High Copy Number Research Articles

Comparative assessment of the copy number of satellite repeats in the genome of Triticeae species.

Satellite repeats are a significant component of the genome of Triticeae and play a crucial role in the speciation. They are a valuable tool for studying these processes. Pseudoroegneria species play a special role among grasses, as they are considered putative donors of the St-genome in many polyploid species. The aim of this study was to compare the copy number of satellite repeats in the genomes of Triticeae species. Quantitative real-time PCR was applied to determine the copy numbers of 22 newly discovered satellite repeats revealed in the whole-genome sequences of Pseudoroegneria species and one additional repeat previously identified in the genome of Aegilops crassa. The study focused on seven species of Pseudoroegneria, three species of Thinopyrum, Elymus pendulinus, Ae. tauschii, Secale cereale, and Triticum aestivum. Based on the copy number level and coefficients of variation, we identified three groups of repeats: those with low variability between species (medium-copy CL82), those with medium variability (low- and medium-copy CL67, CL3, CL185, CL119, CL192, CL89, CL115, CL95, CL168), and those with high coefficients of variation (CL190, CL184, CL300, CL128, CL207, CL69, CL220, CL101, CL262, CL186, CL134, CL251, CL244). CL69 exhibited a specific high copy number in all Pseudoroegneria species, while CL101 was found in both Pseudoroegneria and Th. junceum, CL244 in Th. bessarabicum, CL184 in P. cognata and S. cereale. CL95, CL128, CL168, CL186, CL207, and CL300 exhibited higher copy numbers in P. cognata compared to other species; CL3, CL95, CL115, CL119, CL190, CL220, CL207, and CL300 in P. kosaninii; CL89 in P. libanotica; CL134 in P. geniculata. Our assessment of the copy number of new satellite repeats in the St-genome and the analysis of their amplification specificity between species can contribute to the molecular-genetic and chromosome markers used for evolutionary, phylogenetic, and population studies of Triticeae species.

Generation of a stably transfected mouse embryonic stem cell line for inducible differentiation to excitatory neurons

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.

Food and Environmental Virology: Use of Passive Sampling to Characterize the Presence of SARS-CoV-2 and Other Viruses in Wastewater.

Fecal shedding of SARS-CoV-2 leads to a renaissance of wastewater-based epidemiology (WBE) as additional tool to follow epidemiological trends in the catchment of treatment plants. As alternative to the most commonly used composite samples in surveillance programs, passive sampling is increasingly studied. However, the many sorbent materials in different reports hamper the comparison of results and a standardization of the approach is necessary. Here, we compared different cost-effective sorption materials (cheesecloths, gauze swabs, electronegative filters, glass wool, and tampons) in torpedo-style housings with composite samples. Despite a remarkable variability of the concentration of SARS-CoV-2-specific gene copies, analysis of parallel-deposited passive samplers in the sewer demonstrated highest rate of positive samples and highest number of copies by using cheesecloths. Using this sorption material, monitoring of wastewater of three small catchments in the City of Dresden resulted in a rate of positive samples of 50% in comparison with composite samples (98%). During the investigation period, incidence of reported cases of SARS-CoV-2 in the catchments ranged between 16 and 170 per 100,000 persons and showed no correlation with the measured concentrations of E gene in wastewater. In contrast, constantly higher numbers of gene copies in passive vs. composite samples were found for human adenovirus and crAssphage indicating strong differences of efficacy of methods concerning the species investigated. Influenza virus A and B were sporadically detected allowing no comparison of results. The study contributes to the further understanding of possibilities and limits of passive sampling approaches in WBE.

