Medical treatment selection and outcomes for hospitalized patients with severe ulcerative colitis as defined by the Japanese criteria.

BACKGROUND: Hospitalization for ulcerative colitis (UC) is potentially life-threatening. Severe disease in the Japanese criteria which modifies the Truelove-Witts' criteria might encompass more fulminant cases than the definition for acute severe UC. However, few studies have investigated the predictive factors for clinical remission (CR) after medical treatments for severe hospitalized patients by Japanese criteria. METHODS: Medical treatment selection, CR rates, and factors contributing to CR on day 14 were assessed in severe patients by Japanese criteria. We also investigated whether the reduction rate in patient-reported outcome 2 (PRO2) on day 3 could predict short-term prognosis. RESULTS: Eighty-five severe hospitalized patients were selected. Corticosteroids, tacrolimus, and infliximab were mainly selected as first-line treatments (76/85; 89.4%). The CR rates on day 14 were 26.8%, 21.4%, and 33.3% in patients receiving corticosteroids, tacrolimus, and infliximab, respectively. Extensive disease (odds ratio [OR] 0.022; 95% confidence interval [CI] 0.002-0.198), higher PRO2 (OR 0.306; 95% CI 0.144-0.821), and higher reduction rate in PRO2 on day 3 (OR 1.047; 95% CI 1.019-1.075) were independent factors predicting CR on day 14. If the cutoff value for the reduction rate in PRO2 on day 3 was 18.3%, sensitivity was 0.714 and specificity was 0.731 to predict CR on day 14. A higher reduction rate in PRO2 on day 3 (OR 0.922; 95% CI 0.853-0.995) was a negative factor to predict surgery within 28 days. CONCLUSIONS: Tacrolimus and infliximab in addition to corticosteroids were used as first-line treatment in severe hospitalized patients. PRO2 on day 3 is a useful marker for switching to second-line therapy or colectomy.

Effectiveness of One-Year Pemafibrate Therapy on Non-Alcoholic Fatty Liver Disease Refractory to Long-Term Sodium Glucose Cotransporter-2 Inhibitor Therapy: A Pilot Study.

Background: Both pemafibrate and sodium glucose cotransporter-2 (SGLT2) inhibitor can decrease serum transaminase levels in patients with non-alcoholic fatty liver disease (NAFLD) complicated with dyslipidemia and type 2 diabetes mellitus (T2DM), respectively. However, the effectiveness of combined therapy has been rarely reported. Methods: This is a two-center retrospective observational study. NAFLD patients complicated with T2DM treated with pemafibrate for >1 year were included, in whom prior treatment with SGLT2 inhibitor > 1 year failed to normalize serum alanine aminotransferase (ALT) levels. Hepatic inflammation, function, and fibrosis were assessed by ALT, albumin-bilirubin (ALBI) score, and Mac-2 binding protein glycosylation isomer (M2BPGi) levels, respectively. Results: Seven patients were included. The median duration of prior treatment with SGLT2 inhibitors was 2.3 years. During the one year before starting pemafibrate therapy, the therapy did not significantly change hepatic enzymes. All patients received pemafibrate 0.1 mg twice daily without dose escalations. During one year of pemafibrate therapy, triglyceride, aspartate aminotransferase, ALT, γ-glutamyl transpeptidase, ALBI score, and M2BPGi levels significantly improved (p < 0.05), although weight or hemoglobin A1c did not significantly change. Conclusions: One year of pemafibrate therapy improves markers of hepatic inflammation, function, and fibrosis in NAFLD patients in whom long-term SGLT2 inhibitor therapy failed to normalize serum ALT.

