Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein—Acanthamoeba silent-information regulator 2-like protein (AcSir2)—was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 μM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 μM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.

Background: Postoperative thyroid stimulating hormone (TSH) suppression therapy is recommended for patients with intermediate- and high-risk differentiated thyroid cancer to prevent the recurrence of thyroid cancer. With the recent increase in small thyroid cancer cases, the extent of resection during surgery has generally decreased. Therefore, questions have been raised about the efficacy and long-term side effects of TSH suppression therapy in patients who have undergone a lobectomy. Methods: This is a multicenter, prospective, randomized, controlled clinical trial in which 2,986 patients with papillary thyroid cancer are randomized into a high-TSH group (intervention) and a low-TSH group (control) after having undergone a lobectomy. The principle of treatment includes a TSH-lowering regimen aimed at TSH levels between 0.3 and 1.99 μIU/mL in the low-TSH group. The high-TSH group targets TSH levels between 2.0 and 7.99 μIU/mL. The dose of levothyroxine will be adjusted at each visit to maintain the target TSH level. The primary outcome is recurrence-free survival, as assessed by neck ultrasound every 6 to 12 months. Secondary endpoints include disease-free survival, overall survival, success rate in reaching the TSH target range, the proportion of patients with major cardiovascular diseases or bone metabolic disease, the quality of life, and medical costs. The follow-up period is 5 years. Conclusion The results of this trial will contribute to establishing the optimal indication for TSH suppression therapy in low-risk papillary thyroid cancer patients by evaluating the benefit and harm of lowering TSH levels in terms of recurrence, metabolic complications, costs, and quality of life.

Background/Aims: The mucoprotective drug rebamipide is used to treat gastritis and peptic ulcers. We compared the efficacy of Mucosta Ⓡ (rebamipide 100 mg) and its new formulation, AD-203 (rebamipide 150 mg), in treating erosive gastritis. Methods: This double-blind, active control, noninferiority, multicenter, phase 3 clinical trial randomly assigned 475 patients with endoscopically proven erosive gastritis to two groups: AD-203 twice daily or Mucosta Ⓡ thrice daily for 2 weeks. The intention-to-treat (ITT) analysis included 454 patients (AD-203, n=229; Mucosta Ⓡ , n=225), and the per-protocol (PP) analysis included 439 patients (AD-203, n=224; Mucosta Ⓡ , n=215). The posttreatment assessments included the primary (erosion improvement rate) and secondary endpoints (erosion and edema cure rates; improvement rates of redness, hemorrhage, and gastrointestinal symptoms). Drug-related adverse events were evaluated. Results: According to the ITT analysis, the erosion improvement rates (posttreatment) in AD-203-treated and Mucosta Ⓡ -treated patients were 39.7% and 43.8%, respectively. According to the PP analysis, the erosion improvement rates (posttreatment) in AD-203-treated and Mucosta Ⓡ -treated patients were 39.3% and 43.7%, respectively. The one-sided 97.5% lower limit for the improvement rate difference between the study groups was −4.01% (95% confidence interval [CI], –13.09% to 5.06%) in the ITT analysis and −4.44% (95% CI, –13.65% to 4.78%) in the PP analysis. The groups did not significantly differ in the secondary endpoints in either analysis. Twenty-four AD-203-treated and 20 Mucosta Ⓡ -treated patients reported adverse events but no serious adverse drug reactions; both groups presented similar adverse event rates. Conclusions The new formulation of rebamipide 150 mg (AD-203) twice daily was not inferior to rebamipide 100 mg (Mucosta Ⓡ ) thrice daily. Both formulations showed a similar efficacy in treating erosive gastritis.

Background: Postoperative thyroid stimulating hormone (TSH) suppression therapy is recommended for patients with intermediate- and high-risk differentiated thyroid cancer to prevent the recurrence of thyroid cancer. With the recent increase in small thyroid cancer cases, the extent of resection during surgery has generally decreased. Therefore, questions have been raised about the efficacy and long-term side effects of TSH suppression therapy in patients who have undergone a lobectomy. Methods: This is a multicenter, prospective, randomized, controlled clinical trial in which 2,986 patients with papillary thyroid cancer are randomized into a high-TSH group (intervention) and a low-TSH group (control) after having undergone a lobectomy. The principle of treatment includes a TSH-lowering regimen aimed at TSH levels between 0.3 and 1.99 μIU/mL in the low-TSH group. The high-TSH group targets TSH levels between 2.0 and 7.99 μIU/mL. The dose of levothyroxine will be adjusted at each visit to maintain the target TSH level. The primary outcome is recurrence-free survival, as assessed by neck ultrasound every 6 to 12 months. Secondary endpoints include disease-free survival, overall survival, success rate in reaching the TSH target range, the proportion of patients with major cardiovascular diseases or bone metabolic disease, the quality of life, and medical costs. The follow-up period is 5 years. Conclusion The results of this trial will contribute to establishing the optimal indication for TSH suppression therapy in low-risk papillary thyroid cancer patients by evaluating the benefit and harm of lowering TSH levels in terms of recurrence, metabolic complications, costs, and quality of life.

The first edition of the guidelines for the use of ultrasound contrast agents was published in 2004, dealing with liver applications. The second edition of the guidelines in 2008 reflected changes in the available contrast agents and updated the guidelines for the liver, as well as implementing some nonliver applications. The third edition of the contrast-enhanced ultrasound (CEUS) guidelines was the joint World Federation for Ultrasound in Medicine and Biology-European Federation of Societies for Ultrasound in Medicine and Biology (WFUMB-EFSUMB) venture in conjunction with other regional US societies such as Asian Federation of Societies for Ultrasound in Medicine and Biology, resulting in a simultaneous duplicate on liver CEUS in the official journals of both WFUMB and EFSUMB in 2013. However, no guidelines were described mainly for Sonazoid due to limited clinical experience only in Japan and Korea. The new proposed consensus statements and recommendations provide general advice on the use of Sonazoid and are intended to create standard protocols for the use and administration of Sonazoid in hepatic and pancreatobiliary applications in Asian patients and to improve patient management.