Exploring quantitative traits-associated copy number deletions through reanalysis of UK10K consortium whole genome sequencing cohorts

ObjectivesWe performed comprehensive association analyses of common high-confidence gnomAD-reported copy number deletions (CNDs) with 60 quantitative traits from UK10K consortium WGS data.MethodsThe study made use of data generated by the UK10K Consortium. UK10K consortium WGS data consist of TwinsUK (n = 1754, middle-aged females) and ALSPAC (n = 1867, birth to adolescence) cohorts. UK10K consortium called 18,739 CNDs (hg19) with GenomeSTRiP software. After filtering out variants with minor allele frequency < 0.05 or HWE P < 1.0 × 10− 6, 1222 (TwinsUK) and 1211 (ALSPAC) CNDs remained for association analyses with 60 normalized quantitative traits.ResultsWe identified 23 genome-wide significant associations at 13 loci, among which 2 associations reached experiment-wide significance. We found that two common deletions in chromosome 4, located between WDR1 and ZNF518B (23.3 kb, dbVar ID:nssv15888957, 4:10211262–10,234,569 and 9.8 kb, dbVar ID:nssv15888975, 4:10392422–10,402,191), were associated with uric acid levels (P = 5.23 × 10− 11 and 2.29 × 10− 8, respectively). We also discovered a novel deletion spanning chromosome 18 (823 bp, dbVar ID: nssv15841628, 8:74347187–74,348,010) associated with low HDL cholesterol levels (P = 4.15 × 10− 7). Additionally, we observed two red blood cell traits-associated loci with genome-wide significance, a 13.2 kb deletion in 7q22.1 (nssv15922542) and a 3.7 kb deletion in 12q24.12 (nssv15813226), both of which were located in regions previously reported to be associated with red blood cell traits. Two deletions in 11q11 (nssv15803200 and nssv15802240), where clusters of multiple olfactory receptor genes exist, and a deletion (nssv15929560) upstream to DOCK5 were associated with childhood obesity. Finally, when defining Trait-Associated copy number Deletions (TADs) as CNDs with phenotype associations at sub-threshold significance (P < 10− 3), we identified 157 (97.5%) out of 161 TADs in non-coding regions, with a mean size of 4 kb (range: 209 − 47,942 bp).ConclusionWe conducted a reanalysis of the UK10K Whole Genome Sequencing cohort, which led to the identification of multiple high confidence copy number deletions associated with quantitative traits. These deletions have standard dbVar IDs and replicate previous findings, as well as reveal novel loci that require further replication studies.

Acute Kidney Injury and BK Polyomavirus in Urine Sediment Cells.

Polyomaviruses are widespread, with BK viruses being most common in humans who require immunosuppression due to allotransplantation. Infection with BK polyomavirus (BKV) may manifest as BK virus-associated nephropathy and hemorrhagic cystitis. Established diagnostic methods include the detection of polyomavirus in urine and blood by PCR and in tissue biopsies via immunohistochemistry. In this study, 79 patients with pathological renal retention parameters and acute kidney injury (AKI) were screened for BK polyomavirus replication by RNA extraction, reverse transcription, and virus-specific qPCR in urine sediment cells. A short fragment of the VP2 coding region was the target of qPCR amplification; patients with (n = 31) and without (n = 48) a history of renal transplantation were included. Urine sediment cell immunofluorescence staining for VP1 BK polyomavirus protein was performed using confocal microscopy. In 22 patients with acute renal injury, urinary sediment cells from 11 participants with kidney transplantation (KTX) and from 11 non-kidney transplanted patients (nonKTX) were positive for BK virus replication. BK virus copies were found more frequently in patients with AKI stage III (n = 14). Higher copy numbers were detected in KTX patients having experienced BK polyoma-nephropathy (BKPyVAN) in the past or diagnosed recently by histology (5.6 × 109-3.1 × 1010). One patient developed BK viremia following delayed graft function (DGF) with BK virus-positive urine sediment. In nonKTX patients with BK copies, decoy cells were absent; however, positive staining of cells was found with epithelial morphology. Decoy cells were only found in KTX patients with BKPyVAN. In AKI, damage to the tubular epithelium itself may render the epithelial cells more permissive for polyoma replication. This non-invasive diagnostic approach to assess BK polyomavirus replication in urine sediment cells has the potential to identify KTX patients at risk for viremia and BKPyVAN during AKI. This method might serve as a valuable screening tool for close monitoring and tailored immunosuppression decisions.

