Low DNA Levels Research Articles

New and rapid visual detection assay for Trogoderma granarium Everts based on recombinase polymerase amplification and CRISPR/Cas12a.

Khapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a system. We designed and selected the first khapra beetle-specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA=100 nM/500 nM). With only a 37°C-heat-source and a blue light torch, RPA and CRISPR/CAS12a-based visualization assays can be completed within 40 min to differentiate among khapra beetle and other nine similar Dermestidae species. After DNA extraction using a kit (4-5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min-RPA-reaction at 37°C, followed by a 15 min-color-reaction under 37°C in dark using CRISPR/CAS12a system and a fluorescent probe (5'-FAM/3'-BHQ1 labeled). This method is ingenious to low levels of DNA (10-1 ng/μL) and meets the sensitivity requirements for detecting a single khapra beetle's egg (about 0.7 mm). Our specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37°C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management and provides value as a reference for monitoring and identification of other pests. This article is protected by copyright. All rights reserved.

Biomimetic affinity chromatography for antibody purification: Host cell protein binding and impurity removal

Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.

LAMP (Loop-mediated isothermal amplification) assay for rapid identification of Varroa mites

Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V. destructor and V. jacobsoni, providing the ability to tell them apart from other non-target beehive associated mite and insect species. This LAMP assay is specific in detecting these Varroa species and has been tested on specimens originating from multiple countries. It produces amplification of V. destructor and V. jacobsoni in 16 ± 3.4 min with an anneal derivative of 78 ± 0.5 °C whilst another Varroa species,V. underwoodi, showed late amplification. A gBlock gene fragment, used here as a positive control has a different anneal derivative of 80 °C. Three non-destructive DNA extraction methods (HotShot, QuickExtract and Xtract) were tested and found to be suitable for use in the field. The LAMP assay was sensitive to very low levels of Varroa DNA, down to 0.24 picogram (~ 1 × 10 copies/µL of Varroa gBlock). This is a new molecular tool for rapid and accurate detection and identification of Varroa mites for pest management, in areas where these mites do not occur.

Identification of Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., two new members of the genus Nocardioides.

A polyphasic taxonomic characterization of two novel strain pairs (designated zg-579T/zg-578 and zg-536T/zg-ZUI104) isolated from the faeces of Marmota himalayana was conducted based on phylogenetic analysis of the nearly full-length 16S rRNA gene and genome, digital DNA-DNA hybridization, ortho-average nucleotide identity (Ortho-ANI), and phenotypic and chemotaxonomic traits. Comparative analysis of the nearly full-length 16S rRNA gene sequences showed that strain zg-579T was most closely related to Nocardioides dokdonensis FR1436T (97.57 %) and Nocardioides deserti SC8A-24T (97.36 %), whereas strain zg-536T had the highest similarity to Nocardioides caeni MN8T (98.33 %), Nocardioides convexus W2-2-3T (98.26 %) and Nocardioides daeguensis 2C1-5T (98.19 %). Low levels of DNA-DNA relatedness and Ortho-ANI values (19.8-31.0 %/78.6-88.2 %, zg-579T; 19.9-31.3 %/78.8-86.2 %, zg-536T) between the two new type strains and previously known species within the genus Nocardioides support the hypothesis that the four newly characterized strains could be considered to represent two novel species within this genus. The dominant cellular fatty acids found in strain pair zg-536T/zg-ZUI104 were iso-C16 : 0 and C18 : 1 ω9c, whereas C17 : 1 ω8c was major component in zg-579T/zg-578. Galactose and ribose were the main cell-wall sugars in these two new strain pairs. Diphosphatidylglycerol (DPG), phosphatidylcholine, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were the major polar lipids in zg-579T, whereas DPG, PG and PI predominated in zg-536T. Both strain pairs had MK8(H4) as the major respiratory quinone and ll-diaminopimelic acid as the major cell-wall peptidoglycan. The optimal growth conditions for the two novel strain pairs were 30 °C, pH 7.0 and 0.5 % NaCl (w/v). Based on these polyphasic characterizations, two novel species within the genus Nocardioides are proposed, i.e. Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., with zg-579T (=CGMCC 4.7663T=JCM 33892T) and zg-536T (=CGMCC 4.7662T=JCM 33891T) as the type strains.

