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.

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.

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.