Low DNA Research Articles

Abstract 556: The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes

Abstract Background: Mutations in the genes encoding isocitrate dehydrogenase (IDH1 and IDH2) are frequently detected in various cancers, including acute myeloid leukemia (~20%) and glioma (up to ~80%). They commonly involve a single amino acid substitution at codon R132 in IDH1, and R140 or R172 in IDH2 and are important for diagnosis and prognosis as well as therapeutic decisions. With advancements in molecular diagnostics and precision therapy, IDH inhibitors such as ivosidenib, vorasidenib and enasidenib are now mainstay in management of patients with a susceptible mutation. Methods: The Idylla™ IDH1-2 Mutation Assay Kit (RUO) is a fully automated qPCR assay for the qualitative detection of 15 common mutations in IDH1 (R132C/H/G/S/L) and IDH2 (R140Q/L/G/W, R172K/M/G/W/S) at codon level. The assay consists of a ready-for-use Idylla™ DNA Cartridge and an assay specific Idylla™ Vial. It runs on the Idylla™ Platform and covers the entire process from sample to result including specimen processing, real-time PCR amplification, detection, data analysis, and result reporting, within 100 minutes.Five clinical sites in US and Europe participated in an early access program to evaluate performance of the assay and establish a final classification algorithm by testing a total of 241 specimens, including FFPE, human whole blood and bone marrow samples, and extracted DNA from these sample types. Reference results were obtained using a range of established methods with different mutations in scope, such as Sanger sequencing, immunohistochemistry, NGS and qPCR. Results: Of 241 Cartridges, one invalid result was obtained, while 13 results were flagged as having low DNA input. Compared to available reference methods, overall concordance was 98.3% (235/239), 100% (140/140) and 99.6% (230/231) for IDH1 R132, IDH2 R140 and R172, respectively (see table). Conclusion: The Idylla™ IDH1-2 Mutation Assay Kit demonstrates a high concordance with established reference methods across a range of different specimen types. Reference method(s) PPA (%) NPA (%) OPA (%) MUT WT Total Idylla™ IDH1-2 Mutation Assay Kit IDH1_R132x 111 3 114 99.1 97.6 98.3 WT 1 124 125 Total 112 127 239 IDH2_R140x 10 0 10 100.0 100.0 100.0 WT 0 130 130 Total 10 130 140 IDH2_R172x 25 0 25 96.2 100.0 99.6 WT 1 205 206 Total 26 205 231 Total 148 462 610 Citation Format: Bram De Craene, Katrien Trappeniers, Ana Marcelino, Lee Robertson, Phillippe Taniere, Jo Van Dorpe, Malaïka Van der Linden, Maria Arcila, Khedoudja Nafa, Jochen K. Lennerz, Richard Craig Carson, Richard D. Ledding, Sofie Metsu. The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 556.

Abstract 7022: Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. Traditionally, sodium bisulfite conversion was used to distinguish 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) from cytosines. However, this method damages DNA and introduces significant sequencing bias. NEBNext® Enzymatic Methyl-seq (EM-seq™) is an enzymatic conversion approach that minimizes DNA damage therefore enabling longer insert sizes, lower duplication rates and a more accurate quantification of methylation in DNA samples with inputs ranging from 10 ng to 200 ng. An improved enzymatic conversion method, EM-seq v2, has a more streamlined workflow and an expanded DNA input range of 100 pg to 200 ng. Libraries made using NA12878 genomic DNA show improved yields, consistent global methylation and an even GC bias representation. The increased library yields subsequently reduces sequencing duplications and costs. Sequencing costs are an important consideration when performing whole genome methylome analysis as this often needs high sequencing depth to attain meaningful coverage to accurately call methylation. Reduced representation libraries, a commonly used method to investigate DNA methylation, relies on enrichment of CpGs within libraries digested using MspI. These CpGs are commonly found in regulatory regions. Although lacking comprehensive CpG coverage, for many researchers this method provides enough cost effective CpG methylation coverage for their studies. Reduced Representation EM-seq libraries (RREM-seq) and Reduced Representation Bisulfite libraries (RRBS) were made using 200 ng to 100 pg of NA12878 DNA. EM-seq adaptors were ligated to MspI digested DNA followed by conversion using either EM-seq v2 or sodium bisulfite. Libraries were PCR amplified and sequenced on an Illumina NovaSeq platform. RREM-seq resulted in higher yields and reproducible results regardless of input amounts compared to RRBS libraries. RREM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. RREM-seq libraries have superior sequencing metrics resulting in robust methylation profiling. Focusing on a subset of the genome generates higher coverage DNA methylation data at a lower DNA sequencing cost. Citation Format: Vaishnavi Panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Ariel Erijman, Bradley Langhorst, Louise Williams. Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7022.