High-grade squamous intraepithelial lesion cervicovaginal paps with negative high-risk HPV testing, a prospective study with histological follow-up.

High-risk human papillomavirus (hrHPV) testing has become integral in the screening and treatment protocols for cervical neoplasia. Stand-alone HPV testing is advocated as a screening tool for cervical neoplasia. However, negative hrHPV tests with diagnosis of high-grade squamous intraepithelial lesion or worse (≥HSIL) have been reported. We report our experience with paps diagnosed as HSIL, negative hrHPV testing, and subsequent follow-up. Of 303 women with HSIL diagnosed on ThinPrep pap between 2019 and 2023, 84 (28%) were tested for hrHPV by Roche Cobas. Repeat testing was performed at a referral center. Immunohistochemistry (IHC) for p16 was performed on follow-up biopsies and hrHPV in-situ hybridization. Of 84 HSIL cases, 8 were hrHPV negative. Follow-up biopsies available in 7 cases were ≥HSIL (1 with invasive squamous cell carcinoma, 1 endocervical adenocarcinoma in situ). Follow-up HSIL was found on additional cases of HPV negative atypical glandular cells favor neoplastic and atypical squamous cells favor HSIL. IHC for p16 was positive on all biopsies. hrHPV FISH was negative. Our experience with hrHPV testing by Roche Cobas demonstrates that some morphologically diagnostic HSIL paps are hrHPV negative: 8.3% of HSIL paps with subsequent histological HSIL were HPV negative. The index case caused concern among our clinical colleagues. Positive staining for p16 is highly suggestive of HPV induced disease. Possible reasons for negative hrHPV testing could include non-hrHPV types, low HPV DNA levels, or HPV types not included in the Cobas testing panel.

Comparison of Real-Time PCR and Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma

Plasma Epstein-Barr virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma. However, existing real-time quantitative PCR (qPCR) assays are limited by poor interlaboratory reproducibility. This is a barrier to biomarker integration into staging systems and management. It was hypothesized that EBV digital PCR (dPCR) would have similar sensitivity but improved precision relative to qPCR. Using the World Health Organization EBV standard and patient specimens, the NRG-HN001 BamHI-W qPCR, two commercial EBNA-1 qPCR assays, and two laboratory-developed dPCR assays amplifying the BamHI-W, EBNA-1, and EBER targets were compared. Testing was conducted in the North American reference laboratory for the NRG-HN001 randomized trial. The EBV dPCR assays achieved similar performance compared with qPCR. Although dPCR does not require quantitation standards, different dPCR thresholding algorithms yielded significant qualitative and quantitative variation. This was most evident with low levels of EBV DNA. No-template control-informed thresholding (ddpcRquant) mitigated false-positive/false-negative findings. The NRG-HN001 BamHI-W qPCR and laboratory-developed BamHI-W droplet dPCR offered higher sensitivity, lower limit of blank, higher precision at low plasma EBV DNA levels (≤1500 IU/mL), and higher overall agreement with clinical specimens versus single-copy qPCR/dPCR targets (EBNA-1/EBER). These data confirm the rationale for using the BamHI-W target to define prognostic thresholds and indicate that both qPCR and dPCR methods harmonized to the World Health Organization standard can provide the necessary analytical performance.