Pemafibrate therapy for non-alcoholic fatty liver disease is more effective in lean patients than obese patients.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, with an increasing incidence. Pemafibrate is a novel selective peroxisome proliferator-activated receptor-a (PPAR-a) modulator which is expected to improve NAFLD. The aim of this study is to identify predictors of improvement of hepatic inflammation and fibrosis after pemafibrate therapy in patients with NAFLD. Material and methods: Seventy-one non-diabetic patients with NAFLD treated with pemafibrate for more than six months were included in this retrospective review. Hepatic inflammation and fibrosis were evaluated by alanine aminotransferase (ALT) and Mac-2 binding protein glycosylation isomer (M2BPGi) levels, respectively. Results: During six months of pemafibrate therapy, significant improvements were observed in ALT and M2BPGi levels regardless of the body mass index (BMI) compared to baseline. Lean NAFLD was identified as a significant positive predictor for > 50% reduction of ALT showing reduced hepatic inflammation. Subsequent multivariate analysis confirmed this result. Reduction of ALT in the lean NAFLD group (BMI < 25) was significantly greater than in the obese NAFLD group (BMI > 30) (p = 0.034). Lean NAFLD and age > 50 years were identified as significant positive predictors for > 20% reduction of M2BPGi showing reduced hepatic fibrosis. Subsequent multivariate analysis confirmed these results. Reduction of M2BPGi in the lean NAFLD group was significantly greater than in the obese NAFLD group (p = 0.022). Conclusions: Pemafibrate therapy improves markers of hepatic inflammation and fibrosis regardless of BMI. Patients with lean NAFLD have a greater response to pemafibrate therapy compared to those with obese NAFLD.

Pemafibrate improves hepatic inflammation, function and fibrosis in patients with non-alcoholic fatty liver disease: a one-year observational study.

AIM OF THE STUDY: To optimize the long-term outcomes of patients with non-alcoholic fatty liver disease (NAFLD), long-term therapy is important to prevent cirrhosis and hepatocellular carcinoma. Pemafibrate, a novel selective peroxisome proliferator-activated receptor-α modulator, is a promising therapeutic agent for patients with NAFLD. However, only short-term clinical studies are currently available. The aim of this study is to evaluate the long-term outcomes of patients with NAFLD treated with pemafibrate. MATERIAL AND METHODS: This is a retrospective observational study. Patients with NAFLD treated with pemafibrate 0.1 mg twice daily for one year were retrospectively reviewed. RESULTS: Twenty-two patients without diabetes mellitus were included and analyzed. Regarding hepatic inflammation markers, alanine aminotransferase (ALT) significantly decreased during the first three months and was maintained. Low-density lipoprotein and triglycerides significantly decreased at three months and were maintained. Regarding markers of hepatic function, the albumin-bilirubin score decreased significantly during one year of therapy due to significantly elevated serum albumin and decreased total bilirubin levels. Regarding markers of fibrosis, Mac-2 binding protein glucosylation isomer (M2BPGi) significantly decreased, and platelet count increased significantly. Next, we performed correlation analysis between changes in M2BPGi and other parameters. Changes in aspartate aminotransferase, ALT and triglycerides positively correlated with the change in M2BPGi. CONCLUSIONS: One-year pemafibrate therapy improves markers of hepatic inflammation, function and fibrosis in non-diabetic patients with NAFLD. Improvement of hepatic fibrosis markers significantly correlates with improvement of hepatic inflammation markers and triglyceride levels.

Real-world efficacy and safety of 12-week sofosbuvir/velpatasvir treatment for patients with decompensated liver cirrhosis caused by hepatitis C virus infection.

AIM: This study aimed to evaluate the real-world efficacy and safety of 12-week sofosbuvir/velpatasvir (SOF/VEL) treatment for patients with decompensated liver cirrhosis caused by hepatitis C virus (HCV) infection. METHODS: A total 72 of patients with Child-Pugh (CP) class B or C were enrolled. We evaluated the sustained virologic response at 12 weeks after the end of treatment (SVR12), adverse events (AEs), and changes in the liver function. RESULTS: All participants had genotype 1 or 2 HCV infection. At baseline, the numbers of patients with CP class B and C were 59 and 13, respectively. The overall SVR12 rate was 95.8% (69/72); 94.9% (56/59) in CP class B and 100% (13/13) in CP class C. The serum albumin level, prothrombin time and ascites were significantly improved (P < 0.01); however, the serum bilirubin level and encephalopathy did not improve. Among patients who achieved SVR12, 75.0% showed an improvement in their CP score, while 5.9% showed a worsening. The presence of large portosystemic shunt (diameter ≥6 mm) and hyperbilirubinemia (≥2.0 mg/dL) were independent factors that interfered with the improvement in the CP score (P < 0.05). The most common AEs were encephalopathy (15.3%) and skin symptoms (7.9%). Two patients discontinued SOF/VEL due to AEs. CONCLUSIONS: Treatment with SOF/VEL for 12 weeks was relatively safe and effective for patients with decompensated cirrhosis. An SVR provided an improvement of the liver function in the majority of patients. However, large portosystemic shunt and hyperbilirubinemia were independent factors that interfered with the improvement in the CP score.