Extrachromosomal DNA: Biogenesis and Functions in Cancer

In cancer, oncogenes can untether themselves from chromosomes onto circular, extrachromosomal DNA (ecDNA) particles. ecDNA are common in many of the most aggressive forms of cancer of women and men and of adults and children, and they contribute to treatment resistance and shorter survival for patients. Hiding in plain sight and missing from cancer genome maps, ecDNA was not, until recently, widely recognized to be an important feature of cancer pathogenesis. However, extensive new data demonstrate that ecDNA is a frequent and potent driver of aggressive cancer growth and treatment failure that can arise early or late in the course of the disease. The non-Mendelian genetics of ecDNA lies at the heart of the problem. By untethering themselves from chromosomes, ecDNA are randomly distributed to daughter cells during cell division, promoting high oncogene copy number, intratumoral genetic heterogeneity, accelerated tumor evolution, and treatment resistance due to rapid genome change. Further, the circular shape of ecDNA, and its high level of chromatin accessibility, promotes oncogene transcription and generates unique enhancer–promoter interactions in cis, as well as cooperative regulatory interactions between ecDNA particles in trans. In this review, we discuss the state of the field and its implications for patients with oncogene-amplified cancers.

Hepatitis E virus infection of transplanted kidneys

Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy numbers led us to study HEV infection within renal transplants. To investigate the frequency of renal HEV infection in transplanted patients, 15 samples from patients with contemporaneous diagnoses of HEV infection were identified at our center. Ten samples had sufficient residual paraffin tissue for immunofluorescence (IF) and RNA-fluorescence-in-situ-hybridization (RNA-FISH). The biopsy of the pediatric index patient was additionally sufficient for tissue polymerase chain reaction and electron microscopy. HEV RNA was detected in paraffin tissue of the index patient by tissue polymerase chain reaction. Subsequently, HEV infection was localized in tubular epithelial cells by IF, RNA-FISH, and electron microscopy. One additional biopsy from an adult was positive for HEV by RNA-FISH and IF. Focal IF positivity for HEV peptide was observed in 7 additional allografts. Ribavirin therapy was not successful in the pediatric index patient; after relapse, ribavirin is still administered. In the second patient, successful elimination of HEV was achieved after short-course ribavirin therapy. HEV infection is an important differential diagnosis for T cell rejection within transplanted kidneys. Immunostaining of HEV peptide does not necessarily prove acute infection. RNA-FISH seems to be a reliable method to localize HEV.

Application of multiple binding sites for LAMP primers across P. falciparum genome improves detection of the parasite from whole blood samples

IntroductionMalaria remains a significant health concern, particularly in regions with widespread prevalence. As the transmission rates decrease, there is a rise in low-density infections with the causative parasite, P. falciparum, that often escape detection through standard point-of-care diagnostic tools. In-low transmission areas, even few undetected cases can trigger outbreaks, necessitating rapid and sensitive diagnostics. Loop-mediated isothermal Amplification (LAMP) stands out as a nucleic acid technique that can easily utilizes un-processed samples such of saliva, urine, and lysed whole blood templates for a sensitive detection. However, most nucleic acid tests detect genes with few copies per parasite making it difficult to detect low-density parasitaemia.MethodsWe selected Pfr364 multi-copy repeats of the P. falciparum genome as a target for amplification due to their higher copy number, ideal for rapid amplification, addressing amplification drawbacks of limited parasites DNA. We used a sequence clustering approach to design a novel set of LAMP primers, capable of binding to multiple sites. Subsequently, we developed a hydroxynaphthol blue (HNB) colorimetric LAMP assay, using genomic DNA obtained from the 3D7 strain cultivated in vitro. This assay’s performance was validated using archived clinical samples of both whole blood and matched saliva, ensuring accuracy through comparative analysis against gold standard, nested PCR, targeting the 18S RNA gene.ResultsThe HNB-LAMP assay achieved rapid amplification within 15 minutes and exhibited high sensitivity with a limit of detection of 1 parasite. Further, the LAMP assay was robust in whole blood lysed with Triton X-100 and heat-treated saliva clinical samples. Against nested PCR, the assay showed sensitivity of 100% for whole blood and 40% for saliva samples. Moreover, co-analysis with the nested PCR showed a perfect agreement between the two techniques. (K = 0.99 for whole blood, and 0.66 for saliva).ConclusionOur study presents a method for detecting P. falciparum using LAMP, which results in increased sensitivity, shorter assay times, and a simpler workflow than nucleic acid tests relying on conventional DNA extraction and additional equipment for result interpretation. These findings hold great promise for improved malaria diagnosis, especially in settings where low-density parasitaemia is prevalent and rapid and accurate malaria detection is crucial.

