoint was the rate of SSAL within 2 years after the primary resection, whereas the secondary endpoint was the rate of natural defecation via the anus at 2 years after the primary resection. Using various statistical analyses, such as propensity score matching, the rate of SSAL was compared between groups. RESULTS: A total of 263 patients were analyzed [177 males and 86 females, median age 61 (27-84) years]. Prior to propensity score matching (n = 263), NIR-FI was performed in 70 patients (26.6%) The rates of SSAL were 1.4% (1/70) in the NIR-FI group and 10.4% (20/193) in the control group (p = 0.02). After propensity score matching (n = 163), the rates of SSAL were 1.5% (1/66) in the NIR-FI group and 11.7% (12/103) in the control group (p = 0.02). Propensity score analyses, as well as simple regression analyses, revealed that NIR-FI was associated with a significantly lower risk of SSAL (OR 0.10-0.13; p = 0.03-0.05). CONCLUSIONS: NIR-FI is useful in reducing the rate of SSAL after laparoscopic ISR.

Development of a performance rubric for transanal endoscopic rectal purse-string sutures.

BACKGROUND: Placing a transanal endoscopic rectal purse-string suture (taEPS) is the crucial first component of transanal total mesorectal excision (taTME). However, no structured training is available to improve the procedure-specific skills for taEPS. The aim of this study was to create a performance rubric to improve taEPS skills and provide preliminary evidence for its validity. METHODS: A performance rubric was created based on technical considerations for taEPS, identified by consulting with taTME surgical and performance assessment experts. Ten independent, blinded raters assessed 10 videotaped taEPS procedures of consecutive taTME cases, at National Cancer Center Hospital East (NCCHE), Chiba, Japan, in January 2018-March 2019 using the rubric and the Global Operative Assessment of Laparoscopic Skills (GOALS). Internal consistency and inter-rater reliabilities were calculated. Videotaped taEPS procedures were timed and assessed by the rubric. Correlation between rubric scores and suturing times were analyzed. RESULTS: The rubric consists of four items: loading the needle (LN), atraumatic needle passage (AP), planned suture path (PS), and overall performance (OA). Videotaped performances were graded on a 3-point Likert scale; scores were calculated as sums of the points. Cronbach's α for internal consistency was 0.713. Inter-rater reliabilities were LN: 0.73, AP: 0.76, PS: 0.71, and OA: 0.70. Rubric and GOALS scores were strongly correlated (r = 0.964, p < 0.001). In 112 consecutive taEPS performances, rubric scores were strongly correlated with suturing time (r = - 0.69, p < 0.001). Surgeons' experience with taTME was associated with rubric scores and suturing time. CONCLUSIONS: This study provides preliminary validation for the taEPS skill performance rubric. The rubric's structured training may facilitate skill acquisition by providing trainees with critical clinical considerations.

Recurrence of rectal anastomotic leakage following stoma closure: assessment of risk factors.

AIM: In patients with a previous history of rectal anastomotic leakage (AL), the surgical indications and timing for closure of a diverting stoma have to be carefully judged. Even if AL has apparently healed before stoma closure, re-leakage may occur after closure. The aim of this study was to determine the incidence and risk factors for recurrent AL following stoma closure. We also examined the treatment strategies aiming to minimize the risk of recurrent AL. METHODS: From January 2009 to December 2016, 1008 patients underwent sphincter-saving surgery [low anterior resection, all-sphincter-preserving rectal resection with hand-sewn coloanal anastomosis (CAA) and intersphincteric resection (ISR)] for primary rectal cancer with curative intent at our hospital. A total of 69 patients with AL with a Clavien-Dindo Grade III or more who subsequently underwent closure of a diverting stoma were retrospectively reviewed for this study. RESULTS: The incidence of recurrent leakage after stoma closure in this series was 13% overall with an incidence of 25% in the CAA/ISR group and 5% in the low anterior resection group. Significant risk factors included hand-sewn anastomosis (P = 0.0257) compared to stapled anastomosis, ischaemia at the anastomotic site as the cause of initial AL (P < 0.001) and a shorter interval between confirmation of healing and stoma closure (P = 0.00952). CONCLUSION: Ischaemia at the anastomotic site was the main risk factor for recurrent leakage, particularly after CAA/ISR. Additional treatment options before stoma closure should be considered to avoid re-leakage in such cases.

