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: 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: 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.

Purpose: Although the combination of androgen deprivation therapy with docetaxel or abiraterone acetate and prednisone has become a standard treatment for metastatic hormone-sensitive prostate cancer (mHSPC) and has shown improved overall survival, a subset of patients still progress to castration-resistant disease. However, the underlying molecular features in these patients remain poorly understood. Materials and Methods: We performed single-cell RNA sequencing (scRNA-seq) on 12 tissue samples, including 2 mHSPC samples, 7 primary prostate cancer (PCa) samples, and 3 matched normal samples from 7 patients. Raw sequencing data was processed by Cell Ranger software (10X Genomics) and aligned to the human reference genome (GRCh38). A comprehensive analysis of samples from patients with mHSPC was also conducted and validated using a cohort of 52 patients with mHSPC. Results: Our results identified distinct subpopulations within luminal and mononuclear phagocyte (MNP) clusters characterized by proliferative activation associated with unfavorable clinical outcomes. Furthermore, we observed that the MNP cluster exhibited significant proliferation activity. To understand the underlying mechanisms associated with aggressiveness, we conducted cell-cell interaction, copy number variation and pseudotime analysis. Utilizing 87 network genes from scRNA-seq, we classified 52 mHSPC patients into 2 molecular subtypes and demonstrated their correlation with distinct transcriptomic profiles and survival outcomes. Importantly, a 14-gene signature derived from 3 distinct gene sets exhibited a strong association with patient survival and drug response. The prognostic value of our findings was further validated in large-scale cohorts comprising localized PCa, metastatic PCa, mHSPC, and metastatic castration-resistant PCa patients. Conclusion This study provides valuable insights into the identification of high-risk patients, novel biomarkers, and potential therapeutic targets for individuals with mHSPC. Furthermore, the results in this study can serve as a basis for future investigations aimed at refining prognostic strategies and developing targeted therapies for patients with mHSPC.

Purpose: This study explored the feasibility of utilizing a deep learning artificial intelligence (AI) model to detect ileocolic intussusception on grayscale ultrasound images. Methods: This retrospective observational study incorporated ultrasound images of children who underwent emergency ultrasonography for suspected ileocolic intussusception. After excluding video clips, Doppler images, and annotated images, 40,765 images from two tertiary hospitals were included (positive-to-negative ratio: hospital A, 2,775:35,373; hospital B, 140:2,477). Images from hospital A were split into a training set, a tuning set, and an internal test set (ITS) at a ratio of 7:1.5:1.5. Images from hospital B comprised an external test set (ETS). For each image indicating intussusception, two radiologists provided a bounding box as the ground-truth label. If intussusception was suspected in the input image, the model generated a bounding box with a confidence score (0-1) at the estimated lesion location. Average precision (AP) was used to evaluate overall model performance. The performance of practical thresholds for the modelgenerated confidence score, as determined from the ITS, was verified using the ETS. Results: The AP values for the ITS and ETS were 0.952 and 0.936, respectively. Two confidence thresholds, CTopt and CTprecision, were set at 0.557 and 0.790, respectively. For the ETS, the perimage precision and recall were 95.7% and 80.0% with CTopt, and 98.4% and 44.3% with CTprecision. For per-patient diagnosis, the sensitivity and specificity were 100.0% and 97.1% with CTopt, and 100.0% and 99.0% with CTprecision. The average number of false positives per patient was 0.04 with CTopt and 0.01 for CTprecision. Conclusion The feasibility of using an AI model to diagnose ileocolic intussusception on ultrasonography was demonstrated. However, further study involving bias-free data is warranted for robust clinical validation.

Purpose: This study explored the feasibility of utilizing a deep learning artificial intelligence (AI) model to detect ileocolic intussusception on grayscale ultrasound images. Methods: This retrospective observational study incorporated ultrasound images of children who underwent emergency ultrasonography for suspected ileocolic intussusception. After excluding video clips, Doppler images, and annotated images, 40,765 images from two tertiary hospitals were included (positive-to-negative ratio: hospital A, 2,775:35,373; hospital B, 140:2,477). Images from hospital A were split into a training set, a tuning set, and an internal test set (ITS) at a ratio of 7:1.5:1.5. Images from hospital B comprised an external test set (ETS). For each image indicating intussusception, two radiologists provided a bounding box as the ground-truth label. If intussusception was suspected in the input image, the model generated a bounding box with a confidence score (0-1) at the estimated lesion location. Average precision (AP) was used to evaluate overall model performance. The performance of practical thresholds for the modelgenerated confidence score, as determined from the ITS, was verified using the ETS. Results: The AP values for the ITS and ETS were 0.952 and 0.936, respectively. Two confidence thresholds, CTopt and CTprecision, were set at 0.557 and 0.790, respectively. For the ETS, the perimage precision and recall were 95.7% and 80.0% with CTopt, and 98.4% and 44.3% with CTprecision. For per-patient diagnosis, the sensitivity and specificity were 100.0% and 97.1% with CTopt, and 100.0% and 99.0% with CTprecision. The average number of false positives per patient was 0.04 with CTopt and 0.01 for CTprecision. Conclusion The feasibility of using an AI model to diagnose ileocolic intussusception on ultrasonography was demonstrated. However, further study involving bias-free data is warranted for robust clinical validation.

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.

Background: A healthcare system’s collapse due to a pandemic, such as the coronavirus disease 2019 (COVID-19), can expose healthcare workers (HCWs) to various mental health problems. This study aimed to investigate the impact of the COVID-19 pandemic on the depression and anxiety of HCWs. Methods: A nationwide questionnaire-based survey was conducted on HCWs who worked in healthcare facilities and public health centers in Korea in December 2020. Patient Health Questionnaire-9 (PHQ-9) and Generalized Anxiety Disorder-7 (GAD-7) were used to measure depression and anxiety. To investigate factors associated with depression and anxiety, stepwise multiple logistic regression analysis was performed. Results: A total of 1,425 participating HCWs were included. The mean depression score (PHQ-9) of HCWs before and after COVID-19 increased from 2.37 to 5.39, and the mean anxiety score (GAD-7) increased from 1.41 to 3.41. The proportion of HCWs with moderate to severe depression (PHQ-9 ≥ 10) increased from 3.8% before COVID-19 to 19.5% after COVID-19, whereas that of HCWs with moderate to severe anxiety (GAD-7 ≥ 10) increased from 2.0% to 10.1%. In our study, insomnia, chronic fatigue symptoms and physical symptoms after COVID-19, anxiety score (GAD-7) after COVID-19, living alone, and exhaustion were positively correlated with depression. Furthermore, post-traumatic stress symptoms, stress score (Global Assessment of Recent Stress), depression score (PHQ-9) after COVID-19, and exhaustion were positively correlated with anxiety. Conclusion In Korea, during the COVID-19 pandemic, HCWs commonly suffered from mental health problems, including depression and anxiety. Regularly checking the physical and mental health problems of HCWs during the COVID-19 pandemic is crucial, and social support and strategy are needed to reduce the heavy workload and psychological distress of HCWs.