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Objective: This study aimed to assess the diagnostic value of metagenomic nextgeneration sequencing (mNGS) across synovial fluid, prosthetic sonicate fluid, and periprosthetic tissues among patients with periprosthetic joint infection (PJI), intending to optimize specimen selection for mNGS in these patients. Methods: This prospective study involved 61 patients undergoing revision arthroplasty between September 2021 and September 2022 at the First Affiliated Hospital of Zhengzhou University. Among them, 43 cases were diagnosed as PJI, and 18 as aseptic loosening (AL) based on the American Musculoskeletal Infection Society (MSIS) criteria. Preoperative or intraoperative synovial fluid, periprosthetic tissues, and prosthetic sonicate fluid were collected, each divided into two portions for mNGS and culture. Comparative analyses were conducted between the microbiological results and diagnostic efficacy derived from mNGS and culture tests. Furthermore, the variability in mNGS diagnostic efficacy for PJI across different specimen types was assessed. Results: The sensitivity and specificity of mNGS diagnosis was 93% and 94.4% for all types of PJI specimens; the sensitivity and specificity of culture diagnosis was 72.1% and 100%, respectively. The diagnostic sensitivity of mNGS was significantly higher than that of culture (χ² = 6.541, P=0.011), with no statistically significant difference in specificity (χ² = 1.029, P=0.310). The sensitivity of the synovial fluid was 83.7% and the specificity was 94.4%; the sensitivity of the prosthetic sonicate fluid was 90.7% and the specificity was 94.4%; and the sensitivity of the periprosthetic tissue was 81.4% and the specificity was 100%. Notably, the mNGS of prosthetic sonicate fluid displayed a superior pathogen detection rate compared to other specimen types. Conclusion: mNGS can function as a precise diagnostic tool for identifying pathogens in PJI patients using three types of specimens. Due to its superior ability in pathogen identification, prosthetic sonicate fluid can replace synovial fluid and periprosthetic tissue as the optimal sample choice for mNGS. [ABSTRACT FROM AUTHOR]

Objective: There is still a lack of highly sensitive methods for monitoring recurrence of colorectal cancer patients after liver metastasis surgery. The aim of this study was to evaluate the prognostic value of tumor-naive ctDNA detection after resection of colorectal liver metastases (CRLM). Methods: Patients with resectable CRLM were prospectively enrolled. Based on the tumor-naive strategy, NGS panels containing 15 colorectal cancer hotspot mutated genes were used to detect ctDNA 3-6 weeks after surgery. Results: A total of 67 patients were included in the study, and the positive rate of postoperative ctDNA was 77.6% (52/67). Patients with positive ctDNA had a significantly higher risk of recurrence after surgery (HR 3.596, 95% CI 1.479 to 8.744, P = 0.005), and a higher proportion relapsed within 3 months after surgery (46.7% vs 3.8%). The C-index of postoperative ctDNA in predicting recurrence was higher than that of CRS and postoperative CEA. The nomogram combining CRS and postoperative ctDNA can improve the accuracy of recurrence prediction. Conclusion: Tumor-naive ctDNA detection can detect molecular residual lesions in patients with colorectal cancer after liver metastasis, and its prognostic value is superior to conventional clinical factors. [ABSTRACT FROM AUTHOR]