Assessment of Immune Responses to a Trivalent Pichinde Virus-Vectored Vaccine Expressing Hemagglutinin Genes from Three Co-Circulating Influenza A Virus Subtypes in Pigs.

Pichinde virus (PICV) can infect several animal species and has been developed as a safe and effective vaccine vector. Our previous study showed that pigs vaccinated with a recombinant PICV-vectored vaccine expressing the hemagglutinin (HA) gene of an H3N2 influenza A virus of swine (IAV-S) developed virus-neutralizing antibodies and were protected against infection by the homologous H3N2 strain. The objective of the current study was to evaluate the immunogenicity and protective efficacy of a trivalent PICV-vectored vaccine expressing HA antigens from the three co-circulating IAV-S subtypes: H1N1, H1N2, and H3N2. Pigs immunized with the trivalent PICV vaccine developed virus-neutralizing (VN) and hemagglutination inhibition (HI) antibodies against all three matching IAV-S. Following challenge infection with the H1N1 strain, five of the six pigs vaccinated with the trivalent vaccine had no evidence of IAV-S RNA genomes in nasal swabs and bronchoalveolar lavage fluid, while all non-vaccinated control pigs showed high number of copies of IAV-S genomic RNA in these two types of samples. Overall, our results demonstrate that the trivalent PICV-vectored vaccine elicits antibody responses against the three targeted IAV-S strains and provides protection against homologous virus challenges in pigs. Therefore, PICV exhibits the potential to be explored as a viral vector for delivering multiple vaccine antigens in swine.

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B092.

Abstract B082: A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC)

