Background: The 16S rRNA-targeted next-generation sequencing (NGS) has been widely used as the primary tool for microbiome analysis. However, whether the sequenced microbial diversity absolutely represents the original sample composition remains unclear. This study aimed to evaluate whether 16S rRNA gene-targeted NGS accurately captures bacterial community composition. Methods: Mock communities were constructed using equal amounts of DNA from 18 bacterial strains in three formats: genomic DNA, recombinant plasmids, and polymerase chain reaction (PCR) templates. The V3V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq. Results: Data regression analysis revealed that the recombinant plasmid produced more accurate and precise correlation curve than that by the gDNA and PCR products, with a slope closest to 1 (1.0082) and the highest R² value (0.9975). Despite the same input amount of bacterial DNA, the NGS read distribution varied across all three mock communities. Using multiple regression analysis, we found that the guanine-cytosine (GC) content of the V3V4 region, 16S rRNA gene, size of gDNA, and copy number of 16S rRNA were significantly associated with the NGS output of each bacterial species. Conclusion This study demonstrated that recombinant plasmids are the preferred option for quality control and that NGS output is biased owing to certain bacterial characteristics, such as %GC content, gDNA size, and 16S rRNA gene copy number. Further research is required to develop a system that compensates for NGS process biases using mock communities.

Background: This study analyzed the epidemiology and treatment outcomes of germ cell tumorpatients at a single institution. Methods: A retrospective analysis was conducted on intracranial germ cell tumor (iGCT) pa-tients treated at a single tertiary hospital from 2004 to 2019. Patients were categorized based on treatment modality: Korean Society for Pediatric Neuro-Oncology (KSPNO) protocol or bleomycin, etoposide, and cisplatin with radiation therapy. Results: Forty-nine iGCT patients treated with combined chemotherapy and radiotherapywere analyzed. The median age was 19 years (range: 6–40), with a median follow-up duration of 148.0 months (range: 10.5–265.5). Tumors were most common in the pineal gland (51.0%). Although no significant differences in outcomes were observed between treatment modalities, outcomes varied significantly by pathological type. The 10-year progression-free survival rates for germinoma and non-germinomatous germ cell tumors (NGGCTs) were 88.1% and 32.7%, respectively (p=0.003), while the 10-year overall survival rates were 92.9% and 67.5%, respectively (p<0.001). Fourteen patients experienced CTCAE (Common Terminology Criteria for Adverse Events) grade ≥3 adverse events, with one eventrelated death. Conclusion Pure germinoma demonstrated higher survival and lower recurrence rates comparedto NGGCT. The KSPNO protocol appears to be an acceptable and safe treatment option for iGCT patients. Further multi-institutional studies with larger cohorts are warranted.

Background: The 16S rRNA-targeted next-generation sequencing (NGS) has been widely used as the primary tool for microbiome analysis. However, whether the sequenced microbial diversity absolutely represents the original sample composition remains unclear. This study aimed to evaluate whether 16S rRNA gene-targeted NGS accurately captures bacterial community composition. Methods: Mock communities were constructed using equal amounts of DNA from 18 bacterial strains in three formats: genomic DNA, recombinant plasmids, and polymerase chain reaction (PCR) templates. The V3V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq. Results: Data regression analysis revealed that the recombinant plasmid produced more accurate and precise correlation curve than that by the gDNA and PCR products, with a slope closest to 1 (1.0082) and the highest R² value (0.9975). Despite the same input amount of bacterial DNA, the NGS read distribution varied across all three mock communities. Using multiple regression analysis, we found that the guanine-cytosine (GC) content of the V3V4 region, 16S rRNA gene, size of gDNA, and copy number of 16S rRNA were significantly associated with the NGS output of each bacterial species. Conclusion This study demonstrated that recombinant plasmids are the preferred option for quality control and that NGS output is biased owing to certain bacterial characteristics, such as %GC content, gDNA size, and 16S rRNA gene copy number. Further research is required to develop a system that compensates for NGS process biases using mock communities.