Anatomy of the smooth muscle structure in the female anorectal anterior wall: convergence and anterior extension of the internal anal sphincter and longitudinal muscle.

AIM: The anatomy of the region between the vagina and anal canal plays an essential role when performing a proctectomy for low-lying tumours. However, the anatomical characteristics of this area remain unclear. The purpose of the present study was to clarify the configuration, and both lateral and inferior extensions, of the muscle bundles in the anorectal anterior wall in females. METHODS: Using cadaveric specimens, macroscopic anatomical and histological evaluations were conducted at the anatomy department of our institute. Macroscopic anatomical specimens were obtained from six female cadavers. Histological specimens were obtained from eight female cadavers. RESULTS: The smooth muscle fibres of the internal anal sphincter and longitudinal muscle extended anteriorly in the anorectal anterior wall of females and the muscle bundles showed a convergent structure. The anterior extending smooth muscle fibres merged into the vaginal smooth muscle layer, distributed subcutaneously in the vaginal vestibule and perineum and spread to cover the anterior surface of the external anal sphincter and the levator ani muscle. Relatively sparse space was observed in the region anterolateral to the rectum on histological analysis. CONCLUSION: Smooth muscle fibres of the rectum and vagina are intermingled in the median plane, and there is relatively sparse space in the region anterolateral to the rectum. Therefore, when detaching the anorectal canal from the vagina during proctectomy, an approach from both the lateral sides should be used.

Clinical benefit of high resolution anorectal manometry for the evaluation of anal function after intersphincteric resection.

AIM: Intersphincteric resection (ISR) is an advanced anus-preserving operation for treating low rectal cancer while avoiding colostomy. High-resolution anorectal manometry (HR-ARM) allows objective and accurate evaluation of anal function. However, correlations between anal function after ISR and HR-ARM parameters are unknown. The aim of the study was to evaluate HR-ARM for objective evaluation of anal function after ISR. METHOD: A total of 81 patients who underwent ISR at our hospital between October 2014 and March 2016 were identified from our prospectively collected database and electronic medical records. Of these, 68 patients who had been evaluated using HR-ARM both before and after ISR were included in the study. Faecal incontinence (FI) was assessed by Wexner score. Multivariate analysis was performed to determine risk factors for severe FI after ISR. RESULTS: Maximum resting pressure (MRP) (P < 0.001) and maximum squeeze pressure (P = 0.04) were significantly lower after ISR, and MRP (P < 0.001) and maximum squeeze pressure (P = 0.02) were significantly lower after total (or subtotal) ISR than after partial ISR. The overall incidence of severe FI after ISR was 18% (12/68), and a high pressure zone before ISR ≤ 3 cm (P = 0.007) and MRP before ISR > 60 mmHg (P = 0.02) were independently associated with an elevated incidence of severe FI after ISR. Decreased preoperative MRP also correlated with severe FI after ISR (P = 0.008). CONCLUSION: HR-ARM is reliable for the evaluation of anal function after ISR, and the high pressure zone and MRP may be useful preoperative predictors of severe FI after ISR.

Safety and efficacy of high-dose ranimustine, cytarabine, etoposide and CY (MCVAC) regimen followed by autologous peripheral blood stem cell transplantation for high-risk diffuse large B-cell lymphoma.