Abstract Introduction: KRASG12C inhibitors are a new therapeutic approach to treat KRAS mutant-driven cancers, but as with other oncogene directed therapies, adaptive resistance develops quickly and frequently. The predominant resistance mechanisms are 1) secondary mutations in KRAS and/or other mitogen-activated protein kinase (MAPK) signaling genes and 2) amplification of KRAS or MAPK genes. Extrachromosomal DNA (ecDNA) are the predominant site of focal high copy number oncogene or resistance gene amplifications in human cancers. Previous studies have demonstrated KRASG12C amplification on ecDNA to be an acquired resistance mechanism after treatment with approved KRASG12C inhibitors. Cancers enabled by ecDNA remain underserved by current therapeutic options, including targeted therapies, and have poor prognosis. An in silico “loss of function” screen revealed ribonucleotide reductase (RNR) as a critical target required for the survival of ecDNA amplified tumor cells. RNR is the rate-limiting enzyme in the biosynthesis of deoxyribonucleotide triphosphates (dNTP), the building blocks of DNA and ecDNA replication and repair. While non-selective drugs with RNR inhibitory activity (e.g., gemcitabine) indirectly validate RNR as a cancer target, no selective small molecules targeting RNR have been approved. Methods: We have developed BBI-825, a potent, selective, and orally bioavailable RNR inhibitor (RNRi) for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers. Results: BBI-825 inhibits RNR with an IC50 of 20 nM in vitro and reduces deoxyadenosine triphosphate (dATP) levels in ecDNA amplified cancer cells, with an EC50 of less than 100 nM. On target selectivity of BBI-825 was confirmed using SAFETYscan cellular screening assay investigating target selectivity over 78 targets spanning hormone receptors, kinases, calcium, GPCR, ion channel, and transporter signaling. Importantly, BBI-825 prevented resistance-conferring ecDNA-enabled amplification of KRASG12C in KRASG12C mutated colorectal cancer cells treated with the KRAS inhibitor adagrasib in vitro. Prevention of ecDNA amplification correlated with antiproliferation of BBI-825-treated tumor cells in long-term cultures. Treatment of KRASG12C mutated CRC tumor xenograft models with single agent adagrasib resulted in rapid acquired resistance through KRASG12C amplification on ecDNA. In concordance with in vitro observations, co-treatment with BBI-825 delayed the development of adagrasib resistance concurrent with an abrogation of ecDNA-enabled KRASG12C amplification (P=0.001). Furthermore, BBI-825 significantly inhibited the growth of pre-established adagrasib-resistant KRASG12C ecDNA-amplified tumors in vivo (tumor growth inhibition (TGI) = 92%, p&amp;lt;0.002), reinforcing clinical actionability in the KRAS inhibitor-refractory setting. Conclusions: These findings support the advancement of BBI-825 as a selective RNR inhibitor for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers, including KRASG12C mutant CRC. Citation Format: Sudhir Chowdhry, Ryan Hansen, Jacques Mauger, AnneMarie Pferdekamper, Edison Tse, Ben Norman, Jafar Moininazeri, Salvador Garcia, Yen Truong, Debbie Liao, Joshua Lange, Evan Holmes, Anthony Celeste, Dave Solis, Auzon Steffy, Joshua Plum, Julie Wiese, Julius Apuy, Ardalan Ardeshiri, Snezana Milutinovic, Deepti Wilkinson, Anthony Pinkerton, Christian Hassig, Shailaja Kasibhatla. A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B082.

The Clinical Significance and Prognostic Value of Serum Beta-2 Microglobulin in Adult Lymphoma-Associated Hemophagocytic Lymphohistiocytosis: A Multicenter Analysis of 326 Patients

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome characterized by an excessive immune response. Beta-2 microglobulin (B2M) forms the light chain section of the major histocompatibility complex (MHC) class Ⅰ antigen. The prognostic role of B2M in lymphoma-associated HLH remains to be determined. This study was to investigate the prognostic role of serum B2M in adult lymphoma-associated hemophagocytic lymphohistiocytosis (HLH). Methods: The clinical and laboratory characteristics of adult patients in a multicenter cohort with lymphoma-associated HLH who had baseline serum B2M levels between August 2009 and June 2023 were retrospectively analyzed. The diagnosis of HLH was established according to the HLH-2004 criteria. Results: A total of 326 cases were included and the median serum B2M level was 5.19 mg/L. The most common subtype of lymphoma was T/NK cell lymphoma (51.8%, 169/326), followed by B-cell non-Hodgkin lymphoma (45.4%, 148/227) and Hodgkin lymphoma 8/326 (2.5). The optimal cut-off value of serum B2M for predicting overall survival was 8.73 mg/L (p &amp;lt; 0.0001). Patients were divided into two subgroups by the optimal cut-off value. The median survival was 21 days for the high level subgroup, and 236 days for the low level subgroup (Figure 1). The 6-month survival rates were 22% and 54%, respectively. The subgroup with B2M level &amp;gt; 8.73 mg/L was older and had a higher proportion of stage IV. Patients with higher levels of B2M showed lower levels of platelets, albumin, and fibrinogen, and high levels of creatinine. A moderate correlation between creatinine and serum B2M was found (Spearman's ρ = 0.417, p &amp;lt; 0.001). The multivariate analysis showed that the high levels of serum B2M (&amp;gt; 8.73 mg/L) and creatinine (≥ 133 μmol/L), decreased fibrinogen (≤ 1.5 g/L), agranulocytosis (&amp;lt; 0.5×10 9/L), severe thrombocytopenia (&amp;lt; 50×10 9/L), and increased Epstein-Barr virus DNA were found to have significant prognostic values in all patients. The high serum B2M level (&amp;gt; 8.73 mg/L), severe thrombocytopenia (&amp;lt; 50×10 9/L), agranulocytosis (&amp;lt; 0.5×10 9/L), and high Epstein-Barr virus DNA copy number were independent prognostic factors in patients with creatinine &amp;lt; 133 μmol/L. Finally, a prognostic scoring system was established based on serum B2M levels as well as five other independent prognostic factors and divided the patients into three groups with significant prognostic differences (median survival: low-risk [score ≤ 1] 428 days vs. intermediate-risk [score = 2] 102 days vs. high-risk [score ≥ 3] 18 days, P &amp;lt; 0.0001) (Figure 2). The 6-month survival rates were 67%, 46% and 14%, respectively. Conclusions: This study confirmed that the serum B2M level could be an independent prognostic factor in lymphoma-associated HLH and established a prognostic scoring system to predict patients' survival.