Abstract 5207: Hypermethylation of SOX17 is an important epigenetic mark in Afro-Brazilian breast cancers

Abstract Afro-Brazilian women suffer from higher excess breast cancer mortality after adjusting for age, level of education and stage at diagnosis. Limited studies have researched disease characteristics in this population, which can benefit from the identification of molecular markers that can provide an insight into who is at greater risk of mortality. Epigenetic biomarkers represent the combined effect of environmental and genetic factors. Particularly DNA methylation biomarkers can be valuable as biomarkers of breast cancer risk and prognosis. While studies in other populations have identified DNA methylation biomarkers associated with breast cancer and its clinicopathological outcomes, research on Brazilian cohorts has been limited in scope and focused on women of European descent. The goal of this work was to determine the epigenetic makeup of breast cancer in Brazilian women of African descent. To identify epigenetic biomarkers in Brazilian women who self-identified as Black or Brown, we conducted a case-series analysis investigating DNA methylation patterns in tumor (T) and adjacent non-tumor (NT) tissue. For this, we used the Illumina EPIC (850k) arrays to carry out DNA methylation profiling in 48 frozen tissue paired T and NT samples of breast cancer patients treated at the National Cancer Institute Breast Cancer Hospital in Rio de Janeiro, Brazil. We identified a total of 7,749 probes with Benjamini-Hochberg adjusted p-value < 0.0001 and differences between T and NT tissue above |0.3|. Of these, 5,976 or 77.12% had lower DNA methylation in T when compared to NT tissue; while 1,773 (22.88%) had higher DNA methylation. These were used to identify 21 differentially methylated regions (DMRs) that contained a minimum of seven cg sites, a mean difference larger than |0.2| and a maximum difference above |0.4|. DMRs were mostly (12) hypermethylated in T, and included PCDHGA, NKAPL, ZKSCAN4, SPAG6, CA3, DPP6, RGS22, NXPH1, ANK3, OTX2, SOX17, and CDO1. We used pyrosequencing to validate the levels of SOX17 in eighty-eight tumor and non-adjacent tumor tissue DNAs. We found SOX17 to be hypermethylated in T (44.5±19.1% vs 10.2±10.6, p<0.0001). Differences in DNA methylation in T and NT tissue we still observed by race/ethnicity categories, 43.9±18.6% vs 11.0.±13.2% (p<0.0001) for Brown, and 45.1±20.7% vs 8.8±3.0% for Black women (p<0.0001), respectively. Similarly to previously reported, SOX17 hypermethylation was found to be associated with Luminal A and B molecular subtypes (p<0.0001). To our knowledge, this is the first study to use a discovery approach to identify epigenetic biomarkers in Black and Brown Brazilian women. Our findings confirming previously reported data on SOX17 hypermethylation in breast cancer are encouraging. We expect to validate other epigenetic markers unique to this population that can ultimately aid in the molecular characterization of Brazilian breast cancers. Citation Format: Lissette Delgado-Cruzata, Diego J. Gomes de Paula, Jennifer G. Vieira, André L. Christianes, Tatiana Simao, Leonor B. Gusmao, Luis Felipe Ribeiro Pinto, Sheila Coelho Soares Lima. Hypermethylation of SOX17 is an important epigenetic mark in Afro-Brazilian breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5207.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Single-cell transcriptomic sequencing data reveal aberrant DNA methylation in SMAD3 promoter region in tumor-associated fibroblasts affecting molecular mechanism of radiosensitivity in non-small cell lung cancer