Abstract 2287: Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA

Abstract Purpose: Extracellular vesicles (EVs) have been shown to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform circulating cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated whether EVs could be a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. Methods: evDNA from both DNase-treated and untreated circulating EVs, enriched from patients with advanced pancreatic cancer (n=10), was analyzed by fluorometry and digital droplet PCR to determine the level of evDNA inside and outside (surface-bound) the EVs. To also determine if choice of methodology affected the results, EVs were isolated using four different small EV isolation methods (differential ultracentrifugation, size-exclusion chromatography, affinity purification and precipitation) and differential centrifugation for isolation of large EVs. Western blotting, dynamic light scattering and transmission electron microscopy confirmed the enrichment of EVs. Results: Our results indicated that EVs enriched from patient blood samples contained significantly lower amounts of DNA compared to cfDNA (p < 0.001); >50-fold lower for untreated sEVs and >1000-fold lower for DNase-treated EVs. Thus, most of the detected vesicle-associated DNA seemed to be located outside of the vesicles. Further, tumor-derived DNA in EVs was barely detectable and the tumor-derived fraction in all evDNA samples was similar to that of cfDNA. Conclusion: Our results demonstrated that evDNA do not add any extra information compared with cfDNA as a source of tumor-derived DNA, at least not in patients with advanced pancreatic cancer. Citation Format: Morten Lapin, Kjersti Tjensvoll, Karoline Nedrebø, Eline Taksdal, Hans Janssen, Bjørnar Gilje, Oddmund Nordgard. Circulating extracellular vesicles from patients with advanced pancreatic cancer contain low levels of tumor-derived DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2287.

Diagnosis of Fusarium oxysporum f. sp. ciceris causing Fusarium wilt of chickpea using loop-mediated isothermal amplification (LAMP) and conventional end-point PCR

Fusarium oxysporum (Fo) is ubiquitous in soil and forms a species complex of pathogenic and putatively non-pathogenic strains. Pathogenic strains cause disease in over 150 plant species. Fusarium oxysporum f. sp. ciceris (Foc) is a major fungal pathogen causing Fusarium wilt in chickpeas (Cicer arietinum). In some countries such as Australia, Foc is a high-priority pest of biosecurity concern. Specific, sensitive, robust and rapid diagnostic assays are essential for effective disease management on the farm and serve as an effective biosecurity control measure. We developed and validated a novel and highly specific PCR and a LAMP assay for detecting the Indian Foc race 1 based on a putative effector gene uniquely present in its genome. These assays were assessed against 39 Fo formae speciales and found to be specific, only amplifying the target species, in a portable real-time fluorometer (Genie III) and qPCR machine in under 13 min with an anneal derivative temperature ranging from 87.7 to 88.3 °C. The LAMP assay is sensitive to low levels of target DNA (> 0.009 ng/µl). The expected PCR product size is 143 bp. The LAMP assay developed in this study was simple, fast, sensitive and specific and could be explored for other Foc races due to the uniqueness of this marker to the Foc genome.

Merkel cell polyomavirus (MCPyV) DNA prevalence in Brazilian blood donors.

Merkel cell polyomavirus (MCPyV) is an oncogenic virus that has been etiologically linked to Merkel cell carcinoma. Low levels of MCPyV DNA have been detected in blood donors with unclear impact on transfusion. The prevalence of MCPyV DNA in Brazilian blood donors is unclear. Therefore, the objective of this study was to evaluate the MCPyV DNA prevalence among Brazilian blood donors. We examined the presence of MCPyV DNA by real-time PCR (qPCR) in a total of 450 serum samples obtained from blood donors from three Brazilian regions (North, Central-West and South). The overall estimated MCPyV DNA prevalence was 1.1% (CI=95%, 0.16-2.06%). Divided by region, in North Brazil (city of Macapa, state of Amapá) and South Brazil (city of Santa Maria, state of Rio Grande do Sul), the MCPyV prevalence was the same: 1.33% (CI=95%, range 0.0-3.14%). In Central-West Brazil (city of Brasilia), the MCPyV prevalence was 0.6% (CI=95%, 0.0-1.96%). All MCPyV positive samples showed a high cycle threshold (median Ct=35.5), most probably related to the low viral load. More studies are necessary to unveil the impact of this oncogenic virus on transfusion medicine and if such exists, especially in regards of its infectivity and transmission potential.