Failure and out of Specification Manufacturing of Autologous CAR-T Cells Could be Associated with a High Concentration of Total Nucleated Cells, CD3 + Cells and Neutrophils in the Apheresis Product

Manufacturing capacities of commercial autologous chimeric antigen receptor (CAR)-T cells have been significantly increased during the last years, allowing treatment of a larger patient cohort. However, this was associated with the emergence of several failures and out of specifications (OOS), resulting in treatment delay. The aim of our study was to identify early indicators related to apheresis products that can predict CAR-T cells manufacturing failure or OOS. To this end, we analyzed manipulation processes of cellular starting materials (conservation, freezing, cryopreservation) and quality controls (QC) including count and/or concentration of total nucleated cells (TNC), CD3 +, CD4 +, CD8 + cells, platelets, monocytes and neutrophils. We analyzed 802 apheresis products that were collected for the manufacturing of axicabtagene ciloleucel (axi-cel, n=303), tisagenlecleucel (tisa-cel, n=403), brexucatagene autoleucel (brexu-cel, n=44) and idecabtagene vicleucel (ide-cel, n=52). This cohort was classified into 3 groups: failure (CAR-T cells not produced), OOS (CAR-T cells produced but non-compliant) and compliant. We identified 21 failures and 24 OOS, resulting in either no treatment or a significant delay when the OOS were injected (57.6±17.5d versus 45.9±16.3d in compliant group, p=0.0405). Among each CAR-T cells product, 13 (4.3%) axi-cel, 30 (7.4%) tisa-cel, 1 (2.3%) brexu-cel and 1 (1.9%) ide-cel were concerned. The main causes of failures were low viability (33.3%), non-compliant dose (28.6%), microbiological contamination (23.8%) and formulation concerns (14.3%). In OOS, low viability (41.7%), low dose (29.2%), microbiological contamination (4.2%), formulation (12.5%) and safety concerns (8.3%; i.e high vector copy number) were identified. 17 products (13 failures and 4 OOS) were successfully remanufactured without an increased delay comparing to compliant products (47.5±15.9 and 47.3±15.1 respectively versus 45.9±16.3 d). To identify risk factors of manufacturing failure or OOS due to non-compliant viability and/or dose, we analyzed processing and QC of collected cellular starting materials. OOS were associated with a high concentration of TNC (146.7±94.3 vs 61.6±36.1 x10 6/mL, p=0.009) and CD3 + cells (88.6±76.5 vs 26.0±21.9 x10 6/mL, p=0.0112) in cell collections, while failures were observed in apheresis with high neutrophils concentration (14.3±15.03 x10 3/µL vs 6.0±7.6 x10 3/µL, p=0.0195). Interestingly, neutrophils were significantly decreased in apheresis collected for CAR-T cells remanufacturing which succeed (p=0.0156). This preliminary study showed that high TNC, CD3 + cells and neutrophils concentration in the apheresis product might be predictive factors of failures and/or OOS of commercial autologous CAR-T cells manufacturing.