Pemafibrate decreases markers of hepatic inflammation in patients with non-alcoholic fatty liver disease.

AIM OF THE STUDY: Non-alcoholic fatty liver disease (NAFLD) is frequently complicated by dyslipidemia and is considered to be a hepatic manifestation of metabolic syndrome. Pemafibrate is a novel selective peroxisome proliferator-activated receptor-α modulator. There are no reports of the clinical effects of pemafibrate in patients with NAFLD. The aim of this study is to determine the effect of pemafibrate on patients with NAFLD. MATERIAL AND METHODS: This is an observational study of patients with NAFLD complicated by dyslipidemia treated with pemafibrate for three months. Patient medical records were retrospectively reviewed. RESULTS: Thirty-eight patients were included, and all patients had dyslipidemia without diabetes. Changes in parameters after three months of pemafibrate therapy were evaluated. Weight was not significantly changed. Alanine aminotransferase, a marker of hepatic inflammation, significantly improved. Remarkably, alkaline phosphatase and γ-glutamyl transpeptidase decreased in all patients. The albumin-bilirubin score, a marker of hepatic function, improved due to significant elevation of serum albumin and decrease in total bilirubin. Lipid profiles including high-density lipoprotein cholesterol and triglycerides significantly decreased. Low-density lipoprotein cholesterol did not significantly change. The NAFLD fibrosis score significantly improved, but the FIB-4 index did not significantly change. CONCLUSIONS: Three months of pemafibrate treatment of patients with NAFLD improves markers of hepatic inflammation, function and fibrosis. This is the first clinical study evaluating the effect of pemafibrate in patients with NAFLD.

Long-term empagliflozin therapy improves levels of hepatic fibrosis marker in patients with non-alcoholic fatty liver disease complicated by type 2 diabetes mellitus.

The long-term outcomes of patients with non-alcoholic fatty liver disease (NAFLD) treated with sodium-glucose cotransporter-2 inhibitors remain indeterminate. Empagliflozin improves hyperglycemia by increasing glucose excretion in the urine, and it reduces fat volume and insulin resistance. The aim of this study is to assess the effect of long-term empagliflozin therapy on hepatic inflammation, function and fibrosis in patients with NAFLD. This is a two-center retrospective observational study including patients with NAFLD complicated by type 2 diabetes mellitus. We retrospectively reviewed the medical records. Changes in parameters were investigated over one-year empagliflozin treatment. Twenty-four patients treated with empagliflozin were evaluated. Weight, body mass index, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, fasting plasma glucose, hemoglobin A1c, serum insulin and homeostasis model assessment insulin resistance significantly decreased during treatment (p < 0.05). Albumin-bilirubin (ALBI) score, a marker of hepatic function, was significantly improved (p < 0.01). The FIB-4 index and Mac-2 Binding Protein Glucosylation Isomer, markers of hepatic fibrosis, significantly improved (p < 0.01). One-year empagliflozin treatment of patients with NAFLD complicated by type 2 diabetes mellitus significantly improves markers of hepatic inflammation, function and fibrosis. J. Med. Invest. 67 : 280-284, August, 2020.

Efficacy and safety of glecaprevir and pibrentasvir combination therapy in old-aged patients with chronic hepatitis C virus infection.