Development of an Efficient Dye-Based qPCR System Still Functional for Low Levels of Transgenic DNA in Food Products

Development of an Efficient Dye-Based qPCR System Still Functional for Low Levels of Transgenic DNA in Food Products

Examining the transfer of male DNA onto female underwear in simulated non sexual social interaction versus digital penetration to assist in the evaluation of results in casework scenarios

Experiments have been carried out by the UK and Ireland Association of Forensic Science Providers Body Fluid Forum (AFSP BFF) to determine the levels of male DNA, detected during Y-STR analysis, that may be expected on female underwear from non-sexual social interaction and digital penetration, versus non-sexual social interaction only. The data obtained strongly supports the existing assumptions made: whilst low levels of DNA may be inadvertently transferred to the inside surface of a female’s underwear during social interaction with a male, there is a low expectation of detecting a matching Y-STR profile to that male, which is suitable for statistical evaluation, unless he is a co-habitant of that female.

Natural history of decompensated cirrhosis with serum hepatitis B DNA < 2000 IU/mL: a retrospective study

Background and aimsPatients with low HBV DNA levels (< 2000 IU/mL), HBV DNA negative, and HBsAg-negative hepatitis B virus(HBV)infection can still progress to decompensated cirrhosis; however, clinical research data in such patients, especially treatment-naïve patients, are currently insufficient. This study assessed the natural history of aforementioned patients.MethodsWe retrospectively reviewed the data of 250 patients with HBV-associated decompensated cirrhosis(HBV DNA < 2000 IU/mL) who had not been treated with antiviral medication.ResultsThe mean age of the 250 patients was 53.90 ± 11.73 years and 183 patients (73.2%) were male. HBV DNA, HBsAg, and HBeAg positivity was detected in 77 (30.8%), 200 (80%), and 137 (54.8%) patients, respectively. HBsAg (odds ratio [OR], 3.303; 95% confidence interval [CI], 1.338–8.152; P = 0.010) and HBeAg (OR, 0.200; 95% CI, 0.107–0.376; P < 0.001) positivity were independent factors for low HBV DNA levels. The incidence of hepatocellular carcinoma (HCC) (P < 0.001) and portal vein thrombosis (P = 0.001) was higher in the low HBV DNA levels group. Multivariate analysis showed that HBV DNA positivity (OR, 3.548; 95% CI, 1.463–8.604; P = 0.005), HBeAg positivity (OR, 0.080; 95% CI, 0.022–0.289; P < 0.001), and glutamyltransferase (GGT) (OR, 1.003; 95% CI, 1.000–1.006; P = 0.040) were independent factors for HCC. Age was not related to the occurrence of cirrhosis complications.ConclusionPatients with decompensated cirrhosis with HBV DNA < 2000 IU/mL still had severe liver damage and could develop severe cirrhosis complications. HCC risk was higher in low HBV DNA levels patients. HBsAg positivity and HBeAg negativity may be associated to the occurrence of low HBV DNA levels.

227MO Characterization of minimal residual disease (MRD) post-radiotherapy (RT) in nasopharyngeal carcinoma (NPC) patients identifies a favorable subgroup with low risk of relapse

Detectable plasma Epstein-Barr virus (EBV) DNA post-RT implies a poor prognosis, but Hui et al. reported a model that could sub-stratify these patients by an EBV DNA cut-off of 500 copies/mL. Detection limits however vary between different EBV DNA assays, which could lead to false positives at low EBV DNA levels. We investigated the EBV DNA kinetics and survival in patients harboring a detectable, but non-quantifiable EBV DNA post-RT (≤265 copies/mL; termed as MRD).

Association of hepatitis B virus DNA levels with overall survival for advanced hepatitis B virus-related hepatocellular carcinoma under immune checkpoint inhibitor therapy.