Relationship between clinical features and droplet digital PCR copy number in non-HIV patients with pneumocystis pneumonia

ObjectiveDroplet digital PCR (ddPCR) is a novel assay to detect pneumocystis jjrovecii (Pj) which has been defined to be more sensitive than qPCR in recent studies. We aimed to explore whether clinical features of pneumocystis pneumonia (PCP) were associated with ddPCR copy numbers of Pj.MethodsA total of 48 PCP patients were retrospectively included. Pj detection was implemented by ddPCR assay within 4 h. Bronchoalveolar fluid (BALF) samples were collected from 48 patients with molecular diagnosis as PCP via metagenomic next generation sequencing (mNGS) or quantitative PCR detection. Univariate and multivariate logistic regression were performed to screen out possible indicators for the severity of PCP. The patients were divided into two groups according to ddPCR copy numbers, and their clinical features were further analyzed.ResultsPj loading was a pro rata increase with serum (1,3)-beta-D glucan, D-dimmer, neutrophil percentage, procalcitonin and BALF polymorphonuclear leucocyte percentage, while negative correlation with albumin, PaO2/FiO2, BALF cell count, and BALF lymphocyte percentage. D-dimmer and ddPCR copy number of Pj were independent indicators for moderate/severe PCP patients with PaO2/FiO2 lower than 300. We made a ROC analysis of ddPCR copy number of Pj for PaO2/FiO2 index and grouped the patients according to the cut-off value (2.75). The high copy numbers group was characterized by higher level of inflammatory markers. Compared to low copy number group, there was lower level of the total cell count while higher level of polymorphonuclear leucocyte percentage in BALF in the high copy numbers group. Different from patients with high copy numbers, those with high copy numbers had a tendency to develop more severe complications and required advanced respiratory support.ConclusionThe scenarios of patients infected with high ddPCR copy numbers of Pj showed more adverse clinical conditions. Pj loading could reflect the severity of PCP to some extent.

Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

A Novel Multiplex LAMP Assay for the Rapid and Accurate Diagnosis of Visceral Leishmaniasis Caused by Leishmania infantum from Iran.

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per μL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per μL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries.

The Expansion of House Mouse Major Urinary Protein Genes Likely Did Not Facilitate Commensalism with Humans.

Mouse wild-derived strains (WDSs) combine the advantages of classical laboratory stocks and wild animals, and thus appear to be promising tools for diverse biomedical and evolutionary studies. We employed 18 WDSs representing three non-synanthropic species (Mus spretus, Mus spicilegus, and M. macedonicus) and three house mouse subspecies (Mus musculus musculus, M. m. domesticus, M. m. castaneus), which are all important human commensals to explore whether the number of major urinary protein (MUP) genes and their final protein levels in urine are correlated with the level of commensalism. Contrary to expectations, the MUP copy number (CN) and protein excretion in the strains derived from M. m. castaneus, which is supposed to be the strongest commensal, were not significantly different from the non-commensal species. Regardless of an overall tendency for higher MUP amounts in taxa with a higher CN, there was no significant correlation at the strain level. Our study thus suggests that expansion of the Mup cluster, which appeared before the house mouse diversification, is unlikely to facilitate commensalism with humans in three house mouse subspecies. Finally, we found considerable variation among con(sub)specific WDSs, warning against generalisations of results based on a few strains.