ObjectiveNon-small cell lung cancer (NSCLC) often exhibits resistance to radiotherapy, posing significant treatment challenges. This study investigates the role of SMAD3 in NSCLC, focusing on its potential in influencing radiosensitivity via the ITGA6/PI3K/Akt pathway.MethodsThe study utilized gene expression data from the GEO database to identify differentially expressed genes related to radiotherapy resistance in NSCLC. Using the GSE37745 dataset, prognostic genes were identified through Cox regression and survival analysis. Functional roles of target genes were explored using Gene Set Enrichment Analysis (GSEA) and co-expression analyses. Gene promoter methylation levels were assessed using databases like UALCAN, DNMIVD, and UCSC Xena, while the TISCH database provided insights into the correlation between target genes and CAFs. Experiments included RT-qPCR, Western blot, and immunohistochemistry on NSCLC patient samples, in vitro studies on isolated CAFs cells, and in vivo nude mouse tumor models.ResultsFifteen key genes associated with radiotherapy resistance in NSCLC cells were identified. SMAD3 was recognized as an independent prognostic factor for NSCLC, linked to poor patient outcomes. High expression of SMAD3 was correlated with low DNA methylation in its promoter region and was enriched in CAFs. In vitro and in vivo experiments confirmed that SMAD3 promotes radiotherapy resistance by activating the ITGA6/PI3K/Akt signaling pathway.ConclusionHigh expression of SMAD3 in NSCLC tissues, cells, and CAFs is closely associated with poor prognosis and increased radiotherapy resistance. SMAD3 is likely to enhance radiotherapy resistance in NSCLC cells by activating the ITGA6/PI3K/Akt signaling pathway.

Optimal filter materials for protist quantification via droplet digital PCR

The use of droplet digital polymerase chain reaction (ddPCR) has greatly improved the quantification of harmful protists, outperforming traditional methods like quantitative PCR. Notably, ddPCR provides enhanced consistency and reproducibility at it resists PCR inhibitors commonly found in environmental DNA samples. This study aimed to determine the most effective filter material for ddPCR protocols by assessing the reproducibility of species-specific gene copy numbers and filtration time across six filter types: cellulose acetate (CA), mixed cellulose ester (MCE), nylon (NY), polycarbonate (PC), polyethersulfone (PES), and polyvinylidene fluoride (PVDF). The study used two species of Chattonella marina complexes as a case study. Filtration rates were slower for NY, PC, and PVDF filters. Moreover, MCE, NY, PES, and PVDF yielded lower DNA amounts than other filters. Importantly, the CA filter exhibited the lowest variance (38–39%) and the highest determination coefficients (R2 = 0.92–0.96), indicating superior performance. These findings suggest that the CA filter is the most suitable for ddPCR quantification of marine protists, offering quick filtration and reliable reproducibility.

Deconvoluting multi-person biological mixtures and accurate characterization and identification of separated contributors using non-targeted single-cell DNA sequencing