High hepatitis B virus (HBV) DNA level is an independent risk factor for postoperative HBV-associated liver cancer recurrence. We sought to examine whether HBV DNA level and antiviral therapy are associated with survival outcomes in patients with advanced hepatocellular carcinoma (HCC) treated with anti-programmed cell death protein 1 (PD-1)based immunotherapy. This single-institution retrospective analysis included 217 patients with advanced HBV-related HCC treated from 1 June 2018, through 30 December 2020. Baseline information was compared between patients with low and high HBV DNA levels. Overall survival (OS) and progression-free survival (PFS) were compared, and univariate and multivariate analyses were applied to identify potential risk factors for oncologic outcomes. The 217 patients included in the analysis had a median survival time of 20.6months. Of these HBV-associated HCC patients, 165 had known baseline HBV DNA levels. Baseline HBV DNA level was not significantly associated with OS (P = 0.59) or PFS (P = 0.098). Compared to patients who did not receive antiviral therapy, patients who received antiviral therapy had significantly better OS (20.6 vs 11.1months, P = 0.020), regardless of HBV DNA levels. Moreover, antiviral status (adjusted HR = 0.24, 95% CI 0.094-0.63, P = 0.004) was an independent protective factor for OS in a multivariate analysis of patients with HBV-related HCC. HBV viral load does not compromise the clinical outcome of patients with HBV-related HCC treated with anti-PD-1-based immunotherapy. The use of antiviral therapy significantly improves survival time of HBV-related HCC patients.

Dynamic enhancer transcription associates with reprogramming of immune genes during pattern triggered immunity in Arabidopsis

BackgroundEnhancers are cis-regulatory elements present in eukaryote genomes, which constitute indispensable determinants of gene regulation by governing the spatiotemporal and quantitative expression dynamics of target genes, and are involved in multiple life processes, for instance during development and disease states. The importance of enhancer activity has additionally been highlighted for immune responses in animals and plants; however, the dynamics of enhancer activities and molecular functions in plant innate immunity are largely unknown. Here, we investigated the involvement of distal enhancers in early innate immunity in Arabidopsis thaliana.ResultsA group of putative distal enhancers producing low-abundance transcripts either unidirectionally or bidirectionally are identified. We show that enhancer transcripts are dynamically modulated in plant immunity triggered by microbe-associated molecular patterns and are strongly correlated with open chromatin, low levels of methylated DNA, and increases in RNA polymerase II targeting and acetylated histone marks. Dynamic enhancer transcription is correlated with target early immune gene expression patterns. Cis motifs that are bound by immune-related transcription factors, such as WRKYs and SARD1, are highly enriched within upregulated enhancers. Moreover, a subset of core pattern-induced enhancers are upregulated by multiple patterns from diverse pathogens. The expression dynamics of putative immunity-related enhancers and the importance of WRKY binding motifs for enhancer function were also validated.ConclusionsOur study demonstrates the general occurrence of enhancer transcription in plants and provides novel information on the distal regulatory landscape during early plant innate immunity, providing new insights into immune gene regulation and ultimately improving the mechanistic understanding of the plant immune system.

Abstract 3405: Characterizing treatment response in head and neck cancer through viral ctDNA quantification and fragment length

