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.

Objective: Anterior cervical discectomy and fusion (ACDF) with anterior plating is a commonly performed procedure for cervical disc diseases. While the clinical outcomes of most reported multilevel ACDF cases are excellent, symptomatic pseudarthrosis remains a challenge, often requiring revision surgeries. This study aims to present the radiological characteristics of multilevel ACDF constructs, which can be considered during intraoperative management to prevent pseudarthrosis. Methods: This retrospective cohort study included patients who underwent multilevel (3 or 4 levels) ACDF with anterior plating between June 2010 and August 2022. Patients were regularly followed at 4 months, 12 months, and then annually postoperation. Fusion rates and characteristic radiological patterns, such as the formation of bony buttresses underneath the anterior plate, were graded and evaluated. Results: A total of 163 patients were included in the study. Overall fusion rates were 26.38%, 64.34%, and 81.58% at 4-month, 1-year, and the final follow-up, respectively. Nonunions at 4-month follow-up with tightly engaged anterior plate with bony buttress formation were more likely to fuse in the later period (Buttress grade 0 vs. 1; p=0.01, odds ratio [OR], 5.70, Buttress grade 1 vs. >2; p<0.01, OR, 12.00). Conclusion This study emphasizes the significance of pseudarthrosis following multilevel ACDF. Pseudarthrosis predominantly occurs in the caudal-most segment of the construct, particularly when it terminates at C7. Constructs that are not tightly engaged and lack bony buttress formation in the caudal part of multilevel ACDF are more likely to develop pseudarthrosis.

Objective: Anterior cervical discectomy and fusion (ACDF) with anterior plating is a commonly performed procedure for cervical disc diseases. While the clinical outcomes of most reported multilevel ACDF cases are excellent, symptomatic pseudarthrosis remains a challenge, often requiring revision surgeries. This study aims to present the radiological characteristics of multilevel ACDF constructs, which can be considered during intraoperative management to prevent pseudarthrosis. Methods: This retrospective cohort study included patients who underwent multilevel (3 or 4 levels) ACDF with anterior plating between June 2010 and August 2022. Patients were regularly followed at 4 months, 12 months, and then annually postoperation. Fusion rates and characteristic radiological patterns, such as the formation of bony buttresses underneath the anterior plate, were graded and evaluated. Results: A total of 163 patients were included in the study. Overall fusion rates were 26.38%, 64.34%, and 81.58% at 4-month, 1-year, and the final follow-up, respectively. Nonunions at 4-month follow-up with tightly engaged anterior plate with bony buttress formation were more likely to fuse in the later period (Buttress grade 0 vs. 1; p=0.01, odds ratio [OR], 5.70, Buttress grade 1 vs. >2; p<0.01, OR, 12.00). Conclusion This study emphasizes the significance of pseudarthrosis following multilevel ACDF. Pseudarthrosis predominantly occurs in the caudal-most segment of the construct, particularly when it terminates at C7. Constructs that are not tightly engaged and lack bony buttress formation in the caudal part of multilevel ACDF are more likely to develop pseudarthrosis.

Differentiated thyroid cancer demonstrates a wide range of clinical presentations, from very indolent cases to those with an aggressive prognosis. Therefore, diagnosing and treating each cancer appropriately based on its risk status is important. The Korean Thyroid Association (KTA) has provided and amended the clinical guidelines for thyroid cancer management since 2007. The main changes in this revised 2024 guideline include 1) individualization of surgical extent according to pathological tests and clinical findings, 2) application of active surveillance in low-risk papillary thyroid microcarcinoma, 3) indications for minimally invasive surgery, 4) adoption of World Health Organization pathological diagnostic criteria and definition of terminology in Korean, 5) update on literature evidence of recurrence risk for initial risk stratification, 6) addition of the role of molecular testing, 7) addition of definition of initial risk stratification and targeting thyroid stimulating hormone (TSH) concentrations according to ongoing risk stratification (ORS), 8) addition of treatment of perioperative hypoparathyroidism, 9) update on systemic chemotherapy, and 10) addition of treatment for pediatric patients with thyroid cancer.

Purpose: This study evaluated the effectiveness of different surveillance intensities on morbidity and mortality in women with breast cancer. Methods: This retrospective study included patients who had undergone breast cancer surgery in the Republic of Korea between 2009 and 2011. The patients were divided into two groups based on the intensity of their postsurgical surveillance: intensive surveillance group (ISG) and less-intensive surveillance group. Surveillance intensity was measured based on the frequency and type of follow-up diagnostic tests conducted, including mammography, ultrasonography, computed tomography, magnetic resonance imaging, bone scans, and positron emission tomography scans. Results: We included 1,356 patients with a median follow-up period of 121.2 months (range, 12.8–168.0 months). The analysis revealed no significant difference in the overall survival (OS) between the two groups within five years of surgery. However, patients with ISG exhibited significantly better breast cancer-specific survival (BCSS) and distant metastasisfree survival (DMFS) within the same period. Five years after surgery, the differences in survival outcomes between the groups were not statistically significant. Conclusion Intensive surveillance did not demonstrate a significant improvement in OS for patients with breast cancer beyond five years postoperatively. However, within the first five years, intensive surveillance was associated with better BCSS and DMFS. These findings suggest that personalized surveillance strategies may benefit specific patient subsets, particularly in the early years after treatment. Further nationwide randomized studies are warranted to refine surveillance guidelines and optimize outcomes in patients with breast cancer.

Objective: Anterior cervical discectomy and fusion (ACDF) with anterior plating is a commonly performed procedure for cervical disc diseases. While the clinical outcomes of most reported multilevel ACDF cases are excellent, symptomatic pseudarthrosis remains a challenge, often requiring revision surgeries. This study aims to present the radiological characteristics of multilevel ACDF constructs, which can be considered during intraoperative management to prevent pseudarthrosis. Methods: This retrospective cohort study included patients who underwent multilevel (3 or 4 levels) ACDF with anterior plating between June 2010 and August 2022. Patients were regularly followed at 4 months, 12 months, and then annually postoperation. Fusion rates and characteristic radiological patterns, such as the formation of bony buttresses underneath the anterior plate, were graded and evaluated. Results: A total of 163 patients were included in the study. Overall fusion rates were 26.38%, 64.34%, and 81.58% at 4-month, 1-year, and the final follow-up, respectively. Nonunions at 4-month follow-up with tightly engaged anterior plate with bony buttress formation were more likely to fuse in the later period (Buttress grade 0 vs. 1; p=0.01, odds ratio [OR], 5.70, Buttress grade 1 vs. >2; p<0.01, OR, 12.00). Conclusion This study emphasizes the significance of pseudarthrosis following multilevel ACDF. Pseudarthrosis predominantly occurs in the caudal-most segment of the construct, particularly when it terminates at C7. Constructs that are not tightly engaged and lack bony buttress formation in the caudal part of multilevel ACDF are more likely to develop pseudarthrosis.