Objective: Combination therapy with glecaprevir and pibrentasvir (G/P) has been shown to provide a sustained virologic response (SVR) rate of >97% in patients with chronic hepatitis C virus (HCV) infection in the first published real-world Japanese data. However, a recently published study showed that the treatment was often discontinued in patients ≥75 years old, resulting in low SVR in intention-to-treat (ITT) analysis. Thus, our aim was to evaluate real-world data for G/P therapy in patients ≥75 years of age, the population density of which is high in "rural" regions. Patients and Methods: We conducted a multicenter study to assess the efficacy and safety of G/P therapy for chronic HCV infection, in the North Kanto area in Japan. Results: Of the 308 patients enrolled, 294 (95.5%) completed the treatment according to the protocol. In ITT and per-protocol analyses, the overall SVR12 rate was 97.1% and 99.7%, respectively. The old-aged patients group consisted of 59 participants, 56 of whom (94.9%) completed the scheduled protocol. Although old-aged patients tended to have non-SVR factors such as liver cirrhosis, history of HCC, and prior DAA therapies, the SVR12 rates in old-aged patients were 98.3% and 100% in the ITT and PP analyses, respectively. Of 308 patients enrolled, adverse events were observed in 74 patients (24.0%), with grade ≥3 events in 8 patients (2.6%). There was no significant difference in any grade and grade ≥3 adverse events between the old-aged group and the rest of the study participants. Only one patient discontinued the treatment because of adverse events. Conclusion: G/P therapy is effective and safe for old-aged patients.

Diabetes mellitus exacerbates citrin deficiency via glucose toxicity.

AIMS: Citrin is an aspartate/glutamate carrier that composes the malate-aspartate reduced nicotinamide adenine dinucleotide (NADH) shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD) and adult-onset type II citrullinemia (CTLN2). Hepatic glycolysis is essentially impaired in citrin deficiency and a low-carbohydrate diet was recommended. The lethal effect of infusion of glycerol- and fructose-containing osmotic agents was reported in these patients. Hyperalimentation was also reported to exacerbate CTLN2; however, glucose toxicity was unclear in citrin deficiency. METHODS: We studied two CTLN2 patients complicated with type 2 diabetes mellitus (DM), Case 1 presented with hyperammonemic encephalopathy accompanied with DM, while Case 2 presented with hyperammonemic encephalopathy relapse upon the onset of DM after several years' remission following supplementation with medium-chain triglycerides (MCT) and adherence to a low-carbohydrate diet. RESULTS: Insulin therapy with MCT supplementation and a low-carbohydrate diet improved hyperammonemia and liver function in Case 1. Additional insulin therapy improved hyperammonemia in Case 2. CONCLUSION: Glucose is not toxic for citrin deficiency in normoglycemia because glucose uptake and metabolism by hepatocytes are limited in normoglycemia. However, glucose becomes toxic during persistent hyperglycemia and antidiabetic therapy is indispensable for CTLN2 patients with DM.

No Regional Disparities in Sofosbuvir Plus Ribavirin Therapy for HCV Genotype 2 Infection in Tochigi Prefecture and Its Vicinity.

Objective Regional disparities were observed in the outcomes of interferon (IFN)-based therapy for chronic hepatitis C virus (HCV) infection in a Japanese nationwide study. However, whether or not these regional disparities are observed in the outcomes of direct-acting antiviral drugs, including sofosbuvir (SOF) plus ribavirin (RBV) therapy, remains unclear. Methods We conducted a multicenter study to assess the efficacy of SOF plus RBV therapy for HCV genotype 2 infection in Tochigi Prefecture and its vicinity, in which IFN-based therapy yielded a low sustained virologic response (SVR) rate. In addition, we divided Tochigi Prefecture into six regions to examine regional disparities in the SVR. Patients We enrolled patients with chronic HCV genotype 2 infection. Results Of the 583 patients enrolled, 569 (97.6%) completed the treatment, and 566 (97.1%) also complied with post-treatment follow-up for 12 weeks. The overall SVR12 rate was 96.1% by per protocol and 93.7% by intention-to-treat analyses. No marked differences were observed in the SVR12 between subjects ≥65 and <65 years of age. Although large gaps were observed in the characteristics of patients and accessibility to medical resources, there was no significant difference in the SVR12 rate among the six regions in Tochigi Prefecture. Conclusion SOF plus RBV therapy was effective for HCV genotype 2 infection in an area where IFN-based therapy had previously shown unsatisfactory results. In addition, no regional disparities in the SVR12 were observed in Tochigi Prefecture.

Medium-chain triglycerides supplement therapy with a low-carbohydrate formula can supply energy and enhance ammonia detoxification in the hepatocytes of patients with adult-onset type II citrullinemia.