IntroductionThe northern Red Sea has been coined a refuge for reef corals due to the exceptional thermal tolerance of these organisms. With ocean warming threatening coral reefs worldwide, a panoptic characterization of corals living in extreme conditions may provide insights into future responses of corals to environmental change. Among other factors, the genotype of the endosymbiotic algae in the family Symbiodiniaceae has been shown to have major implications on the distribution and resilience of their coral hosts. In this study, we aim at genotyping the Symbiodiniaceae communities associated with three depth generalist and one depth specialist coral species, characterized by the ability to withstand environmental conditions that are apparently limiting for other corals and occurring in a unique geographical region.MethodsWe sampled 50 corals from the northern Saudi Arabian Red Sea and the Gulf of Aqaba, covering a 97 m bathymetric gradient. We used high-throughput ITS2 gene sequencing and recovered different patterns of host–algal associations.Results and discussionThe majority of the recovered algal genotypes appeared host- and environment-specific, while others were more widely distributed. At large, coral specimens were overwhelmingly associated with symbionts from the genus Cladocopium and specifically with many previously undescribed genotypes. This suggests the selection of specific genotypes, which might confer resistance and/or resilience to their host counterparts. Interestingly, we found a limited association with Durusdinium spp. and other known tolerant taxa in mesophotic corals in the northern Red Sea, but not in the Gulf of Aqaba. The broad absence of Durusdinium spp., typically ascribed to be stress tolerant, warrants further investigation into Symbiodiniaceae species that convey environmental resilience. Our data will serve as a baseline to explore the occurrence of specific symbionts that might be contributing to coral acclimation and adaptation and to assay how biodiversity might be impacted if subjected to increasing stressors.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Nextgeneration sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes. [ABSTRACT FROM AUTHOR]

While it is considered one of the most common cancers and the leading cause of death in men worldwide, prognostic stratification and treatment modalities are still limited for patients with prostate cancer (PCa). Recently, the introduction of genomic profiling and the use of new techniques like next-generation sequencing (NGS) in many cancers provide novel tools for the discovery of new molecular targets that might improve our understanding of the genomic aberrations in PCa and the discovery of novel prognostic and therapeutic targets. In this study, we investigated the possible mechanisms through which Dickkopf-3 (DKK3) produces its possible protective role in PCa using NGS in both the DKK3 overexpression PCa cell line (PC3) model and our patient cohort consisting of nine PCa and five benign prostatic hyperplasia. Interestingly, our results have shown that DKK3 transfection-modulated genes are involved in the regulation of cell motility, senescence-associated secretory phenotype (SASP), and cytokine signaling in the immune system, as well as in the regulation of adaptive immune response. Further analysis of our NGS using our in vitro model revealed the presence of 36 differentially expressed genes (DEGs) between DKK3 transfected cells and PC3 empty vector. In addition, both CP and ACE2 genes were differentially expressed not only between the transfected and empty groups but also between the transfected and Mock cells. The top common DEGs between the DKK3 overexpression cell line and our patient cohort are the following: IL32, IRAK1, RIOK1, HIST1H2BB, SNORA31, AKR1B1, ACE2, and CP. The upregulated genes including IL32, HIST1H2BB, and SNORA31 showed tumor suppressor functions in various cancers including PCa. On the other hand, both IRAK1 and RIOK1 were downregulated and involved in tumor initiation, tumor progression, poor outcome, and radiotherapy resistance. Together, our results highlighted the possible role of the DKK3-related genes in protecting against PCa initiation and progression.

To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies' enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to assess the diagnostic value of metagenomic next-generation sequencing (mNGS) across synovial fluid, prosthetic sonicate fluid, and periprosthetic tissues among patients with periprosthetic joint infection (PJI), intending to optimize specimen selection for mNGS in these patients., Methods: This prospective study involved 61 patients undergoing revision arthroplasty between September 2021 and September 2022 at the First Affiliated Hospital of Zhengzhou University. Among them, 43 cases were diagnosed as PJI, and 18 as aseptic loosening (AL) based on the American Musculoskeletal Infection Society (MSIS) criteria. Preoperative or intraoperative synovial fluid, periprosthetic tissues, and prosthetic sonicate fluid were collected, each divided into two portions for mNGS and culture. Comparative analyses were conducted between the microbiological results and diagnostic efficacy derived from mNGS and culture tests. Furthermore, the variability in mNGS diagnostic efficacy for PJI across different specimen types was assessed., Results: The sensitivity and specificity of mNGS diagnosis was 93% and 94.4% for all types of PJI specimens; the sensitivity and specificity of culture diagnosis was 72.1% and 100%, respectively. The diagnostic sensitivity of mNGS was significantly higher than that of culture (X 2 = 6.541, P =0.011), with no statistically significant difference in specificity (X 2 = 1.029, P =0.310). The sensitivity of the synovial fluid was 83.7% and the specificity was 94.4%; the sensitivity of the prosthetic sonicate fluid was 90.7% and the specificity was 94.4%; and the sensitivity of the periprosthetic tissue was 81.4% and the specificity was 100%. Notably, the mNGS of prosthetic sonicate fluid displayed a superior pathogen detection rate compared to other specimen types., Conclusion: mNGS can function as a precise diagnostic tool for identifying pathogens in PJI patients using three types of specimens. Due to its superior ability in pathogen identification, prosthetic sonicate fluid can replace synovial fluid and periprosthetic tissue as the optimal sample choice for mNGS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tan, Wu, Zhan, Sheng, Tang, Xu and Ma.)