The efficacy of high-dose chemotherapy followed by autologous hematopoietic SCT for relapsed diffuse large B-cell lymphoma (DLBCL) has been reported, but an optimal conditioning regimen has not been determined. This study was conducted to evaluate the safety and efficacy of the MCVAC regimen (consisting of ranimustine (MCNU), cytarabine, etoposide and CY) followed by autologous peripheral blood stem cell transplantation (PBSCT) for patients with high-risk or relapsed DLBCL. A total of 40 patients with DLBCL who received the MCVAC regimen followed by autologous PBSCT were retrospectively evaluated. Median follow-up duration of the surviving patients was 51.2 months (range, 5.4-151.2 months). At 5-year OS and PFS were 73.7% (95% confidence interval (CI), 58.6-88.8) and 62.5% (95% CI, 46.8-78.2), respectively. Although relapse remained the most frequent cause of treatment failure, late-onset adverse events were observed, including two cases of severe pulmonary impairment, and two cases of therapy-related myelodysplastic syndromes (MDS)/AML. In conclusion, the MCVAC regimen would be an effective and tolerable conditioning regimen without TBI for autologous PBSCT for high-risk or relapsed DLBCL. However, late-onset pulmonary toxicity and MDS/AML should be monitored.

High gene expression of the mutant adenovirus vector, both in vitro and in vivo, with the insertion of integrin-targeting peptide into the fiber.

In the present study, a first-generation adenovirus (Ad) vector was modified with the RGD peptide inserted into the fiber. The insertion of an integrin-targeting sequence into the Ad vector notably enhanced the luciferase expression in the Coxsackie virus and Adenovirus Receptor-deficient A2058 and B16BL6 melanoma cells. The results of an in vivo study with tumor-bearing mice also showed that Ad-RGD-Luc had enhanced gene expression in many organs and in the B16BL6 tumor compared to that induced by a conventional Ad vector after intravenous injection.

Granulocyte transfusion as a treatment for enterococcal meningoencephalitis after allogeneic bone marrow transplantation from an unrelated donor.

Bacterial meningoencephalitis occurring in the pre-engraftment period after bone marrow transplantation (BMT) is a rare complication, and the feasibility of granulocyte transfusion (GTX) in such cases remains to be elucidated. A 37-year-old man developed enterococcal meningoencephalitis during a severely granulocytopenic pre-engraftment period after BMT. Despite therapy with appropriate antibiotics, cultures of blood and cerebro-spinal fluid (CSF) continued to grow Enterococcus faecalis, and he developed rapid mental deterioration and seizure. Granulocytes were collected from his HLA-mismatched, ABO-matched sibling with subcutaneous injection of granulocyte colony-stimulating factor (G-CSF) and oral dexamethazone. Transfusion of 4.4 x 10(10) granulocytes resulted in a 12-h post-transfusion granulocyte increment of 2.0 x 10(9)/l, and maintained peripheral blood granulocyte counts above 0.5 x 10(9)/l for 3 days. A rapid increase of granulocytes in CSF was also observed, and cultures of blood and CSF became negative after GTX. A transient worsening of seizure was observed as a potential side effect of GTX. The patient subsequently developed septic shock because of Pseudomonas aeruginosa and died. Further studies are warranted to evaluate the clinical efficacy of GTX for the treatment of uncontrolled infections in granulocytopenic stem cell transplant recipients.

Extremely high levels of C-reactive protein in patients with acute lupus serositis.

Abstract We present the cases of two patients (19- and 40-year-old women) with systemic lupus erythematosus (SLE) who showed marked elevation of C-reactive protein (CRP). In both patients, pleural and/or peritoneal effusions were caused by lupus serositis. Methylprednisolone pulse therapy was effective in improving the serositis and normalizing CRP. Although it is generally considered that the CRP response is relatively weak in lupus patients, these cases suggest that a strong CRP response can occur in a subset of SLE.

Successful treatment of patients with rheumatic disorders and acquired factor VIII inhibitors with cyclophosphamide and prednisolone combination therapy: two case reports.