High overall copy number variation burden by genome-wide methylation profiling holds negative prognostic value in surgically treated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer with an overall 5-year survival of around 10 %. New prognostic tools to stratify patients are needed. Our main aim was to evaluate the prognostic value of overall copy number variation (CNV) burden in surgically treated PDAC. DNA extracted from 108 surgical PDAC specimens was examined to collect data on the genome-wide DNA methylation status of >850,000 CpG sites in promoter, gene body, and enhancer regions (Illumina Infinium Methylation EPIC BeadChip Kit). CNV profiles were obtained and all PDACs were stratified into one of three groups: Low, moderate, or high overall CNV burden. Tumors histologically showing a dominant conventional and/or tubulopapillary pattern in 60 %–100 % and 0–59 % were categorized as Group A and Group B as per Kalimuthu. We also performed targeted next-generation sequencing (NGS) and immunohistochemistry. High overall CNV burden held independent negative prognostic value with poor survival (HR 4.01 (95%CI 1.96–8.19), p = 0.00014) and was more frequent in Group B (p = 0.0003). Most frequent chromosomal arm-level aberrations were gains of 8q (29 %) and 1q (19 %) and losses of 17p (55 %), 18q (43 %), 6q (37 %), 9p (36 %), 6p (26 %), 19p (26 %), and 8p (25 %). Most frequent mutations found were in KRAS (95 %), TP53 (62 %), CDKN2A (24 %), SMAD4 (23 %), ATM (9 %), ARID1A (7 %), RNF43 (7 %), GNAS (6 %), and KDM6A (6 %). Group A PDACs showed more frequently KRAS variants other than Gly12Val and Gly12Asp (p = 0.012). Our data indicate that overall CNV burden using genome-wide methylation profiling may be a useful prognostic tool in surgically treated PDAC. Importantly, our approach, using data from genome-wide methylation profiling for analysis of overall CNV burden, can be performed on formalin-fixed and paraffin embedded PDAC tissues. Future studies should examine the prognostic value of overall CNV burden in unresectable PDAC.

Wide reference databases for typing Trypanosoma cruzi based on amplicon sequencing of the minicircle hypervariable region.

Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing. Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed. We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples.

A High Copy Number from a Pharyngeal Swab Is Not Associated with Different Presenting Features in 100 Children with Acute Adenovirus Infection from a Cluster in Italy.

Human mastadenoviruses, frequently denominated adenoviruses (HAdVs), may cause respiratory tract, gastrointestinal or, less frequently, other involvements. Epidemics of HAdV infections occur globally, in communities, and in closed or crowded settings. In our institution, a cluster of infants and children admitted for HAdV infection was recently observed. The aim of this study was to describe the pattern of their presenting features and investigate the possible correlation between the HAdV copy number and the clinical picture. Two main patterns of clinical presentation were observed: 68 patients had mainly respiratory symptoms (pharyngitis n = 67, cough n = 44; tonsillar exudate n = 17; other respiratory signs n = 4) while 26 patients showed prevalent gastrointestinal involvement (diarrhea n = 26, vomiting n = 8). Patients with respiratory symptoms had a significantly higher count of WBC, PMN, and platelets, while CRP level approached statistical significance (p = 0.07) for higher values in the patients with diarrhea. In order to explore the impact of selected presenting features, the possible association between the level of CRP and the presence of pharyngeal exudate, cough, vomiting, diarrhea, duration of fever, number of neutrophils, and administration of antibiotics was analyzed. Patients falling in the tertile with more elevated CRP values had tonsillar exudate and diarrhea significantly more often, while those in the lower tertile had a 4.4-day duration fever vs. ≥5.0 days in the remaining patients. Antibiotic therapy was administered more frequently to patients with higher values of CRP (p = 0.006). The duration of hospitalization was not associated with the CRP level. The median time from the receipt of a positive HAdV PCR test result to patient discharge was 1 day in 73% of cases. The number of copies of HAdV detected via PCR ranged between 47 million and 15/μL. Falling in the highest tertile of copy number was significantly associated with pharyngitis. The 24 patients with evidence of viral coinfection had no difference in the demographics or presenting features, with the only exception being a significantly higher leukocyte count. The rapid turn-around of the results of the molecular testing of the HAdV genome on a pharyngeal swab allowed us to rapidly diagnose HAdV infection, allowing us to stop antibiotic therapy and immediately discharge the patients, with reduced discomfort for the families and more appropriate use of hospital beds. A high copy number of HAdV from a pharyngeal swab should not be taken as an indicator of worse prognosis, thus allowing for the preferential use of qualitative rather than quantitative assay.