Background: The 16S rRNA-targeted next-generation sequencing (NGS) has been widely used as the primary tool for microbiome analysis. However, whether the sequenced microbial diversity absolutely represents the original sample composition remains unclear. This study aimed to evaluate whether 16S rRNA gene-targeted NGS accurately captures bacterial community composition. Methods: Mock communities were constructed using equal amounts of DNA from 18 bacterial strains in three formats: genomic DNA, recombinant plasmids, and polymerase chain reaction (PCR) templates. The V3V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq. Results: Data regression analysis revealed that the recombinant plasmid produced more accurate and precise correlation curve than that by the gDNA and PCR products, with a slope closest to 1 (1.0082) and the highest R² value (0.9975). Despite the same input amount of bacterial DNA, the NGS read distribution varied across all three mock communities. Using multiple regression analysis, we found that the guanine-cytosine (GC) content of the V3V4 region, 16S rRNA gene, size of gDNA, and copy number of 16S rRNA were significantly associated with the NGS output of each bacterial species. Conclusion This study demonstrated that recombinant plasmids are the preferred option for quality control and that NGS output is biased owing to certain bacterial characteristics, such as %GC content, gDNA size, and 16S rRNA gene copy number. Further research is required to develop a system that compensates for NGS process biases using mock communities.

Background: This study analyzed the epidemiology and treatment outcomes of germ cell tumorpatients at a single institution. Methods: A retrospective analysis was conducted on intracranial germ cell tumor (iGCT) pa-tients treated at a single tertiary hospital from 2004 to 2019. Patients were categorized based on treatment modality: Korean Society for Pediatric Neuro-Oncology (KSPNO) protocol or bleomycin, etoposide, and cisplatin with radiation therapy. Results: Forty-nine iGCT patients treated with combined chemotherapy and radiotherapywere analyzed. The median age was 19 years (range: 6–40), with a median follow-up duration of 148.0 months (range: 10.5–265.5). Tumors were most common in the pineal gland (51.0%). Although no significant differences in outcomes were observed between treatment modalities, outcomes varied significantly by pathological type. The 10-year progression-free survival rates for germinoma and non-germinomatous germ cell tumors (NGGCTs) were 88.1% and 32.7%, respectively (p=0.003), while the 10-year overall survival rates were 92.9% and 67.5%, respectively (p<0.001). Fourteen patients experienced CTCAE (Common Terminology Criteria for Adverse Events) grade ≥3 adverse events, with one eventrelated death. Conclusion Pure germinoma demonstrated higher survival and lower recurrence rates comparedto NGGCT. The KSPNO protocol appears to be an acceptable and safe treatment option for iGCT patients. Further multi-institutional studies with larger cohorts are warranted.

Background: The 16S rRNA-targeted next-generation sequencing (NGS) has been widely used as the primary tool for microbiome analysis. However, whether the sequenced microbial diversity absolutely represents the original sample composition remains unclear. This study aimed to evaluate whether 16S rRNA gene-targeted NGS accurately captures bacterial community composition. Methods: Mock communities were constructed using equal amounts of DNA from 18 bacterial strains in three formats: genomic DNA, recombinant plasmids, and polymerase chain reaction (PCR) templates. The V3V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq. Results: Data regression analysis revealed that the recombinant plasmid produced more accurate and precise correlation curve than that by the gDNA and PCR products, with a slope closest to 1 (1.0082) and the highest R² value (0.9975). Despite the same input amount of bacterial DNA, the NGS read distribution varied across all three mock communities. Using multiple regression analysis, we found that the guanine-cytosine (GC) content of the V3V4 region, 16S rRNA gene, size of gDNA, and copy number of 16S rRNA were significantly associated with the NGS output of each bacterial species. Conclusion This study demonstrated that recombinant plasmids are the preferred option for quality control and that NGS output is biased owing to certain bacterial characteristics, such as %GC content, gDNA size, and 16S rRNA gene copy number. Further research is required to develop a system that compensates for NGS process biases using mock communities.