Citrin, encoded by SLC25A13, constitutes the malate-aspartate shuttle, the main NADH-shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Citrin deficiency is predicted to impair hepatic glycolysis and de novo lipogenesis, resulting in hepatic energy deficit. Secondary decrease in hepatic argininosuccinate synthetase (ASS1) expression has been considered a cause of hyperammonemia in CTLN2. We previously reported that medium-chain triglyceride (MCT) supplement therapy with a low-carbohydrate formula was effective in CTLN2 to prevent a relapse of hyperammonemic encephalopathy. We present the therapy for six CTLN2 patients. All the patients' general condition steadily improved and five patients with hyperammonemic encephalopathy recovered from unconsciousness in a few days. Before the treatment, plasma glutamine levels did not increase over the normal range and rather decreased to lower than the normal range in some patients. The treatment promptly decreased the blood ammonia level, which was accompanied by a decrease in plasma citrulline levels and an increase in plasma glutamine levels. These findings indicated that hyperammonemia was not only caused by the impairment of ureagenesis at ASS1 step, but was also associated with an impairment of glutamine synthetase (GS) ammonia-detoxification system in the hepatocytes. There was no decrease in the GS expressing hepatocytes. MCT supplement with a low-carbohydrate formula can supply the energy and/or substrates for ASS1 and GS, and enhance ammonia detoxification in hepatocytes. Histological improvement in the hepatic steatosis and ASS1-expression was also observed in a patient after long-term treatment.

Tolvaptan is effective in treating patients with refractory ascites due to cirrhosis.

The treatment of refractory ascites due to cirrhosis is a clinical challenge for hepatologists. Tolvaptan, a novel aquaporin modulator, was made available in Japan in 2013 for the treatment of patients with refractory ascites due to cirrhosis. Despite the potential of this drug, few reports are available regarding its clinical use. The aim of the present study was to clarify the efficacy of tolvaptan in patients with refractory ascites due to cirrhosis and to review the clinical outcomes of treatment. Medical records were retrospectively reviewed for 65 patients with refractory ascites due to cirrhosis who were treated daily with 7.5 mg tolvaptan. The median follow-up time, defined as the period between starting tolvaptan and the last clinic visit or date of mortality, was 175 days (interquartile range 56-406). After one week of tolvaptan treatment, the mean weight reduction was 3.4 kg, with a response rate of 69% (45/65). Subsequently, factors associated with the response to tolvaptan were analyzed. On univariate analysis, maintaining serum sodium (Na) ≥140 mEq/l and an estimated glomerular filtration rate (eGFR) ≥55 ml/min were significant predictors of response (P<0.05). On multivariate analysis, hepatitis C virus etiology, maintaining serum Na ≥140 mEq/l and an eGFR ≥55 ml/min were significant predictors of response (P<0.05). Factors associated with survival were also analyzed using the Cox proportional hazard model. On multivariate analysis, responsiveness to tolvaptan was a predictor of long-term survival (P=0.002), and hyperbilirubinemia was associated with short-term survival (P=0.028). Additionally, Kaplan-Meier analysis with a log-rank test indicated longer survival times in tolvaptan responders than non-responders (P=0.011). In conclusion, tolvaptan was effective in treating patients with refractory ascites due to cirrhosis. In particular, tolvaptan treatment was highly effective for patients with hepatitis C virus etiology and normal serum Na and renal function. Furthermore, response to tolvaptan was associated with longer survival time while hyperbilirubinemia was associated with shorter survival time.

Paraneoplastic Dermatomyositis in Hepatocellular Carcinoma with Colonic Perforation: A Case Report.

BACKGROUND: Dermatomyositis (DM) is an autoimmune disease characterized by cutaneous Gottron papules, heliotrope rash, and proximal myopathy. It may also present as a paraneoplastic syndrome that can complicate a variety of different cancers, such as lung, cervical, and breast cancer. However, the association with hepatocellular carcinoma (HCC) is extremely rare. Moreover, to our knowledge, there are no previous reports of colonic perforation following steroid pulse treatment for a DM patient. CASE SUMMARY: A 61-year-old male complained of a skin rash that began in his neck and spread to his face and abdomen. On physical examination, the patient was also found to have symmetrical proximal muscle weakness, abdominal pain, heliotrope rash in the periorbital skin, and poikiloderma on his face and abdomen. Serum level of muscle enzymes was remarkably increased. Muscle examination revealed symmetrical proximal weakness. The diagnosis of DM was made, and steroid treatment was started for symptomatic relief. A search for causative malignancy revealed HCC. Despite steroid therapy for DM, his symptoms did not improve. Additionally, C-reactive protein elevation was seen along with severe abdominal pain on day 14 of admission. Shortly after this, the patient died of septic shock due to suppurative peritonitis after perforation of the ascending colon. CONCLUSION: Here, we present a rare case of DM caused by non-hepatitis-associated advanced HCC with colonic perforation. The cause of colonic perforation is still unclear. This case demonstrates the need to carefully monitor abdominal pain in DM patients as symptoms can be masked by steroid therapy.