The genetic characterization and identification of individuals who contributed to biological mixtures are complex and mostly unresolved tasks. These tasks are relevant in various fields, particularly in forensic investigations, which frequently encounters crime scene stains generated by more than one person. Currently, forensic mixture deconvolution is mostly performed subsequent to forensic DNA profiling at the level of the mixed DNA profiles, which comes with several limitations. Some previous studies attempted at separating single cells prior to forensic DNA profiling. However, these approaches are biased at selection of the cells and, due to their targeted DNA analysis on low template DNA, provide incomplete and unreliable forensic DNA profiles. We recently demonstrated the feasibility of performing mixture deconvolution prior to forensic DNA profiling through the utilization of a non-targeted single-cell transcriptome sequencing (scRNA-seq). In addition to individual-specific mixture deconvolution, this approach also allowed accurate characterisation of biological sex, biogeographic ancestry and individual identification of the separated mixture contributors. However, RNA has the forensic disadvantage of being prone to degradation, and sequencing RNA - focussing on coding regions - limits the number of single nucleotide polymorphisms (SNPs) utilized for genetic mixture deconvolution, characterization, and identification. These limitations can be overcome by performing single-cell sequencing on the level of DNA instead of RNA. Here, for the first time, we applied non-targeted single-cell DNA sequencing (scDNA-seq) by applying the scATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) technique to address the challenges of mixture deconvolution in the forensic context. We demonstrated that scATAC-seq, together with our recently developed De-goulash data analysis pipeline, is capable of deconvoluting complex blood mixtures of five individuals from both sexes with varying biogeographic ancestries. We further showed that our approach achieved correct genetic characterization of the biological sex and the biogeographic ancestry of each of the separated mixture contributors and established their identity. Furthermore, by analysing in-silico generated scATAC-seq data mixtures, we demonstrated successful individual-specific mixture deconvolution of i) highly complex mixtures of 11 individuals, ii) balanced mixtures containing as few as 20 cells (10 per each individual), and iii) imbalanced mixtures with a ratio as low as 1:80. Overall, our proof-of-principle study demonstrates the general feasibility of scDNA-seq in general, and scATAC-seq in particular, for mixture deconvolution, genetic characterization and individual identification of the separated mixture contributors. Furthermore, it shows that compared to scRNA-seq, scDNA-seq detects more SNPs from fewer cells, providing higher sensitivity, that is valuable in forensic genetics.

The binding affinity-dependent inhibition of cell growth and viability by DNA sulfur-binding domains.

Increasing evidence suggests that DNA phosphorothioate (PT) modification serves several purposes in the bacterial host, and some restriction enzymes specifically target PT-DNA. PT-dependent restriction enzymes (PDREs) bind PT-DNA through their DNA sulfur binding domain (SBD) with dissociation constants (KD) of 5 nM~1 μM. Here, we report that SprMcrA, a PDRE, failed to dissociate from PT-DNA after cleavage due to high binding affinity, resulting in low DNA cleavage efficiency. Expression of SBDs in Escherichia coli cells with PT modification induced a drastic loss of cell viability at 25°C when both DNA strands of a PT site were bound, with one SBD on each DNA strand. However, at this temperature, SBD binding to only one PT DNA strand elicited a severe growth lag rather than lethality. This cell growth inhibition phenotype was alleviated by raising the growth temperature. An invitro assay mimicking DNA replication and RNA transcription demonstrated that the bound SBD hindered the synthesis of new DNA and RNA when using PT-DNA as the template. Our findings suggest that DNA modification-targeting proteins might regulate cellular processes involved in DNA metabolism in addition to being components of restriction-modification systems and epigenetic readers.

Evaluation of Torque Teno Virus DNA Load as a Predictive Biomarker in Kidney Transplant Recipients Converted from Calcineurin Inhibitors to Belatacept

Belatacept is a relevant alternative to calcineurin inhibitors (CNIs) after kidney transplantation (KT). Circulating Torque Teno virus (TTV) DNA load is correlated to infections and rejection risks post-KT in patients treated with CNIs. The aim of this study was to assess the TTV DNA load profile in kidney transplant recipients converted from CNIs to belatacept and explore its use as a predictive biomarker. Sixty-eight single-center kidney transplanted recipients who were converted from CNIs to belatacept between June, 2015 and December, 2020 were included in this study. Whole blood TTV DNA load was measured before, at 3, 6, and 12 months post-belatacept conversion. Our primary end point was to assess the TTV DNA load profile and correlate the results with rejection and opportunistic infection (OPI). TTV DNA load remained stable after belatacept conversion, that is, 3.8 (3.1-4.9), 4.4 (3.2-5.4), 4.0 (3.0-5.7) and 4.2 (3.0-5.2) log10 copies/ml at baseline, 3, 6, and 12 months, respectively. No correlation was found between TTV DNA load and post-KT complications. Chronic allograft dysfunction at 1 year postconversion was associated with a lower TTV DNA load after 6 and 12-months (P= 0.014 and P= 0.021, respectively). A higher TTV DNA load was found in older patients and in those with higher body mass index (BMI) (P= 0.023 and P= 0.005, respectively). Conversion from CNIs to belatacept did not affect TTV DNA load. OPIs or acute rejection occurrences were not associated with TTV DNA load. However, low TTV (lTTV) DNA load after 6 months postconversion may be a promising tool to predict graft dysfunction risk at 1-year postconversion.