Abstract Background: Head and neck cancer (HNC) is the sixth most common cancer type worldwide, and despite decreases in the rates of Human Papillomavirus (HPV)-negative HNC, an alarming increase in HPV+ HNC is occurring. Circulating tumor (ct)DNA isolated from liquid biopsy has been shown to have clinical utility as a biomarker in cancer, with ctDNA levels and fragment size used to monitor treatment response. Methods: The aim of this study was to determine the presence of ctDNA in liquid biopsy (plasma and saliva) of HPV+ HNC patients to non-invasively monitor treatment response and disease progression. Blood and saliva samples were collected from 45 HPV+ HNC patients and 14 HPV-negative controls at the McGill University Health Centre before and/or after surgical resection. Samples were analyzed using droplet digital PCR (ddPCR) with primers and probes for HPV16/HPV18, the most common subtypes of HPV in HNC, accounting for 97% of cases in North America. Analysis was performed for all patients with confirmed follow-up scans and/or pathology. ctDNA levels were correlated with disease course as determined through imaging (&amp;gt;6 months post-sampling) and pathological examination. Results: ctDNA was detectable in 21/23 (91%) patients prior to treatment and 8/36 (22%) post treatment. In the two patients who were not positive for ctDNA prior to treatment, tumor tissue was found to be negative for HPV DNA despite being p16-positive by immunohistochemistry. Patients showed shifts in ctDNA fragment length in longitudinal samples following treatment. All post-treatment patients with no detectable ctDNA had no signs of recurrence/residual disease on follow-up imaging. In contrast, all 8 patients who tested positive for ctDNA post-treatment showed signs of recurrence on follow-up imaging. In our cohort, 14 patients had matched pre- and post-treatment samples and all showed major reductions in ctDNA post treatment compared to pre-treatment, with 12/14 patients having a complete loss of detectable ctDNA. Concordance between the presence of ctDNA in saliva and plasma was found in 86% of patients. ctDNA was only detected in one analyte in 6 patients (3 in plasma only and 3 in saliva only). Conclusion: HPV ctDNA was successfully detected in saliva and blood of patients with HPV-positive HNC, with a sensitivity of 92% for ctDNA prior to treatment, and a sensitivity of 100% when adjusted for tumor HPV status as opposed to p16 staining. The inclusion of both plasma and saliva in analysis increases the sensitivity of assays, with the use of both analytes allowing for the detection of ctDNA in patients with low plasma DNA levels and for patients who did not have salivary ctDNA, potentially due to inability to produce sufficient saliva or tumor location. The presence of ctDNA correlated with treatment response, indicating the potential of HPV ctDNA for monitoring tumor progression in HPV-positive HNC patients. Citation Format: Sarah Tadhg Ferrier, Anthony Zeitouni, Nader Sadeghi, Thupten Tsering, Julia V. Burnier. Characterizing treatment response in head and neck cancer through viral ctDNA quantification and fragment length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3405.

P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

BackgroundP-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown.ResultsHerein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0–250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1.ConclusionsOur results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.

Utilization of the lymph node-to-primary tumor ratio of PET standardized uptake value and circulating Epstein–Barr virus DNA to predict distant metastasis in nasopharyngeal carcinoma

Background and purposeTo determine the clinical impact of integrating Epstein–Barr virus (EBV) DNA and lymph node-to-primary tumor ratio (NTR) of positron emission tomography (PET) standardized uptake value (SUV) in predicting distant metastasis, such as distant metastasis-free survival (DMFS), in patients with nasopharyngeal carcinoma (NPC). Materials and methodsWe retrospectively reviewed patients diagnosed with non-disseminated NPC between 2010 and 2017. The optimal cut-off values of EBV DNA and SUV NTR were determined using receiver operating characteristic analysis. The prognostic values of SUV NTR and EBV DNA on DMFS and overall survival were evaluated using the Kaplan–Meier method. Univariate and multivariable analyses were performed using the Wald Chi-squared test and Cox proportional hazards regression, respectively. ResultsA total of 488 patients were included in the analysis. The median follow-up period was 61.6 months. The optimal cut-off values of EBV DNA and SUV NTR were 3377.5 copies per mL and 0.64, respectively. The five-year DMFS for patients with high vs low EBV DNA and SUV NTR levels were 64.9% vs 86.6% (p < 0.001) and 78.7% vs 87.4% (p = 0.021), respectively. In subgroup analysis, the high-risk group with high levels of pretreatment EBV DNA and SUV NTR had worse DMFS in either American Joint Committee on Cancer (AJCC) stage I–III or IVA–B (p = 0.001 and <0.001, respectively). Univariate and multivariable analyses showed the statistical significance of EBV DNA, SUV NTR, and their composite in DMFS (p < 0.001 for EBV DNA; p = 0.022 for SUV NTR; p < 0.001 for their composite). ConclusionThis study showed that EBV DNA and SUV NTR have independent and additive values as prognosticators for distant metastasis in patients with NPC, suggesting that these two individual factors, except the AJCC staging system, should be included in future studies.