Background: The 16S rRNA-targeted next-generation sequencing (NGS) has been widely used as the primary tool for microbiome analysis. However, whether the sequenced microbial diversity absolutely represents the original sample composition remains unclear. This study aimed to evaluate whether 16S rRNA gene-targeted NGS accurately captures bacterial community composition. Methods: Mock communities were constructed using equal amounts of DNA from 18 bacterial strains in three formats: genomic DNA, recombinant plasmids, and polymerase chain reaction (PCR) templates. The V3V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq. Results: Data regression analysis revealed that the recombinant plasmid produced more accurate and precise correlation curve than that by the gDNA and PCR products, with a slope closest to 1 (1.0082) and the highest R² value (0.9975). Despite the same input amount of bacterial DNA, the NGS read distribution varied across all three mock communities. Using multiple regression analysis, we found that the guanine-cytosine (GC) content of the V3V4 region, 16S rRNA gene, size of gDNA, and copy number of 16S rRNA were significantly associated with the NGS output of each bacterial species. Conclusion This study demonstrated that recombinant plasmids are the preferred option for quality control and that NGS output is biased owing to certain bacterial characteristics, such as %GC content, gDNA size, and 16S rRNA gene copy number. Further research is required to develop a system that compensates for NGS process biases using mock communities.

Background: This study analyzed the epidemiology and treatment outcomes of germ cell tumorpatients at a single institution. Methods: A retrospective analysis was conducted on intracranial germ cell tumor (iGCT) pa-tients treated at a single tertiary hospital from 2004 to 2019. Patients were categorized based on treatment modality: Korean Society for Pediatric Neuro-Oncology (KSPNO) protocol or bleomycin, etoposide, and cisplatin with radiation therapy. Results: Forty-nine iGCT patients treated with combined chemotherapy and radiotherapywere analyzed. The median age was 19 years (range: 6–40), with a median follow-up duration of 148.0 months (range: 10.5–265.5). Tumors were most common in the pineal gland (51.0%). Although no significant differences in outcomes were observed between treatment modalities, outcomes varied significantly by pathological type. The 10-year progression-free survival rates for germinoma and non-germinomatous germ cell tumors (NGGCTs) were 88.1% and 32.7%, respectively (p=0.003), while the 10-year overall survival rates were 92.9% and 67.5%, respectively (p<0.001). Fourteen patients experienced CTCAE (Common Terminology Criteria for Adverse Events) grade ≥3 adverse events, with one eventrelated death. Conclusion Pure germinoma demonstrated higher survival and lower recurrence rates comparedto NGGCT. The KSPNO protocol appears to be an acceptable and safe treatment option for iGCT patients. Further multi-institutional studies with larger cohorts are warranted.

Background: Anterior cruciate ligament (ACL) reconstruction is commonly performed to prevent decreased knee function and restore stability in middle-aged and even older patients. However, few studies have compared the long-term clinical outcomes of ACL reconstruction between older, younger, and middle-aged patients. The purpose of this study was to compare the long-term clinical outcomes of ACL reconstruction in older patients with those in younger and middle-aged patients. Methods: A total of 352 patients who underwent primary ACL reconstruction between January 2003 and March 2008 were retrospectively reviewed and classified into three groups (group A: 246 [age, 20–29 years], group B: 72 [age, 40–49 years], group C: 34 [age, 50–65 years]). The mean follow-up period was 14.2 ± 1.6 years. Clinical outcomes were evaluated and compared between groups. Results: The differences in the range of motion, clinical scores, and stability tests were not statistically significant among the three groups. The difference in the graft failure rate among the three groups was significant (group A: 16 [6.5%], group B: 7 [9.7%], group C: 6 [17.6%]; p = 0.040). In particular, when compared between the two groups, there was a significant difference between group A and group C (p = 0.036). The 10-year survival rates were 93.5%, 90.3%, and 82.4% for groups A, B, and C, respectively (p = 0.048). Conclusions Although graft failure rates were higher in older patients than younger and middle-aged patients, clinical outcomes of ACL reconstruction in older patients were comparable to those of younger and middle-aged patients in terms of the range of motion, clinical scores, and stability tests at a minimum follow-up of 10 years.

Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019.In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virusinfected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105PFU; however, 1×102 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.