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. The study is funded by the Iran University of Medical Sciences and no competing interest exists. N/A.

Impact of live feed substitution with formulated diets on the development, digestive capacity, biochemical composition, and rearing water quality of European lobster (Homarus gammarus, L.) larvae

The lobster aquaculture industry, constrained by limited access to viable and cost-effective feed sources, prompted an investigation into the potential of replacing live feeds with formulated diets for Homarus gammarus larval culture, exploring alternative protein ingredients. Four diets were tested on newly-hatched H. gammarus: live Artemia nauplii (ART), an extruded control diet with krill meal and fishmeal as main protein sources (CTRL), and diets with 15% of dietary protein replaced by shrimp waste meal (SWM) or black soldier fly meal (BSF).Results revealed significant diet-induced effects on digestive enzymes in H. gammarus larvae. ART-fed lobsters exhibited increased trypsin and amylase activities but lower lipase activity than those fed extruded diets, suggesting more efficient utilization of protein and carbohydrate in ART-fed larvae, while formulated diets compensated with efficient lipid utilization. BSF and SWM diets increased exo- and endochitinase activities, likely due to chitin presence. Additionally, CTRL and BSF-fed larvae exhibited elevated body glycogen content. ART-fed larvae displayed a significantly higher ratio of low DNA to high DNA bacteria in their rearing water, indicative of lower nutrient loading. Despite these diet-induced impacts, there was no dietary effect on growth and survival during H. gammarus larval development.The study suggests successful formulated feed utilization by homarid larvae, even with alternative ingredients like BSF and SWM, offering a promising solution to reduce reliance on live feeds in lobster aquaculture. Further research is crucial to assess the long-term implications of the observed physiological responses to the different diets in later life stages.

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil.

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0-3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR's capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA.

Discovery of 3-hydroxymethyl-azetidine derivatives as potent polymerase theta inhibitors

Inhibition of the low fidelity DNA polymerase Theta (Polθ) is emerging as an attractive, synthetic-lethal antitumor strategy in BRCA-deficient tumors. Here we report the AI-enabled development of 3-hydroxymethyl-azetidine derivatives as a novel class of Polθ inhibitors featuring central scaffolding rings. Structure-based drug design first identified A7 as a lead compound, which was further optimized to the more potent derivative B3 and the metabolically stable deuterated compound C1. C1 exhibited significant antiproliferative properties in DNA repair-compromised cells and demonstrated favorable pharmacokinetics, showcasing that 3-hydroxymethyl-azetidine is an effective bio-isostere of pyrrolidin-3-ol and emphasizing the potential of AI in medicinal chemistry for precise molecular modifications.

Evaluation of HIV-1 DNA levels among adolescents living with perinatally acquired HIV-1 in Yaounde, Cameroon: A contribution to paediatric HIV cure research in Sub-Saharan Africa