Presentation of familial Mediterranean fever in a heterozygous MEFV mutation triggered by immunosuppressive therapy for myelodysplastic syndrome.

Familial Mediterranean fever (FMF) is a recessively inherited disease characterized by recurrent episodes of systemic inflammation. The cause of this disease is the mutations affecting both the alleles of MEFV gene. We describe here a case in a heterozygous MEFV mutation complicated with myelodysplastic syndrome (MDS). Clinical symptoms and the effectiveness of colchicines in this patient are typical for FMF. The first attack of FMF in this patient was observed during immunosuppressive therapy for MDS. This case suggests the possibility that certain immunosuppressants may trigger FMF attack in asymptomatic cases carrying MEFV heterozygous mutation.

Clinical significance of microsatellite instability in the inflamed mucosa for the prediction of colonic neoplasms in patients with ulcerative colitis.

BACKGROUND AND AIM: Although molecular mechanisms underlying ulcerative colitis (UC)-associated neoplasms have been studied for years, understanding of these mechanisms remains incomplete and no good predictable marker for development of colonic neoplasms in patients with UC has been established. The aim of this study was to assess if microsatellite instability (MSI) contributes to the development of colonic neoplasms in patients with UC. METHODS: We have examined MSI in chronic inflamed and neoplastic colonic mucosa of UC patients. We have also obtained serial biopsied colonic tissues retrospectively 2-12 years before the final diagnosis from patients with high level MSI (MSI-H+) UC-associated neoplasms, and analyzed MSI using them at different periods. RESULTS: Eight of 12 UC-associated colon cancers (67%), four of six UC-associated high grade dysplasias (67%), and two of six UC-associated low grade dysplasias (33%) revealed MSI-H+ phenotypes. In contrast, 15 of 59 lesions (25%) in inflamed UC mucosa without colonic neoplasm revealed MSI-H +. Interestingly, all four patients with MSI-H+ phenotypes at the final diagnosis of UC-associated colon cancer or dysplasia had already had MSI-H+ at the stage of chronic colitis, 2-12 years before the final diagnosis. CONCLUSION: These results support the notion that MSI contributes to the carcinogenesis of UC-associated neoplasms, and indicate that this analysis in inflamed colonic mucosa at surveillance colonoscopy is useful for identifying UC patients who have high risk for neoplastic progression.

General properties of GFP-display, an electrophoretic analysis for single amino acid changes in target polypeptides.

The migrating position of green fluorescent protein (GFP)-fused polypeptide varied on an SDS/urea gel by a single amino acid change in the fused polypeptide segment. An easy detection method for a single amino acid change based on this observation was called "GFP-display." Using various target polypeptides, staphylococcal protein A (SpA), Ras, p53, and human beta3 adrenergic receptor (AR), and their mobility-shift patterns resulting from the single amino acid changes, several important properties of GFP-display were revealed as follows: (i). since the binding of dodecyl sulfate ions to acidic or hydrophilic amino acids is weaker than that to basic or hydrophobic amino acids, the ions bound weakly to the fused polypeptide segment are forced to come off by high concentrations of urea prior to the ions bound strongly, resulting in the mobility shift, (ii). the mobility shift is estimated to a certain extent using a new parameter called the "GD value" calculated from the isoelectric point, hydrophilicity, and number of fused amino acids, and (iii). the fluorescence intensity of GFP-fused polypeptide tends to increase with the average hydrophilicity of the fused polypeptide segment. GFP-display will be a helpful technique for many kinds of gene or protein studies related to amino acid substitutions such as the random mutagenesis in a gene of interest.