Acquired haemophilia associated with autoimmune disorders can be fatal and has been reported to be refractory to steroid therapy alone. We report two cases of female patients, aged 24 years and 54 years, with acquired haemophilia caused by factor VIII inhibitors. Underlying diseases were systemic lupus erythematosus in the 24-year-old patient and rheumatoid arthritis in the 54-year-old patient. Both conditions were nearly quiescent when the patients manifested haemorrhagic diathesis. In response to combination therapy with prednisolone and cyclophosphamide, coagulation abnormalities were resolved together with complete elimination of factor VIII inhibitors in both patients. Thus, combination therapy with alkylating agents may be recommended as initial therapy for the management of autoimmune patients with factor VIII inhibitors.

Efficient antigen gene transduction using Arg-Gly-Asp fiber-mutant adenovirus vectors can potentiate antitumor vaccine efficacy and maturation of murine dendritic cells.

Dendritic cells (DCs), the most effective antigen-presenting cells, are being studied as adjuvants or antigen delivery vehicles for eliciting T-cell-mediated antitumor immunity. Gene delivery to DCs provides an intracellular source of antigen for efficient and persistent loading to MHC class I molecules capable of activating CD8(+) CTLs, which play a central role in antitumor immunity. We previously reported that the fiber-mutant adenovirus vector (Ad) harboring the Arg-Gly-Asp (RGD) sequence in the HI loop of its fiber knob could more efficiently transduce the LacZ gene into both murine DC lines and normal human DCs than conventional Ad. In the present study, we compared immunological properties and vaccine efficacy of DC2.4 cells, an immature murine DC line, transduced with an ovalbumin (OVA) gene by fiber-mutant Ad (Ad-RGD-OVA) or conventional Ad (Ad-OVA). Ad-RGD-OVA-infected DC2.4 cells could more efficiently present OVA peptides via MHC class I molecules in a vector particle-dependent manner and induce OVA-specific CTL response by vaccination than Ad-OVA-infected DC2.4 cells. This result was correlated with the efficiency of gene transduction into DC2.4 cells by both types of Ad. Moreover, vaccination with Ad-RGD-OVA-infected DC2.4 cells could achieve an equal or greater antitumor effect against challenge with E.G7-OVA tumor cells with lower doses of Ad on infection or fewer cells for immunization than the vaccination procedure using Ad-OVA-infected DC2.4 cells. In addition, the maturation of DC2.4 cells was promoted by efficient expression of the antigen gene by the Arg-Gly-Asp fiber-mutant Ad. Flow cytometric analysis indicated enhanced expression of MHC class I and II molecules as well as CD80, CD86, CD40, and CD54, and reverse transcription-PCR analysis revealed increased levels of interleukin 12 p40 mRNA. However, infection by Ad-OVA or Ad that did not contain the cDNA of interest (Ad-Null and Ad-RGD-Null) contributed little to phenotypical changes in DC2.4 cells. On the basis of these results, we propose that DC manipulation using the Arg-Gly-Asp fiber-mutant Ad system could advance the development of more effective vaccines and allow for more convenient administration of DC-based gene immunotherapy.

High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2.

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. The molecular mechanism of the HGF-induced growth inhibition of tumor cells remains obscure. We have investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. HGF induced strong activation of ERK in HepG2 cells. Although the serum-dependent proliferation of HepG2 cells was inhibited by the MEK inhibitor PD98059 in a dose-dependent manner, 10 microM PD98059 reduced the HGF-induced strong activation of ERK to a weak activation; and as a result, the proliferation inhibited by HGF was completely restored. Above or below this specific concentration, the restoration was incomplete. Expression of constitutively activated Ha-Ras, which induces strong activation of ERK, led to the proliferation inhibition of HepG2 cells, as was observed in HGF-treated HepG2 cells. This inhibition was suppressed by the MEK inhibitor. Furthermore, HGF treatment and expression of constitutively activated Ha-Ras changed the hyperphosphorylated form of the retinoblastoma tumor suppressor gene product pRb to the hypophosphorylated form. This change was inhibited by the same concentration of MEK inhibitor needed to suppress the proliferation inhibition. These results suggest that ERK activity is required for both the stimulation and inhibition of proliferation of HepG2 cells; that the level of ERK activity determines the opposing proliferation responses; and that HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal in HepG2 cells.