BackgroundWith the advent of antiretroviral therapy (ART), most children living with HIV in sub-Saharan Africa (SSA) are growing toward adolescence, with scarcity of evidence on the size of viral reservoirs to enhance paediatric cure research strategies. This study aims to compare HIV-1 proviral DNA levels according to virological response among adolescents living with perinatally acquired HIV-1 (ALPHIV) and identify associated-factors in the Cameroonian context. MethodsIn this observational cohort study, HIV-1 RNA viremia and CD4+ T-cell count were assessed through RT-PCR and flow cytometry respectively at three time-points over 18 months of observation. At the third time-point, 80 randomly-selected participants were classified as with viremia (≥50 HIV-1 copies/mL; n = 40) or without viremia (<50 HIV-1 copies/mL; n = 40); immune-competent (≥500 CD4+ T cells/mm3) or immunocompromised (<500 CD4+ T cells/mm3). Among these participants, total HIV-1 DNA load was quantified through droplet digital PCR using Bio-Rad QX200. ResultsOf the 80 randomly-selected adolescents, median [IQR] age was 15 (13-17) years, 56.2% were female, duration on ART was 9.3 [5.4–12.2] years. Among the 40 viremic ones (median viremia 7312 [283–71482]) HIV-1 copies/ml, 75.0% (30/40) were in virological failure (≥1000 HIV-1 copies/ml), while median of CD4 T cells were 494 [360–793] cell/mm3 with 48.8% (39/80) immunocompromised. No significant variation in HIV-1 RNA viremia and CD4 T cell count was observed between the three time-points, and 13.7% (11/80) adolescents remained aviremic and immune-competent throughout (stable adolescents). A positive and moderate correlation (r = 0.59; p < 0.001) was found between HIV-1 DNA levels and HIV- 1 RNA viremia. Regarding the CD4 T cell count, a negative and weak correlation (r = −0.28; p = 0.014) was found with HIV-1 DNA loads only among adolescents with viremia. Starting ART within the first year of life, ART for over 9 years and aviremia appear as predictors of low HIV-1 DNA loads. ConclusionAmong ALPHIV, high HIV-1 RNA indicates an elevated viral reservoir size, representing a drawback to cure research. Interestingly, early ART initiation and longer ARTduration lead to sustained viral control and limited HIV-1 reservoir size. As limited size of viral reservoir appears consistent with viral control and immune competence, adolescents with sustained viral control (about 14% of this target population) would be candidates for analytical ART interruptions toward establishing paediatric post-treatment controllers in SSA.

Association of dynamics of anellovirus loads with hospital-acquired pneumonia in brain-injured patients during the intensive care unit stay.

Critical illness induces immune disorders associated with an increased risk of hospital-acquired pneumonia (HAP) and acute respiratory distress syndrome (ARDS). Torque Teno Virus (TTV), from the Anelloviridae family, are proposed as a biomarker to measure the level of immunosuppression. Our objective was to describe the kinetics of TTV DNA loads and their association with critical-illness related complications. We performed a longitudinal study in 115 brain-injured patients from a prospective cohort, collected endotracheal and blood samples at three time points (T1, T2, T3) during the two weeks post-admission in intensive care unit, and measured viral DNA loads using the TTV R-gene® kit (Biomerieux) and a pan-Anelloviridae in house qRT-PCR. TTV DNA was detected in the blood of 69, 71, and 64% of brain-injured patients at T1, T2 and T3 respectively. Time-associated variations of TTV and Anellovirus (AV) DNA loads were observed. Using a linear mixed-effects model, we found that HAP and ARDS were associated with lower blood AV DNA loads. Our results show that HAP or ARDS in critically ill patients are associated to changes in AV DNA loads, and should be evaluated further as a biomarker of immune disorders leading to these complications.

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

DNA damage levels in peripheral blood mononuclear cells before and after first cycle of chemotherapy have comparable prognostic values in germ cell tumor patients.

Germ cell tumors (GCTs) represent the most frequent solid malignancy in young men. This malignancy is highly curable by cisplatin (CDDP)-based chemotherapy. However, there is a proportion of patients having a poor prognosis due to refractory disease or its relapse. No reliable biomarkers being able to timely and accurately stratify poor prognosis GCT patients are currently available. Previously, we have shown that chemotherapy-naïve GCT patients with higher DNA damage levels in peripheral blood mononuclear cells (PBMCs) have significantly worse prognosis compared to patients with lower DNA damage levels. DNA damage levels in PBMCs of both chemotherapy-naïve and first cycle chemotherapy-treated GCT patients have been assessed by standard alkaline comet assay and its styrene oxide (SO)-modified version. These levels were correlated with clinico-pathological characteristics. We re-confirm prognostic value of DNA damage level in chemotherapy-naïve GCT patients and reveal that this prognosticator is equally effective in GCT patients after first cycle of CDDP-based chemotherapy. Furthermore, we demonstrate that SO-modified comet assay is comparably sensitive as standard alkaline comet assay in case of patients who underwent first cycle of CDDP-based chemotherapy, although it appears more suitable to detect DNA cross-links. We propose that DNA damage levels in PBMCs before and after first cycle of CCDP-based chemotherapy are comparable independent prognosticators for progression-free and overall survivals in GCT patients. Therefore, their clinical use is highly advised to stratify GCT patients to identify those who are most at risk of developing disease recurrence or relapse, allowing tailoring therapeutic interventions to poor prognosis individuals, and optimizing their care management and treatment regimen.