Association between low levels of HIV-1 DNA and HLA class I molecules in chronic HIV-1 infection.

HLA-B27 and -B57 were found in people with low levels of HIV-1 DNA, suggesting that HLA class I molecules may influence the size of HIV-1 reservoir. Aim of the study was to explore the association between HLA class I molecules and HIV-1 DNA in people with chronic HIV-1 infection. Post-hoc analysis of the APACHE trial, on adults with chronic HIV-1 infection, prolonged suppressive antiretroviral therapy and good immunological profile. HIV-1 DNA was quantified in peripheral blood mononuclear cells (PBMCs); HLA-A, -B and -C were tested on genomic DNA. Crude odds ratios (OR) with their respective 95% Wald confidence intervals (95% CIs) were estimated by univariable logistic regression for HLAs with a p-value <0.10. We found 78 and 18 patients with HIV-1 DNA ≥100 copies/106PBMCs and with HIV-1 DNA <100 copies/106PBMCs, respectively. HLA-A24 was present in 21 (29.6%) participants among subjects with HIV-1 DNA ≥100 copies/106PBMCs and 1 (5.9%) among those with HIV-1 DNA <100 copies/106PBMCs (OR = 5.67, 95%CI = 0.79-46.03; p = 0.105); HLA-B39 was present in 1 (1.4%) with HIV-1 DNA ≥100 copies/106PBMCs and in 3 (17.6%) with HIV-1 DNA <100 copies/106PBMCs (OR = 13.71, 95%CI = 1.33-141.77; p = 0.028) and HLA-B55 in 3 (4.2%) and 3 (17.6%), respectively (OR = 4.43, 95%CI = 0.81-24.29; p = 0.087). All the three patients with HLA-B39 and HIV-1 DNA <100 copies/106PBMCs did not have HLA-A24. In patients with HIV-1 infection who maintained a good virological and immunological profile, HLA-B39 and -B55 may be associated with lower levels of HIV-1 DNA.

Lysobacter antarcticus sp. nov., an SUF-system-containing bacterium from Antarctic coastal sediment

A Gram-stain-negative, heterotrophic, aerobic, non-motile, rod-shaped bacterial strain (GW1-59T) belonging to the genus Lysobacter was isolated from coastal sediment collected from the Chinese Great Wall Station, Antarctica. The strain was identified using a polyphasic taxonomic approach. The strain grew well on Reasoner's 2A media and could grow in the presence of 0-4 % (w/v) NaCl (optimum, 1 %), at pH 9.0-11.0 and at 15-37 °C (optimum, 30 °C). Strain GW1-59T possessed ubiquinone-8 as the sole respiratory quinone. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were summed feature 9 (10-methyl C16 : 0 and/or iso-C17 : 1 ω9c), iso-C15 : 0, iso-C16 : 0, iso-C17 : 0, C16 : 0 and iso-C11 : 0 3-OH. DNA-DNA relatedness with Lysobacter concretionis Ko07T, the nearest phylogenetic relative (98.5 % 16S rRNA gene sequence similarity) was 23.4 % (21.1-25.9 %). The average nucleotide identity value between strain GW1-59T and L. concretionis Ko07T was 80.1 %. The physiological and biochemical results and low level of DNA-DNA relatedness suggested the phenotypic and genotypic differentiation of strain GW1-59T from other Lysobacter species. On the basis of phenotypic, phylogenetic and genotypic data, a novel species, Lysobacter antarcticus sp. nov., is proposed. The type strain is GW1-59T (=CCTCC AB 2019390T=KCTC 72831T).