Objective: There is still a lack of highly sensitive methods for monitoring recurrence of colorectal cancer patients after liver metastasis surgery. The aim of this study was to evaluate the prognostic value of tumor-naive ctDNA detection after resection of colorectal liver metastases (CRLM)., Methods: Patients with resectable CRLM were prospectively enrolled. Based on the tumor-naive strategy, NGS panels containing 15 colorectal cancer hotspot mutated genes were used to detect ctDNA 3-6 weeks after surgery., Results: A total of 67 patients were included in the study, and the positive rate of postoperative ctDNA was 77.6% (52/67). Patients with positive ctDNA had a significantly higher risk of recurrence after surgery (HR 3.596, 95% CI 1.479 to 8.744, P = 0.005), and a higher proportion relapsed within 3 months after surgery (46.7% vs 3.8%). The C-index of postoperative ctDNA in predicting recurrence was higher than that of CRS and postoperative CEA. The nomogram combining CRS and postoperative ctDNA can improve the accuracy of recurrence prediction., Conclusion: Tumor-naive ctDNA detection can detect molecular residual lesions in patients with colorectal cancer after liver metastasis, and its prognostic value is superior to conventional clinical factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jiang, Huang, Yang, Chen, Shen, Zhang, Pan, Wang and Guo.)

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Misu, Yoshikawa, Sugimoto, Takamatsu, Kurosu, Ouji, Yoshikawa, Shimojima, Ebihara and Saijo.)

While it is considered one of the most common cancers and the leading cause of death in men worldwide, prognostic stratification and treatment modalities are still limited for patients with prostate cancer (PCa). Recently, the introduction of genomic profiling and the use of new techniques like next-generation sequencing (NGS) in many cancers provide novel tools for the discovery of new molecular targets that might improve our understanding of the genomic aberrations in PCa and the discovery of novel prognostic and therapeutic targets. In this study, we investigated the possible mechanisms through which Dickkopf-3 (DKK3) produces its possible protective role in PCa using NGS in both the DKK3 overexpression PCa cell line (PC3) model and our patient cohort consisting of nine PCa and five benign prostatic hyperplasia. Interestingly, our results have shown that DKK3 transfection-modulated genes are involved in the regulation of cell motility, senescence-associated secretory phenotype (SASP), and cytokine signaling in the immune system, as well as in the regulation of adaptive immune response. Further analysis of our NGS using our in vitro model revealed the presence of 36 differentially expressed genes (DEGs) between DKK3 transfected cells and PC3 empty vector. In addition, both CP and ACE2 genes were differentially expressed not only between the transfected and empty groups but also between the transfected and Mock cells. The top common DEGs between the DKK3 overexpression cell line and our patient cohort are the following: IL32, IRAK1, RIOK1, HIST1H2BB, SNORA31, AKR1B1 , ACE2 , and CP . The upregulated genes including IL32 , HIST1H2BB , and SNORA31 showed tumor suppressor functions in various cancers including PCa. On the other hand, both IRAK1 and RIOK1 were downregulated and involved in tumor initiation, tumor progression, poor outcome, and radiotherapy resistance. Together, our results highlighted the possible role of the DKK3-related genes in protecting against PCa initiation and progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shareef, Hachim, Talaat, Bhamidimarri, Ershaid, Ilce, Venkatachalam, Eltayeb, Hamoudi and Hachim.)