Excavatolide C/cisplatin combination induces antiproliferation and drives apoptosis and DNA damage in bladder cancer cells.

Excavatolide C (EXCC), a marine coral-derived compound, exhibits an antiproliferation effect on bladder cancer cells. The present study evaluated the improvement in the antiproliferation ability of EXCC by co-treatment with cisplatin in bladder cancer cells. EXCC/cisplatin (12.5 and 1μg/mL) showed higher antiproliferation effects on bladder cancer cells than single treatments (EXCC or cisplatin alone) in the 48h ATP assay. EXCC/cisplatin also enhanced the increase in subG1, annexin V-mediated apoptosis, and activation of poly (ADP-ribose) polymerase (PARP) and several caspases (caspases 3, 8, and 9) compared to the single treatments. Cellular and mitochondrial oxidative stress was enhanced with EXCC/cisplatin compared to the single treatments according to analyses of reactive oxygen species (ROS), mitochondrial superoxide, and mitochondrial membrane potential; in addition, cellular antioxidants, such as glutathione (GSH), and the mRNA expressions of antioxidant signaling genes (catalase and NFE2-like bZIP transcription factor 2) were downregulated. EXCC/cisplatin treatment produced more DNA damage than the single treatments, as indicated by γH2AX and 8-hydroxy-2'-deoxyguanosine levels. Moreover, several DNA repair genes for homologous recombination (HR) and non-homologous end joining (NHEJ) were downregulated in EXCC/cisplatin compared to others. The addition of the GSH precursor N-acetylcysteine, which has ROS scavenging activity, attenuated all EXCC/cisplatin-induced changes. Notably, EXCC/cisplatin showed lower antiproliferation, apoptosis, ROS induction, GSH depletion, and γH2AX DNA damage in normal cells than in bladder cancer cells. Therefore, the co-treatment of EXCC/cisplatin reduces the proliferation of bladder cancer cells via oxidative stress-mediated mechanisms with normal cell safety.

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction(PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having anHPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

MDSC-targeted liposomal all-trans retinoic acid suppresses mMdscs and improves immunotherapy in HBV infection

ABSTRACT Background Myeloid-derived suppressor cells (MDSCs) are evolving as a prominent determinant in cancer occurrence and development and are functionally found to suppress T cells in cancer. Not much research is done regarding its involvement in viral infections. This research was designed to investigate the role of MDSCs in hepatitis B virus (HBV) infection and how targeting these cells with our novel all-trans retinoic acid encapsulated liposomal formulation could improve immunotherapy in C57BL/6 mice. Methods Ten micrograms (10 μg) of plasmid adeno-associated virus (pAAV/HBV 1.2, genotype A) was injected hydrodynamically via the tail vein of C57BL/6 mice. An all-trans retinoic acid encapsulated liposomal formulation (L-ATRA) with sustained release properties was used in combination with tenofovir disoproxil fumarate (TDF), a nucleotide analog reverse transcriptase inhibitor (nRTI) to treat the HBV infection. The L-ATRA formulation was given at a dose of 5 mg/kg intravenously (IV) twice a week. The TDF was given orally at 30 mg/kg daily. Results Our results revealed that L-ATRA suppresses MDSCs in HBV infected mice and enhanced T-cell proliferation in vitro. In vivo studies showed higher and improved immunotherapeutic effect in mice that received L-ATRA and TDF concurrently in comparison with the groups that received monotherapy. Lower HBV DNA copies, lower concentrations of HBsAg and HBeAg, lower levels of ALT and AST and less liver damage were seen in the mice that received the combination therapy of L-ATRA + TDF. Conclusions In effect, targeting MDSCs with the combination of L-ATRA and TDF effectively reduced mMDSC and improved immunotherapy in the HBV infected mice. Targeting MDSCs could provide a breakthrough in the fight against hepatitis B virus infection.