Advanced peritonitis and subclinical disseminated intravascular coagulopathy associated with Chlamydia trachomatis infection.

A 29-y-old woman was admitted to our hospital complaining of slight fullness of the lower abdomen. Ultrasound echographic study and magnetic resonance imaging showed pleural effusion and pelvic ascites. Laboratory investigation revealed anemia and thrombocytopenia (hemoglobin 6 mmol/l; platelets 7 x 10(10)/l), remarkable polyclonal hypergammopathy (gamma immunoglobulin 7.7 g/dl) and subclinical disseminated intravascular coagulopathy (DIC). By laparoscopy, extensive adhesion of the peritoneum and bilateral ovarian tubes was observed. From the appearance of adhesion, we suspected Chlamydia trachomatis infection and performed serologic and molecular studies. Administration of clarithromycin resolved hypergammopathy, DIC and ascites.

Autoantibody to DNA excision repair enzyme hMYH in a patient with rheumatic disease.

We screened a human HepG2 cell cDNA expression library using serum from a patient with rheumatic disease. This serum had immunofluorescence reactivity to nuclei with a homogeneous staining pattern and to punctuate nuclear aggregates, chromosomal metaphase plate, midbody, and cytoplasmic bridge. YT1, the longest cDNA clone isolated, has sequence identity to hMYH, the human homologue of the Escherichia coli excision repair enzyme, DNA adenine glycosylase MutY. YT1 is a truncated cDNA of 1619 bp, encoding amino acids 22-535, and contains a full-length 3'-UTR sequence. We were unable to express a bacterial malE fusion protein incorporating amino acids 22 to 535 of hMYH. Consequently, we generated two additional malE fusion proteins of hMYH encoding amino acids 1-120 (pMAL-c2:hMYH(1-120)) and amino acids 121-535 (pMAL-c2:hMYH(121-535)). The patient serum immunoblotted only pMAL-c2:hMYH(1-120), suggesting that the autoepitope(s) is restricted to amino acids 22-120 of hMYH, and detected a protein of approximately 59-kDa in total HeLa and nuclear extracts consistent with reactivity to hMYH. Affinity-purified autoantibodies to pMAL-c2:hMYH(1-120) reacted by immunoblot to pMAL-c2:hMYH(1-120), with no reactivity to pMAL-c2:hMYH(121-535). By immunofluorescence, these antibodies displayed staining of nuclei. This is the first report of autoantibodies to hMYH in a patient with rheumatic disease. We were able to identify hMYH reactivity in relatively small cohorts of sera collected from rheumatoid factor-positive patients (6 of 18) and dsDNA-positive patients (1 of 18), with no reactivity detected in serum collected from 9 healthy subjects.

Corticosteroid therapy in patients with IgA nephropathy and impaired renal function.

AIM: IgA nephropathy (IgAN) is a common type of primary glomerulonephritis that constitutes a major cause of end-stage renal disease. Oral and/or intravenous glucocorticoid therapy can protect against progression of IgAN in patients with preserved renal function. We evaluated steroid therapy in IgAN with established renal dysfunction. PATIENTS AND METHODS: We retrospectively analyzed the effect of methylprednisolone (MP) pulse therapy in 8 IgAN patients with serum creatinine concentrations (sCr) 2.76 +/- 1.32 mg/dl (mean +/- SD). In each patient renal function had progressively deteriorated in the 12 months preceding treatment, as indicated by negative slopes of 1/sCr plotted against time (regression coefficients). RESULTS: Regression coefficients during the 12 months following therapy improved significantly from -0.02333 +/- 0.00732 to -0.00036 +/- 0.00423 dl/mg/month, respectively. The mean difference in slope was 0.0230 +/- 0.0076 dl/mg/month (95% confidence interval, 0.0165 to 0.0295, p < 0.001). Proteinuria also significantly decreased from a mean urine protein/creatinine ratio of 2.57 +/- 1.12 before therapy to 1.12 +/- 0.84 6 months after therapy (p < 0.005). Other factors that might affect progression of renal dysfunction remained unchanged during the observation periods. CONCLUSION: Corticosteroids may attenuate progression of renal failure and delay the need for dialysis in this patient population, although a large randomized trial is necessary.

Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors.

Recent studies have demonstrated the usefulness of dendritic cells (DCs) genetically modified by adenovirus vectors (Ad) to immunotherapy, while sufficient gene transduction into DCs is required for high doses of Ad. The RT-PCR analysis revealed that the relative resistance of DCs to Ad-mediated gene transfer is due to the absence of Coxsackie-adenovirus receptor expression, and that DCs expressed adequate alpha(v)-integrins. Therefore, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob can efficiently transduce and express high levels of the LacZ gene into DCs. The gene delivery by fiber-mutant Ad was more efficient than that by conventional Ad in both murine DC lines and normal human DCs (NHDC). Furthermore, NHDC transduced with fiber-mutant Ad and conventional Ad at 8000-vector particles/cell resulted in a 70-fold difference in beta-galactosidase activity. We propose that alpha(v)-integrin-targeted Ad is a very powerful tool with which to implement DC-based vaccination strategies.

Histopathology of familial ovarian tumors in women from families with and without germline BRCA1 mutations.

Breast cancers from patients with germline BRCA1 mutations show characteristic histopathologic features. However, similar studies of BRCA1-associated ovarian cancers have reported inconsistent findings. Interobserver differences in histopathologic classification are a significant source of variation, and most studies have obtained histopathologic information from pathology reports rather than from review of histopathology slides. We therefore reviewed the histopathology slides and pathology reports to determine histologic type, grade, and stage for cancers of the ovary or peritoneum in 217 women from 126 families enrolled in the Gilda Radner Familial Ovarian Cancer Registry. Peripheral blood DNA from at least 1 affected member of each family was analyzed for BRCA1 mutations, and tumors from BRCA1 mutation-positive families were compared with those from BRCA1-negative families. Of 66 patients from 36 BRCA1-positive families, 64 had ovarian carcinoma, 1 had an ovarian carcinoma in situ, and 1 had a dysgerminoma. Of 151 patients from 90 BRCA1-negative families, 135 had ovarian carcinoma, 10 had ovarian borderline tumors, 3 had ovarian sex cord/stromal tumors, and 3 had primary peritoneal carcinoma. There were fewer grade 1 (P <.001) and stage I (P =.10) cancers in patients from BRCA1-positive families than in patients from BRCA1-negative families. Neither mucinous nor borderline tumors were found in the BRCA1-positive families. Ovarian cancers arising in women from BRCA1-positive families are more likely to be high grade and nonmucinous than cancers arising in women from BRCA1-negative families. The absence of borderline tumors in patients from BRCA1-positive families adds to accumulating evidence that BRCA1 mutations do not play a role in the development of these tumors. HUM PATHOL 31:1420-1424.

Primary ovarian dysgerminoma in a patient with a germline BRCA1 mutation.

Germline mutations in the BRCA1 tumor suppressor gene are associated with increased risk for the development of ovarian cancer. All such cancers thus far reported have been of the epithelial histologic type. We identified an ovarian dysgerminoma in a 16-year-old woman (proband) with a family history of ovarian cancer during a review of histopathologic characteristics of ovarian cancers from women enrolled in the Gilda Radner Familial Ovarian Cancer Registry. Mutation analysis of DNA from this patient's peripheral blood leukocytes revealed a germline BRCA1 mutation (3312insG). The mutation was also present in the mother with breast cancer, a maternal aunt and a distant cousin with ovarian cancer, and a maternal grandfather and an uncle with skin cancer. The development of the proband's dysgerminoma may be unrelated to her germline BRCA1 mutation. Alternatively, such dysgerminomas may be caused by BRCA1 mutations, but occur so infrequently compared with epithelial cancers that they are seldom identified. Analysis of a larger series of ovarian germ cell tumors may resolve this question.